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WO2006071777A2 - Reparation et regeneration de tissus mous au moyen de cellules derivees du post-partum et produits cellulaires - Google Patents

Reparation et regeneration de tissus mous au moyen de cellules derivees du post-partum et produits cellulaires Download PDF

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Publication number
WO2006071777A2
WO2006071777A2 PCT/US2005/046808 US2005046808W WO2006071777A2 WO 2006071777 A2 WO2006071777 A2 WO 2006071777A2 US 2005046808 W US2005046808 W US 2005046808W WO 2006071777 A2 WO2006071777 A2 WO 2006071777A2
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cells
cell
tissue
derived
protein
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WO2006071777A3 (fr
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Laura Brown
Alexander M. Harmon
Anna Gosiewska
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Ethicon Incorporated
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Priority to EP05855379A priority Critical patent/EP1831355A2/fr
Publication of WO2006071777A2 publication Critical patent/WO2006071777A2/fr
Publication of WO2006071777A3 publication Critical patent/WO2006071777A3/fr

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/21—Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
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    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins

Definitions

  • This invention relates to the field of mammalian cell biology and cell culture.
  • the invention relates to cultured cells derived from postpartum tissue having the potential to support cells of and/or differentiate to cells of a soft tissue lineage, and methods of preparation and use of those postpartum tissue-derived cells.
  • the invention also relates to methods for the use of such postpartum-derived cells in the regeneration and repair of soft tissue, and in cell-based therapies for conditions of soft tissue.
  • Injuries to soft tissue are quite common.
  • Soft tissue injury is damage to the pelvic floor. This is a potentially serious medical condition that may occur during childbirth or from complications thereof which can lead to damage to the vesicovaginal fascia. Such an injury can result in a cystocele, which is a herniation of the bladder.
  • Similar medical conditions include rectoceles (a herniation of the rectum), enteroceles (a protrusion of the intestine through the rectovaginal or vesicovaginal pouch), and enterocystoceles (a double hernia in which both the bladder and intestine protrude).
  • hernia The basic manifestation of a hernia is a protrusion of an organ into a defect within the fascia.
  • Surgical approaches toward hernia repair have focused on reducing the * ⁇ >* " " -pr Tes-e-n IcJeS ofIi thIBe hze 11 r 1 n 1 IiaSl cBonOtenSts i .n t ,he pe ⁇ toneal cavity and , generati .ng a ⁇ firm c 1 losure of the fascial defect either by using prosthetic, allogeneic, or autologous materials.
  • a number of techniques have been used to produce this closure including the movement of autologous tissues and the use of synthetic mesh products.
  • Drawbacks to these current products and procedures include hernia recurrence upon weakening of the closure.
  • ligaments and tendons are viscoelastic structures that mediate normal joint movement and stability and are subject to tear and brittleness with age or injury. These structures are complex, relatively static collagenous structures with functional links to the bone, muscle, menisci, and other nearby tendons and ligaments.
  • Soft tissue conditions further include, for example, conditions of skin (e.g., ischemic wounds, diabetic wounds, scar revision or the treatment of traumatic wounds, severe burns, skin ulcers (e.g., decubitus (pressure) ulcers, venous ulcers, and diabetic ulcers), and surgical wounds such as those associated with the excision of skin cancers); vascular conditions (e.g., vascular disease such as peripheral arterial disease, abdominal aortic aneurysm, carotid disease, and venous disease; vascular injury; and improper vascular development); conditions affecting vocal cords; cosmetic conditions (e.g., those involving repair, augmentation, or beautification); muscle diseases (e.g., congenital myopathies; myasthenia gravis; inflammatory, neurogenic, and myogenic muscle diseases; and muscular dystrophies such as Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, limb-girdle-muscular dystrophy, facio
  • Surgical approaches to correct soft tissue conditions or defects in the body generally involve the implantation of structures made of biocompatible, inert materials that attempt to replace or substitute for the defective function. Implantation of non-biodegradable materials results in permanent structures that remain in the body as a foreign object. Implants that are made of resorbable materials are suggested for use as temporary replacements where the object is to allow the healing process to replace the resorbed material.
  • these approaches have met with limited success for the long-term correction of structures in the body.
  • the invention is generally directed to postpartum-derived cells which are derived from postpartum tissue which is substantially free of blood and which is capable of self- renewal and expansion in culture and having the potential to differentiate into or provide trophic support to a cell of a mesodermal or ectodermal lineage, for example, a soft tissue cell phenotype.
  • the present invention provides cells derived from human postpartum tissue substantially free of blood, capable of self -renewal and expansion in culture, having the ability to differentiate to or provide trophic support to cells of a soft tissue phenotype or to differentiate to cells of a soft tissue phenotype; requiring L-valine for growth; capable of growth in about 5% to about 20% oxygen; and further having at least one of the following characteristics: production of at least one of GCP-2, tissue factor, vimentin, and alpha-smooth muscle actin; lack of production of at least one of lack of production of at least one of NOGO-A, GRO- alpha or oxidized low density lipoprotein receptor, as detected by flow cytometry; production of at least one of CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA- A,B, C; lack of production of at least one of CD31, CD34, CD45, CD80, CD86, CDl 17, CD
  • the postpartum-derived cell is an umbilicus-derived cell. In other embodiments, it is a placenta-derived cell. In specific embodiments, the cell has all identifying features of any one of: cell type PLA 071003 (P8) (ATCC Accession No. PTA- 6074); cell type PLA 071003 (PIl) (ATCC Accession No. PTA-6075); cell type PLA 071003 (P16) (ATCC Accession No. PTA-6079); cell type UMB 022803 (P7) (ATCC Accession No. PTA-6067); or cell type UMB 022803 (P17) (ATCC Accession No. PTA-6068).
  • the postpartum-derived cells of the invention are preferably human cells.
  • the cells may provide trophic support to cells of a soft tissue phenotype, for example, that of fascia, epithelium, endothelium, skin, vasculature, muscles, tendons, and ligaments.
  • the cells themselves may be induced to differentiate to a soft tissue phenotype.
  • Populations of PPDCs are provided by the invention.
  • a population of postpartum-derived cells is mixed with another population of cells.
  • the cell population is heterogeneous.
  • a heterogeneous cell population of the invention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% undifferentiated or differentiation-induced PPDCs of the invention.
  • the heterogeneous cell populations of the invention may further comprise, for example, stem cells, epithelial cells (e.g., mucosal cells, for example, cells of oral mucosa; gastrointestinal tract; nasal epithelium; respiratory tract epithelium; vaginal epithelium; corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), smooth muscle cells, myoblasts, myocytes, stromal cells, bladder urothelial cells, cells of the lary
  • Cell populations of the invention may be substantially homogeneous, i.e., comprise substantially only PPDCs (preferably at least about 96%, 97%, 98%, 99% or more PPDCs).
  • Homogeneous cell populations of the invention may comprise umbilicus- or placenta-derived cells.
  • Homogeneous populations of placenta-derived cells may be of neonatal or maternal lineage.
  • Homogeneity of a cell population may be achieved by any method known in the art, for example, by cell sorting (e.g., flow cytometry), bead separation, or by clonal expansion.
  • the invention also provides heterogeneous and homogeneous cell cultures containing undifferentiated or differentiation-induced postpartum-derived cells of the invention.
  • Some embodiments of the invention provide a matrix for administration to a patient.
  • the matrix is seeded with a population of postpartum-derived cells (PPDCs) of the invention.
  • the matrix is pretreated with a population of postpartum-derived cells of the invention.
  • the PPDCs may be differentiation-induced or undifferentiated.
  • the population of PPDCs may be substantially homogeneous or heterogeneous.
  • the matrix may be inoculated with PPDCs and cells of at least one other desired cell type, for example but not by way of limitation, epithelial cells (e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, melanocytes, dermal fibroblasts, vascular endothelial cells (e.g., aortic endothelial cells, coronary • 'C ⁇ / V S O S / * i: s s o a
  • epithelial cells e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium
  • bone marrow cells e.g., adipocytes, stem cells, keratinocytes, melanocyte
  • artery endothelial cells pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), myoblasts, myocytes, stromal cells, and other soft tissue cells or progenitor cells.
  • the matrix may contain or be pre- treated with one or more bioactive factors including, for example, drugs, anti -inflammatory agents, antiapoptotic agents, and growth factors.
  • the seeded or pre-treated matrices can be introduced into a patient's body in any way known in the art, including but not limited to implantation, injection, surgical attachment, transplantation with other tissue, and the like.
  • the matrices of the invention may be configured in vitro or in vivo to a desired shape and/or size, for example, to the shape and/or size of a tissue or organ in vivo.
  • the scaffolds of the invention may be flat or tubular or may comprise sections thereof.
  • the scaffolds of the invention may be multilayered.
  • PPDC products including extracellular matrix (ECM) of PPDCs, cell fractions (e.g., soluble cell fractions; insoluble cell fractions; cell lysate, supernates of cell fractions; cell membrane-containing fractions) of PPDCs, and PPDC-conditioned medium.
  • ECM extracellular matrix
  • Matrices of the invention may comprise or be pre-treated with any one of the foregoing PPDC-products.
  • compositions of PPDCs or a PPDC product and one or more bioactive factors, for example, but not limited to growth factors, anti-apoptotic agents, anti-inflammatory agents, and/or differentiation-inducing factors.
  • bioactive factors for example, but not limited to growth factors, anti-apoptotic agents, anti-inflammatory agents, and/or differentiation-inducing factors.
  • compositions of the invention comprise PPDCs and one or more other cell types, for example, epithelial cells (e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, melanocytes, dermal fibroblasts, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), myoblasts, myocytes, stromal cells, and other soft tissue cells or progenitor cells.
  • PPDCs provide trophic support to a soft tissue cell.
  • soft tissue cells offered trophic support by PPDCs include cells of cartilage tissue, meniscal tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascia tissue, periosteal tissue, ocular tissue, pericardial tissue, lung tissue, synovial tissue, nerve tissue, kidney tissue, bone marrow, urogenital tissue, intestinal tissue, liver tissue, pancreas tissue, spleen tissue, or adipose tissue.
  • PPDCs are induced to differentiate to a cell of a soft tissue phenotype, for example but not limited to, a phenotype of a cell of cartilage tissue, meniscal tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascia tissue, periosteal tissue, ocular tissue, pericardial tissue, lung tissue, synovial tissue, nerve tissue, kidney tissue, bone marrow, urogenital tissue, intestinal tissue, liver tissue, pancreas tissue, spleen tissue, or adipose tissue.
  • a soft tissue phenotype for example but not limited to, a phenotype of a cell of cartilage tissue, meniscal tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascia tissue, periosteal tissue, ocular tissue, pericardial tissue, lung tissue, synovial tissue, nerve tissue,
  • compositions of the postpartum-derived cells, extracellular matrix produced thereby, cell fractions, and PPDC-conditioned medium are included within the scope of the invention.
  • the pharmaceutical compositions preferably include a pharmaceutically acceptable carrier or excipient.
  • methods of regenerating soft tissue in a patient in need thereof by administering PPDCs, PPDC products, PPDC compositions, or matrices of the invention to a patient are provided.
  • a soft tissue condition in a patient by administering one or more postpartum-derived cell, PPDC population, or PPDC products of the invention (e.g., ECM, matrix, cell fraction, conditioned medium, or composition of the invention).
  • Treatment of a soft tissue condition according to the invention includes but is not limited to trophic support of soft tissue, tissue repair, tissue reconstruction, tissue bulking, cosmetic treatment, therapeutic treatment, tissue augmentation, and tissue sealing.
  • the PPDCs and PPDC products of the invention may be used in the treatment of, for example but not by way of limitation, a hernia, damage to the pelvic floor, a burn, cancer, traumatic injury, scars, skin ulcers (e.g., decubitus (pressure) ulcers, venous ulcers, and diabetic ulcers), ischemic wounds, surgical wounds such as those associated with the excision of skin cancers; vascular disease such as peripheral arterial disease, abdominal aortic aneurysm, carotid disease, and venous disease; muscle disease (e.g., congenital myopathies; myasthenia gravis; inflammatory, neurogenic, and myogenic muscle diseases; and muscular dystrophies such as Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, limb-girdle-muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophies, oculopharyngeal muscular dystrophy
  • the invention further provides methods of providing trophic support to cells such as soft tissue cells by exposing or contacting a cell to a postpartum-derived cell of the invention or a PPDC-product.
  • soft tissue cells for which PPDCs may provide trophic support according to the invention include a stem cell, a myocyte, a myoblast, a keratinocyte, a melanocyte, a dermal fibroblast, a bone marrow cell, an adipocyte, an epithelial cell, a stromal cell, and an endothelial cell (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/
  • Methods of the invention further include methods of inducing angiogenesis by exposing a soft tissue cell to a PPDC or PPDC product.
  • soft tissue cells that form endothelial networks in accordance with the methods of the invention include aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells).
  • Methods of providing trophic support or stimulating angiogenesis of the invention may be effected in vitro or in vivo.
  • Methods of the invention also include methods of treating a patient in need of angiogenic factors by administering to a patient a PPDC or PPDC product of the invention.
  • the methods of producing a vascular network involve exposing or contacting a population of soft tissue cells to a PPDC cell population or PPDC product.
  • the population of soft tissue cells preferably contains at least one soft tissue cell of an aortic endothelial cell, coronary artery endothelial cell, pulmonary artery endothelial cell, iliac artery endothelial cell, microvascular endothelial cell, umbilical artery endothelial cell, and umbilical vein endothelial cell.
  • the method of producing a vascular network may be performed in vitro or in vivo.
  • the invention also encompasses the vascular networks produced by the methods of the invention.
  • Methods of treating a condition such as a soft tissue condition in a patient by administering the vascular networks also are provided.
  • the soft tissue condition is a vascular condition, such as a vascular disease or injury or improper vascular development.
  • the vascular network is administered by transplantation to the patient.
  • kits of the PPDCs and/or PPDC products preferably include at least one component of a matrix, a hydrating pf'f / U S O Eu /" 1 Hi SOB """' agent, a cell culture substrate, a bioactive factor, a second cell type, a differentiation-inducing agent, cell culture media, and instructions, for example, for culture of the cells or administration of the cells and/or cell products.
  • the invention provides methods for identifying compounds that modulate growth and/or differentiation of a postpartum-derived cell comprising contacting a cell of the invention with a compound and monitoring the cell for growth or a marker of differentiation. Also provided are methods for identifying compounds toxic to a postpartum-derived cell of the invention by contacting the cell with a compound and monitoring survival of the cell.
  • Stem cells are undifferentiated cells defined by their ability at the single cell level to both self -renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate hi vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
  • Stem cells are classified by their developmental potential as: (1) totipotent - able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent - able to give rise to all embryonic cell types; (3) multipotent - able to give rise to a subset of cell lineages, but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self-renewal), blood cell-restricted oligopotent progenitors, and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent - able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent - able to give rise to a single cell lineage (e.g., spermatogenic stem cells).
  • HSC hematopoietic stem cells
  • Stem cells are also categorized on the basis of the source from which they may be obtained.
  • An adult stem cell is generally a multipotent undifferentiated cell found in tissue comprising multiple differentiated cell types. The adult stem cell can renew itself and, under normal circumstances, differentiate to yield the specialized cell types of the tissue from which it originated, and possibly other tissue types.
  • An embryonic stem cell is a pluripotent cell from the inner cell mass of a blastocyst-stage embryo.
  • a fetal stem cell is one that originates from fetal tissues or membranes.
  • a postpartum stem cell is a multipotent or pluripotent cell that originates substantially from extraembryonic tissue available after birth, namely, the placenta and the umbilicus.
  • Postpartum stem cells may be blood-derived (e.g., as are those obtained from umbilical cord blood) or non-blood-derived (e.g., as obtained from the non-blood tissues of the umbilical cord and placenta).
  • Embryonic tissue is typically defined as tissue originating from the embryo (which in humans refers to the period from fertilization to about six weeks of development. Fetal tissue refers to tissue originating from the fetus, which in humans refers to the period from about six weeks of development to parturition. Extraembryonic tissue is tissue associated with, but not originating from, the embryo or fetus. Extraembryonic tissues include extraembryonic membranes (chorion, amnion, yolk sac and allantois), umbilical cord and placenta (which itself forms from the chorion and the maternal decidua basalis).
  • Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell, such as a nerve cell or a muscle cell, for example.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell.
  • the term committed, when applied to the process of differentiation refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell.
  • the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to.
  • the lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a lineage-specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
  • a progenitor cell is a cell that has the capacity to create progeny that are more differentiated than itself and yet retains the capacity to replenish the pool of progenitors.
  • stem cells themselves are also progenitor cells, as are the P u T / ' U b O ES / H-B B O K more immediate precursors to terminally differentiated cells.
  • a progenitor cell is often defined as a cell that is intermediate in the differentiation pathway, i.e., it arises from a stem cell and is intermediate in the production of a mature cell type or subset of cell types.
  • This type of progenitor cell is generally not able to self-renew. Accordingly, if this type of cell is referred to herein, it will be referred to as a non- renewing progenitor cell or as an intermediate progenitor or precursor cell.
  • the phrase differentiates into a mesodermal, ectodermal or endodermal lineage refers to a cell that becomes committed to a specific mesodermal, ectodermal or endodermal lineage, respectively.
  • Examples of cells that differentiate into a mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, chondrogenic, cardiogenic, dermatogenic, hematopoietic, endothelial, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or stromal.
  • Examples of cells that differentiate into ectodermal lineage include, but are not limited to epithelial cells, neurogenic cells, and neurogliagenic cells.
  • Examples of cells that differentiate into endodermal lineage include, but are not limited to pleurigenic cells, and hepatogenic cells, cells that give rise to the lining of the intestine, and cells that give rise to pancreogenic and splanchogenic cells.
  • the cells of the invention are referred to herein as postpartum-derived cells or postpartum cells (PPDCs). Subsets of the cells of the present invention are referred to as placenta-derived cells (PDCs) or umbilicus-derived cells (UDCs). In addition, the cells may be described as being stem or progenitor cells, the latter term being used in the broad sense.
  • the term derived is used to indicate that the cells have been obtained from their biological source and grown or otherwise manipulated in vitro (e.g., cultured in a growth medium to expand the population and/or to produce a cell line). The in vitro manipulations of postpartum-derived cells and the unique features of the postpartum-derived cells of the present invention are described in detail below.
  • Cell culture refers generally to cells taken from a living organism and grown under controlled conditions ("in culture").
  • a primary cell culture is a culture of cells, tissues or organs taken directly from organisms and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and/or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is sometimes measured by the amount of time needed for the cells to double in number. This is referred to as doubling time.
  • a cell line is a population of cells formed by one or more subcultivations of a primary cell culture. Each round of subculturing is referred to as & passage.
  • cells When cells are subcultured, they are referred to as having been passaged.
  • a specific population of cells, or a cell line is sometimes referred to or characterized by the number of times it has been passaged.
  • a cultured cell population that has been passaged ten times may be referred to as a PlO culture.
  • the primary culture i.e., the first culture following the isolation of cells from tissue, is designated PO.
  • the cells Following the first subculture, the cells are described as a secondary culture (Pl or passage 1).
  • P2 or passage 2 tertiary culture
  • a conditioned medium is a medium in which a specific cell or population of cells has been cultured, and then removed. While the cells are cultured in the medium, they secrete cellular factors that can provide trophic support to other cells. Such trophic factors include, but are not limited to hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules.
  • the medium containing the cellular factors is the conditioned medium.
  • a trophic factor is defined as a substance that promotes survival, growth, proliferation, maturation, differentiation, and/or maintenance of a cell, or stimulates increased activity of a cell.
  • Trophic support is used herein to refer to the ability to promote survival, growth, proliferation, maturation, differentiation, and/or maintenance of a cell, or to stimulate increased activity of a cell.
  • senescence also replicative senescence or cellular senescence refers to a property attributable to finite cell cultures; namely, their inability to grow beyond a finite number of population doublings (sometimes referred to as Hayflick's limit).
  • cellular senescence was first described using fibroblast-like cells, most normal human cell types that can be grown successfully in culture undergo cellular senescence.
  • the in vitro lifespan of different cell types varies, but the maximum lifespan is typically fewer than 100 population doublings (this is the number of doublings for all the cells in the culture to become senescent and thus render the culture unable to divide).
  • Senescence does not depend on chronological time, but rather is measured by the number of cell divisions, or population doublings, the culture has undergone. Thus, cells made quiescent by removing essential growth factors are able to resume growth and division when the growth factors are reintroduced, and thereafter carry out the same number of doublings as equivalent cells grown continuously. Similarly, when cells are frozen in liquid nitrogen after various numbers of population doublings and then thawed and cultured, they undergo substantially the same number of doublings as cells maintained unfrozen in culture. Senescent cells are not dead or dying cells; they are actually resistant to programmed cell death (apoptosis), and have been maintained in their nondividing state for as long as three years. These cells are very much alive and metabolically active, but they do not divide. The nondividing state of senescent cells has not yet been found to be reversible by any biological, chemical, or viral agent.
  • Growth medium refers to a culture medium sufficient for expansion of postpartum-derived cells.
  • Growth medium preferably contains Dulbecco's Modified Essential Media (DMEM). More preferably, Growth medium contains glucose. Growth medium preferably contains DMEM-low glucose (DMEM-LG) (Invitrogen, Carlsbad, CA). Growth medium preferably contains about 15% (v/v) serum (e.g., fetal bovine serum, defined bovine serum).
  • DMEM Dulbecco's Modified Essential Media
  • DMEM-LG DMEM-low glucose
  • serum e.g., fetal bovine serum, defined bovine serum.
  • Growth medium preferably contains at least one antibiotic agent and/or antimycotic agent (e.g., penicillin, streptomycin, amphotericin B, gentamicin, nystatin; preferably, 50 units/milliliter penicillin G sodium and 50 micrograms/milliliter streptomycin sulfate).
  • antibiotic agent and/or antimycotic agent e.g., penicillin, streptomycin, amphotericin B, gentamicin, nystatin; preferably, 50 units/milliliter penicillin G sodium and 50 micrograms/milliliter streptomycin sulfate.
  • Growth medium preferably contains 2-mercaptoethanol (Sigma, St. Louis MO). Most preferably, Growth medium contains DMEM-low glucose, serum, 2-mercaptoethanol, and an antibiotic agent.
  • standard growth conditions refers to standard atmospheric conditions comprising about 5% CO 2 , a temperature of about 35 ⁇ 39°C, more preferably 37°C, and a relative humidity of about 100%.
  • the term isolated refers to a cell, cellular component, or a molecule that has been removed from its native environment.
  • Soft tissue refers generally to extraskeletal structures found throughout the body and includes but is not limited to cartilage tissue, meniscal tissue, ligament tissue, tendon tissue, intervertebral disc tissue, periodontal tissue, skin tissue, vascular tissue, muscle tissue, fascia tissue, periosteal tissue, ocular tissue, pericardial tissue, lung tissue, synovial tissue, nerve tissue, kidney tissue, bone marrow, urogenital tissue, intestinal tissue, liver tissue, pancreas tissue, spleen tissue, or adipose tissue, and combinations thereof.
  • Soft tissue condition is an inclusive term encompassing acute and chronic conditions, disorders or diseases of soft tissue.
  • the term encompasses conditions caused by disease or trauma or failure of the tissue to develop normally.
  • soft tissue conditions include but are not limited to hernias, damage to the pelvic floor, tear or rupture of a tendon or ligament, skin wounds ⁇ e.g., scars, traumatic wounds, ischemic wounds, diabetic wounds, severe burns, skin ulcers (e.g., decubitus (pressure) ulcers, venous ulcers, and diabetic ulcers), and surgical wounds such as those associated with the excision of skin cancers); vascular conditions ⁇ e.g., vascular disease such as peripheral arterial disease, abdominal aortic aneurysm, carotid disease, and venous disease; vascular injury, improper vascular development); and muscle diseases (e.g., congenital myopathies; myasthenia gravis; inflammatory, neurogenic, and myogenic muscle
  • treating (or treatment of) a soft tissue condition refers to ameliorating the effects of, or delaying, halting or reversing the progress of, or delaying or preventing the onset of, a soft tissue condition as defined herein and includes trophic support of soft tissue, soft tissue repair, reconstruction ⁇ e.g., breast reconstruction), bulking, cosmetic treatment, therapeutic treatment, tissue augmentation (e.g., bladder augmentation), and tissue sealing.
  • the term effective amount refers to a concentration of a reagent or pharmaceutical composition, such as a growth factor, differentiation agent, trophic factor, cell population or other agent, that is effective for producing an intended result, including cell growth and/or differentiation in vitro or in vivo, or treatment of a soft tissue condition as described herein.
  • a reagent or pharmaceutical composition such as a growth factor, differentiation agent, trophic factor, cell population or other agent, that is effective for producing an intended result, including cell growth and/or differentiation in vitro or in vivo, or treatment of a soft tissue condition as described herein.
  • growth factors an effective amount may range from about 1 nanogram/milliliter to about 1 microgram/milliliter.
  • PPDCs as administered to a patient in vivo an effective amount may range from as few as several hundred or fewer to as many as several million or more. In specific embodiments, an effective amount of PPDCs may range from 10 3 -10 ⁇ .
  • effective period (or time) and effective conditions refer to a period of time or other controllable conditions ⁇ e.g., temperature, humidity for in vitro methods), necessary or preferred for an agent or pharmaceutical composition to achieve its intended result.
  • patient or subject refers to animals, including mammals, preferably humans, who are treated with the pharmaceutical compositions or in accordance with the methods described herein.
  • pharmaceutically acceptable carrier which may be used interchangeably with the term biologically compatible carrier or medium, refers to reagents, cells, compounds, materials (including, for example, matrices), compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carriers suitable for use in the present invention include liquids, semi-solid (e.g., gels) and solid materials ⁇ e.g., scaffolds).
  • biodegradable describes the ability of a material to be broken down ⁇ e.g., degraded, eroded, dissolved) in vivo.
  • the term includes degradation in vivo with or without elimination (e.g., by resorption) from the body.
  • the semi-solid and solid materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable).
  • a biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathways. Examples include, but are not limited to, hyaluronic acid and saline.
  • the terms autologous transfer, autologous transplantation, autograft and the like refer to treatments wherein the cell donor is also the recipient of the cell replacement therapy.
  • the terms allogeneic transfer, allogeneic transplantation, allograft and the like refer to treatments wherein the cell donor is of the same species as the recipient of the cell replacement therapy, but is not the same individual.
  • a cell transfer in which the donor's cells have been histocompatibly matched with a recipient is sometimes referred to as a syngeneic transfer.
  • the terms xenogeneic transfer, xenogeneic transplantation, xenograft and the like refer to treatments wherein the cell donor is of a different species than the recipient of the cell replacement therapy.
  • matrix refers to a support for the PPDCs or PPDC product of the invention, for example, a scaffold (e.g., VNW scaffold, foams such as PCL/PGA, ⁇ > C 1 , ' IJi H €1 S ./ ' ⁇ ; i Ii 3 U B or self-assembling peptides such as RAD16) or supporting medium (e.g., hydrogel or a biomaterial such as Collagen/oxidized regenerated cellulose).
  • a scaffold e.g., VNW scaffold, foams such as PCL/PGA, ⁇ > C 1 , ' IJi H €1 S ./ ' ⁇ ; i Ii 3 U B or self-assembling peptides such as RAD16
  • supporting medium e.g., hydrogel or a biomaterial such as Collagen/oxidized regenerated cellulose.
  • ANG2 (or Ang2) for angiopoietin 2
  • BDNF brain-derived neurotrophic factor
  • bFGF basic fibroblast growth factor
  • bid (BID) "bis in die” (twice per day);
  • DMEMdg (or DMEM:Lg, DMEM:LG) for DMEM with low glucose
  • EGF epidermal growth factor
  • FGF FGF (or F) for fibroblast growth factor
  • GDF-5 for growth and differentiation factor 5
  • GFAP for glial fibrillary acidic protein
  • HCAEC Human coronary artery endothelial cells
  • HGF for hepatocyte growth factor
  • hMSC Human mesenchymal stem cells
  • HNF-lalpha for hepatocyte-specific transcription factor
  • HUVEC for Human umbilical vein endothelial cells
  • IGF insulin-like growth factor
  • IL-8 for interleukin 8; f.' t l T / USD ⁇ i / H B BlIB " ' Kl 9 for keratin 19;
  • KGF for keratinocyte growth factor
  • MMP matrix metalloprotease
  • MSC for mesenchymal stem cells
  • OxLDLR for oxidized low density lipoprotein receptor
  • PDGFbb for platelet derived growth factor
  • PDGFr-alpha for platelet derived growth factor receptor alpha
  • Rantes (or RANTES) for regulated on activation, normal T cell expressed and secreted; rb for rabbit; rh for recombinant human;
  • TGFbetal for transforming growth factor beta2 TGFbetal for transforming growth factor beta2
  • TGFbeta-3 for transforming growth factor beta-3; T/ u s o y . ⁇ " ' "+ ⁇ B o B
  • TIMPl for tissue inhibitor of matrix metalloproteinase 1
  • VEGF for vascular endothelial growth factor
  • vWF for von Willebrand factor
  • alphaFP for alpha-fetoprotein
  • the invention provides postpartum-derived cells (PPDCs) derived from postpartum tissue substantially free of blood.
  • the PPDCs may be derived from placenta of a mammal including but not limited to human.
  • the cells are capable of self-renewal and expansion in culture.
  • the postpartum-derived cells have the potential to differentiate into cells of other phenotypes.
  • the invention provides, in one of its several aspects, cells that are derived from umbilicus, as opposed to umbilical cord blood.
  • the invention also provides, in one of its several aspects, cells that are derived from placental tissue.
  • the cells have been characterized as to several of their cellular, genetic, immunological, and biochemical properties.
  • the cells have been characterized by their growth, by their cell surface markers, by their gene expression, by their ability to produce certain biochemical trophic factors, and by their immunological properties.
  • a mammalian placenta and umbilicus are recovered upon or shortly after termination of either a full-term or pre-term pregnancy, for example, after expulsion after birth.
  • Postpartum tissue can be obtained from any completed pregnancy, full-term or less than full-term, whether delivered vaginally, or through other means, for example, cesarean section.
  • the postpartum tissue may be transported from the birth site to a laboratory in a sterile container such as a flask, beaker, culture dish, or bag.
  • the container may have a solution or medium, including but not limited to a salt solution, such as, for example, Dulbecco's Modified Eagle's Medium (DMEM) or phosphate buffered saline (PBS), or any solution used for transportation of organs used for transplantation, such as University of Wisconsin solution or perfluorochemical solution.
  • a salt solution such as, for example, Dulbecco's Modified Eagle's Medium (DMEM) or phosphate buffered saline (PBS), or any solution used for transportation of organs used for transplantation, such as University of Wisconsin solution or perfluorochemical solution.
  • DMEM Dulbecco's Modified Eagle's Medium
  • PBS phosphate buffered saline
  • antibiotic and/or antimycotic agents such as but not limited to penicillin, streptomycin, amphotericin B, gentamicin, and nystatin, may be added to the medium or buffer.
  • the postpartum tissue may be rinse
  • Isolation of PPDCs preferably occurs in an aseptic environment. Blood and debris are preferably removed from the postpartum tissue prior to isolation of PPDCs.
  • the postpartum tissue may be washed with buffer solution, such as but not limited to phosphate buffered saline.
  • the wash buffer also may comprise one or more antimycotic and/or antibiotic agents, such as but not limited to penicillin, streptomycin, amphotericin B, gentamicin, and nystatin.
  • the different cell types present in postpartum tissue are fractionated into subpopulations from which the PPDCs can be isolated.
  • This may be accomplished using techniques for cell separation including, but not limited to, enzymatic treatment to dissociate postpartum tissue into its component cells, followed by cloning and selection of specific cell types, for example but not limited to selection based on morphological and/or biochemical markers; selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); separation based upon differential cell agglutinability in the mixed population as, for example, with soybean agglutinin; freeze-thaw procedures; differential adherence properties of the cells in the mixed population; filtration; conventional and zonal centrifugation; centrifugal elutriation (counter-streaming centrifugation); unit gravity separation; countercurrent distribution; electrophoresis; and flow cytometry, for example, fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • postpartum tissue comprising a whole placenta or a fragment or section thereof is disaggregated by ultrasonic disruption, mechanical force (mincing or shear forces), enzymatic digestion with single or combinatorial proteolytic enzymes, such as a matrix metalloprotease and/or neutral protease, for example, collagenase, trypsin, dispase, LIBERASE (Boehringer Mannheim Corp., Indianapolis, IN), hyaluronidase, and/or pepsin, or a combination of mechanical and enzymatic methods.
  • the cellular component of the postpartum tissue may be disaggregated by methods using collagenase-mediated dissociation.
  • Enzymatic digestion methods preferably employ a combination of enzymes, such as a combination of a matrix metalloprotease and a neutral protease.
  • the matrix metalloprotease is preferably a collagenase.
  • the neutral protease is preferably thermolysin or dispase, and most preferably is dispase. More preferably, enzymatic digestion of postpartum tissue uses a combination of a matrix metalloprotease, a neutral protease, and a mucolytic enzyme for ? ' .
  • I'fn SfiiS digestion of hyaluronic acid such as a combination of collagenase, dispase, and hyaluronidase or a combination of LIBERASE (Boehringer Mannheim Corp., Indianapolis, TN) and hyaluronidase.
  • Collagenase may be type 1, 2, 3, or 4.
  • enzymes known in the art for cell isolation include papain, deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, or elastase, that may be used either on their own or in combination with other enzymes such as matrix metalloproteases, mucolytic enzymes, and neutral proteases.
  • Serine proteases are preferably used consecutively following use of other enzymes. The temperature and period of time tissues or cells are in contact with serine proteases is particularly important. Serine proteases may be inhibited by alpha 2 microglobulin in serum and therefore the medium used for digestion is usually serum-free. EDTA and DNAse are commonly used in enzyme digestion procedures to increase the efficiency of cell recovery.
  • the degree of dilution of the digestion may also greatly affect the cell yield as cells may be trapped within the viscous digest.
  • the LIBERASE (Boehringer Mannheim Corp., Indianapolis, IN) Blendzyme (Roche) series of enzyme combinations are very useful and may be used in the instant methods.
  • Other sources of enzymes are known, and the skilled artisan may also obtain such enzymes directly from their natural sources.
  • the skilled artisan is also well-equipped to assess new, or additional enzymes or enzyme combinations for their utility in isolating the cells of the invention.
  • Preferred enzyme treatments are 0.5, 1, 1.5, or 2 hours long or longer.
  • the tissue is incubated at 37°C during the enzyme treatment of the disintegration step.
  • Postpartum tissue comprising the umbilicus and placenta may be used without separation.
  • the umbilicus may be separated from the placenta by any means known in the art.
  • postpartum tissue is separated into two or more sections, such as umbilicus and placenta.
  • placental tissue is separated into two or more sections, each section consisting of predominantly of either neonatal, neonatal and maternal, or maternal aspect. The separated sections then are dissociated by mechanical and/or enzymatic dissociation according to the methods described herein.
  • Cells of neonatal or maternal lineage may be identified by any means known in the art, for example, by karyotype analysis or in situ hybridization for the Y-chromosome.
  • Karyotype analysis also may be used to identify cells of normal karyotype.
  • Isolated cells or postpartum tissue from which PPDCs grow out may be used to initiate, or seed, cell cultures.
  • Cells are transferred to sterile tissue culture vessels either uncoated or coated with extracellular matrix or ligands such as laminin, collagen, gelatin, fibronectin, ornithine, vitronectin, and extracellular membrane protein (e.g., MATRIGEL (BD Discovery Labware, Bedford, MA)).
  • extracellular matrix or ligands such as laminin, collagen, gelatin, fibronectin, ornithine, vitronectin, and extracellular membrane protein (e.g., MATRIGEL (BD Discovery Labware, Bedford, MA)).
  • PPDCs are cultured in any culture medium capable of I > C T .”" I J S O B / « 4 B E O B sustaining growth of the cells such as, but not limited to, DMEM (high or low glucose), Eagle's basal medium, Ham's FlO medium (FlO), Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM), DMEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), and CELL-GRO FREE.
  • DMEM high or low glucose
  • Eagle's basal medium Eagle's basal medium
  • Ham's FlO medium FlO
  • Ham's F-12 medium F12
  • Iscove's modified Dulbecco's medium Iscove's modified Dulbecco's medium
  • MSCGM Mesenchymal Stem Cell Growth Medium
  • DMEM/F12 RPMI
  • the culture medium may be supplemented with one or more components including, for example, serum (e.g., fetal bovine serum (FBS), preferably about 2-15% (v/v); equine serum (ES); human serum(HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one or more growth factors, for example, platelet-derived growth factor (PDGF), insulin-like growth factor- 1 (IGF- 1), leukemia inhibitory factor (LIF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and erythropoietin (EPO); amino acids, including L-valine; and one or more antibiotic and/or antimycotic agents to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination.
  • the culture medium preferably comprises Growth medium (
  • the cells are seeded in culture vessels at a density to allow cell growth.
  • the cells are cultured at about 0 to about 5 percent by volume CO 2 in air.
  • the cells are cultured at about 2 to about 25 percent O 2 in air, preferably about 5 to about 20 percent O 2 in air.
  • the cells preferably are cultured at about 25 to about 40 0 C, more preferably about 35°C to about 39 0 C, and more preferably are cultured at 37°C.
  • the cells are preferably cultured in an incubator.
  • the medium in the culture vessel can be static or agitated, for example, using a bioreactor.
  • PPDCs preferably are grown under low oxidative stress (e.g., with addition of glutathione, ascorbic acid, catalase, tocopherol, N-acetylcysteine).
  • Low oxidative stress refers to conditions of no or minimal free radical damage to the cultured cells.
  • the culture medium is changed as necessary, for example, by carefully aspirating the medium from the dish, for example, with a pipette, and replenishing with fresh medium. Incubation is continued until a sufficient number or density of cells accumulate in the dish.
  • the original explanted tissue sections may be removed and the remaining cells trypsinized P 1 if : T / u S o 1 F:;; . ⁇ n s a o ⁇ ; using standard techniques or using a cell scraper. After trypsinization, the cells are collected, removed to fresh medium and incubated as above.
  • the medium is changed at least once at approximately 24 hours post-trypsinization to remove any floating cells.
  • the cells remaining in culture are considered to be PPDCs.
  • PPDCs After culturing the isolated cells or tissue fragments for a sufficient period of time, PPDCs will have grown out, either as a result of migration from the postpartum tissue or cell division, or both.
  • PPDCs are passaged, or removed to a separate culture vessel containing fresh medium of the same or a different type as that used initially, where the population of cells can be mitotically expanded.
  • PPDCs are preferably passaged up to about 100% confluence, more preferably about 70 to about 85% confluence. The lower limit of confluence for passage is understood by one skilled in the art.
  • the PPDCs of the invention may be utilized from the first subculture (passage 0) to senescence.
  • the preferable number of passages is that which yields a cell number sufficient for a given application.
  • the cells are passaged 2 to 25 times, preferably 4 to 20 times, more preferably 8 to 15 times, more preferably 10 or 11 times, and most preferably 11 times. Cloning and/or subcloning may be performed to confirm that a clonal population of cells has been isolated.
  • Cells of the invention may be cryopreserved and/or stored prior to use.
  • PPDCs may be characterized, for example, by growth characteristics ⁇ e.g., population doubling capability, doubling time, passages to senescence), karyotype analysis (e.g., normal karyotype; maternal or neonatal lineage), flow cytometry (e.g., FACS analysis), immunohistochemistry and/or immunocytochemistry (e.g., for detection of epitopes including but not limited to vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18, von Willebrand factor, CD34, GROalpha, GCP-2, oxidized low density lipoprotein receptor 1, and NOGO-A), gene expression profiling (e.g., gene chip arrays; polymerase chain reaction (for example, reverse transcriptase PCR, real time PCR, and conventional PCR)), protein arrays, protein secretion (e.g., by plasma clotting assay or analysis of PPDC-conditioned medium, for example, by Enzyme Linked Immunosorb
  • antigens HLA-DP, HLA-DQ, HLA-DR), B7-H2, and PD-L2
  • mixed lymphocyte reaction e.g., as measure of stimulation of allogeneic PBMCs
  • PPDCs can undergo at least 40 population doublings in culture.
  • Population doubling may be calculated as [In (cell final/cell initial)/ln 2].
  • Doubling time may be calculated as (time in culture (h)/population doubling).
  • Undifferentiated PPDCs preferably produce at least one of NOGO-A, GCP-2, tissue factor, vimentin, and alpha-smooth muscle actin; more preferred are cells which produce each of GCP-2, tissue factor, vimentin, and alpha-smooth muscle actin. In some embodiments, two, three, four, or five of these factors are produced by the PPDCs.
  • PPDCs lack production of at least one of NOGO-A, GRO-alpha, or oxidized low density lipoprotein receptor, as detected by flow cytometry. In some embodiments, PPDCs lack production of at least two or three of these factors.
  • PPDCs may comprise at least one cell surface marker of CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A 5 B, C.
  • PPDCs preferably produce each of these surface markers.
  • PPDCs may be characterized in their lack of production of at least one of CD31, CD34, CD45, CD80, CD86, CDl 17, CD141, CD178, B7-H2, HLA-G, and HLA- DR 5 DP ,DQ, as detected by flow cytometry.
  • PPDCs preferably lack production of each of these surface markers.
  • PPDCs exhibit expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, is increased for at least one of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; and tumor necrosis factor, alpha-induced protein 3; or at least one of C-type lectin superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin, oxidized low density lipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671, hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1, and clo
  • Preferred PPDCs express, relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, increased levels of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; and tumor necrosis factor, alpha-induced protein 3; or increased levels of C-type lectin superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin, oxidized low density lipoprotein receptor 1, protein kinase C zeta, clone MAGE:4179671, hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1, and clone DKFZp547K
  • PPDCs wherein expression, relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, is increased for at least one of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; and tumor necrosis factor, alpha- induced protein 3, increased relative levels of at least one of C-type lectin superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin, oxidized low density lipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671, hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1, and clon
  • PPDCs wherein expression, relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, is increased for at least one of C-type lectin superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin, oxidized low density lipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671, hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1, and clone DKFZp547Kl 113, increased relative levels of at least one of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; and
  • PPDCs may have expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, is reduced for at least one of: short stature homeobox 2; heat shock 27kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1); elastin; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox 2; sine oculis homeobox homolog 1; crystallin, alpha B; dishevelled associated activator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin; src homology three (SH3) and cysteine rich domain; B-cell translocation gene 1, anti-proliferative; cholesterol 25-hydroxylase; runt-related transcription factor 3; hypothetical protein FLJ23
  • PPDCs may secrete a variety of biochemically active factors, such as growth factors, chemokines, cytokines and the like. Preferred cells secrete at least one of MCP-I, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIPIa, RANTES, and TIMPl. PPDCs may be characterized in their lack of secretion of at least one of TGF-beta2, ANG2, PDGFbb, MIPIb, 1309, MDC, and VEGF, as detected by ELISA. These and other characteristics are available to identify and characterize the cells, and distinguish the cells of the invention from others known in the art.
  • the cell comprises two or more of the foregoing characteristics. More preferred are those cells comprising, three, four, or five or more of the characteristics. Still more preferred are those postpartum-derived cells comprising six, seven, or eight or more of the characteristics. Still more preferred presently are those cells comprising all nine of the claimed characteristics.
  • cells that produce at least two of GCP-2, NOGO-A, tissue factor, vimentin, and alpha-smooth muscle actin. More preferred are those cells producing three, four, or five of these proteins.
  • cell markers are subject to vary somewhat under vastly different growth conditions, and that generally herein described are characterizations in Growth Medium, or variations thereof.
  • Postpartum-derived cells that produce at least one, two, three, or four of CDlO, CD 13, CD44, CD73, CD90, PDGFr-alpha, PD- L2 and HLA-A 5 B, C are preferred. More preferred are those cells producing five, six, or seven of these cell surface markers. Still more preferred are postpartum-derived cells that can produce eight, nine, or ten of the foregoing cell surface marker proteins.
  • PPDCs that lack production of at least one, two, three, or four of the proteins CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA- DR,DP,DQ, as detected by flow cytometry are preferred.
  • PPDCs lacking production of at least T/' USOIHS /' 1 MMB I 1 IOa rive, six, seven, or eight or more of these markers are preferred. More preferred are cells which lack production of at least nine or ten of the cell surface markers. Most highly preferred are those cells lacking production of eleven, twelve, or thirteen of the foregoing identifying proteins.
  • Presently preferred cells produce each of CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-A,B,C, and do not produce any of CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.
  • postpartum-derived cells exhibit expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, is increased for at least one of at least one, two, or three of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; and tumor necrosis factor, alpha-induced protein 3; or at least one, two, or three of C-type lectin superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin, oxidized low density lipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671, hypothetical protein DK
  • the cells exhibit expression, which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, is increased for a combination of interleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3; tumor necrosis factor, alpha-induced protein 3 or a combination of C-type lectin superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin, oxidized low density lipoprotein receptor 1, protein kinase C
  • cells which relative to a human cell that is a fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, have reduced expression for at least one of the genes corresponding to: short stature homeobox 2; heat shock 27kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1); elastin; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox 2; sine oculis homeobox homolog 1; crystallin, alpha B; dishevelled associated activator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin; src homology three (SH3) and cysteine rich domain; B-cell translocation gene 1, anti -proliferative; cholesterol P 3 C r / IJ S
  • cells that have, relative to human fibroblasts, mesenchymal stem cells, or ileac crest bone marrow cells, reduced expression of at least 5, 10, 15 or 20 genes corresponding to those listed above.
  • cell with reduced expression of at least 25, 30, or 35 of the genes corresponding to the listed sequences are also more preferred.
  • those postpartum-derived cells having expression that is reduced, relative to that of a human fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell, of genes corresponding to 35 or more, 40 or more, or even all of the sequences listed.
  • secretion of certain growth factors and other cellular proteins can make cells of the invention particularly useful.
  • Preferred postpartum-derived cells secrete at least one, two, three or four of MCP-I, EL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIPIa, RANTES, and TIMPl.
  • Cells which secrete five, six, seven or eight of the listed proteins are also preferred.
  • Cells which can secrete at least nine, ten, eleven or more of the factors are more preferred, as are cells which can secrete twelve or more, or even all thirteen of the proteins in the foregoing list.
  • PPDCs can also be characterized by their lack of secretion of factors into the medium.
  • Postpartum-derived cells that lack secretion of at least one, two, three or four of TGF-beta2, ANG2, PDGFbb, MIPIb, 1309, MDC, and VEGF, -* C Tf/ U B O B / ll !MB B O fcf as detected by ELISA, are presently preferred for use.
  • Cells that are characterized in their lack secretion of five or six of the foregoing proteins are more preferred. Cells which lack secretion of all seven of the factors listed above are also preferred.
  • placenta-derived cells of the invention were deposited with the American Type Culture Collection (ATCC, Manassas, VA) and assigned ATCC Accession Numbers as follows: (1) strain designation PLA 071003 (P8) was deposited June 15, 2004 and assigned Accession No. PTA-6074; (2) strain designation PLA 071003 (PIl) was deposited June 15 , 2004 and assigned Accession No. PTA-6075; and (3) strain designation PLA 071003 (P16) was deposited June 16, 2004 and assigned Accession No. PTA-6079.
  • ATCC American Type Culture Collection
  • VA American Type Culture Collection
  • Examples of umbilicus-derived cells of the invention were deposited with the American Type Culture Collection (ATCC, Manassas, VA) on June 10, 2004, and assigned ATCC Accession Numbers as follows: (1) strain designation UMB 022803 (P7) was assigned Accession No. PTA-6067; and (2) strain designation UMB 022803 (P17) was assigned Accession No. PTA-6068.
  • PPDCs can be isolated.
  • the invention also provides compositions of PPDCs, including populations of PPDCs.
  • the cell population is heterogeneous.
  • a heterogeneous cell population of the invention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% PPDCs of the invention.
  • the heterogeneous cell populations of the invention may further comprise epithelial cells (e.g., mucosal cells, for example, cells of oral mucosa; gastrointestinal tract; nasal epithelium; respiratory tract epithelium; vaginal epithelium; corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), smooth muscle cells, myoblasts, myocytes, stromal cells, bladder urothelial cells, cells of the larynx, esoph
  • cell populations of the invention may include PPDCs and at least one cell type of bone marrow cells, adipocytes, stem cells, keratinocytes, vascular endothelial cells, myoblasts, myocytes, stromal cells, and other soft tissue progenitor cells.
  • the cell population is substantially homogeneous, i.e., comprises substantially only PPDCs (preferably at least about 96%, 97%, 98%, 99% or more PPDCs).
  • the homogeneous cell population of the invention may comprise umbilicus- or placenta-derived cells. Homogeneous populations of umbilicus-derived cells may be free of cells of maternal lineage.
  • Homogeneous populations of placenta-derived cells may be of neonatal or maternal lineage. Homogeneity of a cell population may be achieved by any method known in the art, for example, by cell sorting (e.g., flow cytometry), bead separation, or by clonal expansion.
  • Methods of the invention further include methods for producing a population of postpartum-derived cells by expanding a cell of the invention in culture.
  • the postpartum-derived cells of the invention preferably expand in the presence of from about 5% to about 20% oxygen.
  • the postpartum-derived cells of the invention preferably are expanded in culture medium such as but not limited to Dulbecco's modified Eagle's medium (DMEM), mesenchymal stem cell growth medium, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), RPMI1640, CELL- GRO FREE, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), Ham's FlO medium, Ham's F12 medium, DMEM/F12, Iscove's modified Dulbecco's medium, or Eagle's basal medium.
  • DMEM Dulbecco's modified Eagle's medium
  • mesenchymal stem cell growth medium e.g., DMEM/MCDB201 (Sigma)
  • the culture medium preferably contains low or high glucose, about 2%-15% (v/v) serum, betamercaptoethanol, and an antibiotic agent.
  • the culture medium may contain at least one of fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, and epidermal growth factor.
  • the cells of the invention may be grown on an uncoated or coated surface. Surfaces for growth of the cells may be coated for example with gelatin, collagen (e.g., native or denatured), fibronectin, laminin, ornithine, vitronectin, or extracellular membrane protein (e.g., MATRIGEL).
  • a population of postpartum- derived cells is mixed with another population of cells.
  • the cells of the invention can be induced to differentiate to an ectodermal, endodermal, or mesodermal lineage, preferably a mesodermal or ectodermal lineage.
  • PPDCs may be induced to differentiate into a given lineage by subjecting them to differentiation-inducing cell culture conditions.
  • populations of cells incubated in the presence of one or more factors, or under conditions, that stimulate cell differentiation along a desired pathway e.g., toward a soft tissue phenotype such as a muscular, endothelial, or epithelial phenotype).
  • Methods to characterize differentiation-induced cells of the invention include, but are not limited to, histological, morphological, biochemical and immunohistochemical methods, or using cell surface markers, or genetically or molecularly, or by identifying factors secreted by the differentiation-induced cell, and by the inductive qualities of the differentiation- induced PPDCs.
  • Methods of using PPDCs or PPDC products include, but are not limited to, histological, morphological, biochemical and immunohistochemical methods, or using cell surface markers, or genetically or molecularly, or by identifying factors secreted by the differentiation-induced cell, and by the inductive qualities of the differentiation- induced PPDCs.
  • the cells of the invention can be engineered using any of a variety of vectors including, but not limited to, integrating viral vectors, e.g., retrovirus vector or adeno-associated viral vectors; non-integrating replicating vectors, e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors; or replication-defective viral vectors.
  • integrating viral vectors e.g., retrovirus vector or adeno-associated viral vectors
  • non-integrating replicating vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • replication-defective viral vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • Other methods of introducing DNA into cells include the use of liposomes, electroporation, a particle gun, or by direct DNA injection.
  • Hosts cells are preferably transformed or transfected with a nucleic acid of interest controlled by or in operative association with, one or more appropriate expression control elements such as promoter or enhancer sequences, internal ribosomal entry sites (IREs), transcription terminators, polyadenylation sites, among others, and a selectable marker.
  • expression control elements such as promoter or enhancer sequences, internal ribosomal entry sites (IREs), transcription terminators, polyadenylation sites, among others, and a selectable marker.
  • engineered cells may be allowed to grow in enriched media and then switched to selective media.
  • a selectable marker in the nucleic acid of interest may confer resistance to a selection agent or allow cells to grow in the absence of an otherwise required factor.
  • Cells may stably integrate the DNA of interest into their chromosomes.
  • Cells expressing the DNA of interest may be cloned and expanded into cell lines.
  • This method can be advantageously used to engineer cell lines which express the DNA of interest.
  • any promoter may be used to drive the expression of the DNA of interest.
  • viral promoters include, but are not limited to, the CMV promoter/enhancer, SV40, papillomavirus, Epstein-Barr virus or elastin gene promoter.
  • the control elements used to control expression of the gene of interest allow for the regulated expression of the gene so that the product is synthesized only when desired in vivo.
  • constitutive promoters are preferably used in a non-integrating and/or replication-defective vector.
  • inducible promoters could be used to drive the expression of the gene of interest when necessary.
  • Inducible promoters include, but are not limited to, those associated with metallothionein and heat shock proteins.
  • An expression control element may be tissue-specific.
  • An example of a transcriptional control region that exhibits tissue specificity is the myosin light chain-2 gene control region, which is active in skeletal muscle (Shani, 1985, Nature 314:283). > C T , ' O S !LI & . " ' • Hi B O H
  • the cells of the invention may be genetically engineered to "knock out” or
  • “knock down” expression of factors that promote inflammation or rejection at the implant site Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below.
  • Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below.
  • “Negative modulation,” as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
  • the expression of a native gene can be reduced or knocked out using a number of techniques including, for example, inhibition of expression by inactivating the gene completely (commonly termed "knockout"), for example using the homologous recombination technique.
  • an exon encoding an important region of the protein is interrupted by a positive selectable marker, e.g., neo, preventing the production of normal mRNA from the target gene and resulting in inactivation of the gene.
  • a gene may also be inactivated by creating a deletion in part of a gene or by deleting the entire gene. By using a construct with two regions of homology to the target gene, for example, the intervening sequence can be deleted (Mombaerts et ah, 1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084).
  • Antisense, small interfering RNA, DNAzymes, and ribozyme molecules which inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene activity.
  • antisense RNA molecules which inhibit the expression of major histocompatibility gene complexes (HLA) have been shown to be most versatile with respect to immune responses.
  • triple helix molecules can be utilized in reducing the level of target gene activity.
  • the expression of EL-I can be knocked out or knocked down in the cells of the invention to reduce the production of inflammatory mediators by the cells of the invention.
  • the expression of MHC class II molecules can be knocked out or knocked down in order to reduce the risk of rejection of the implanted tissue.
  • the cells of the invention may be administered to a patient to allow for the treatment of a soft tissue condition or to produce an anti-inflammatory gene product such as, for example, peptides or polypeptides corresponding to the idiotype of neutralizing antibodies for GM-CSF, TNF, DL-I, IL-2, or other inflammatory cytokines.
  • an anti-inflammatory gene product such as, for example, peptides or polypeptides corresponding to the idiotype of neutralizing antibodies for GM-CSF, TNF, DL-I, IL-2, or other inflammatory cytokines.
  • the genetically engineered cells may be used to produce new tissue in vitro, which is then administered to a subject, as described herein.
  • PPDCs may secrete, for example, at least one of monocyte chemotactic protein 1 (MCP-I), interleukin-6 (IL6), interleukin 8 (IL-8), GCP-2, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), heparin binding epidermal growth factor (HB-EGF), brain-derived neurotrophic factor (BDNF), thrombopoietin (TPO), macrophage inflammatory protein 1 alpha (MlPIa), RANTES, and tissue inhibitor of matrix metalloproteinase 1 (TIMPl), which can be augmented by a variety of techniques, including ex vivo cultivation of the cells in chemically defined medium.
  • MCP-I monocyte chemotactic protein 1
  • IL6 interleukin-6
  • IL-8 interleukin 8
  • GCP-2 GCP-2
  • HGF hepatocyte growth factor
  • KGF keratinocyte growth factor
  • FGF
  • PPDCs have the ability to support survival, growth, and differentiation of other cell types in co-culture.
  • the methods of the invention thus also include methods of providing trophic support to a soft tissue cell.
  • the methods may include a step of exposing a soft tissue cell to a PPDC or PPDC product, such as PPDC conditioned medium.
  • Examples of cells which may be supported by PPDCs or PPDC products include but are not limited to stem cells, myocytes, myoblasts, keratinocytes, melanocytes, dermal fibroblasts, bone marrow cells, adipocytes, epithelial cells, endothelial cells, stromal cells, and endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors). Exposure to or co-culture of PPDC or PPDC products with endothelial cells may stimulate angiogenesis by the endothelial cells.
  • PPDCs or PPDC products are co-cultured or exposed in vitro or are administered in vivo to provide trophic support to another cell type, including but not limited to epithelial cells (e.g., mucosal cells, for example, cells of oral mucosa; gastrointestinal tract; nasal epithelium; respiratory tract epithelium; vaginal epithelium; corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), smooth muscle cells, myothelial
  • the PPDCs For co-culture, it may be desirable for the PPDCs and the which the two cell types are in contact. This can be achieved, for example, by seeding the cells as a heterogeneous population of cells in culture medium or onto a suitable culture substrate.
  • the PPDCs can first be grown to confluence and employed as a substrate for the second desired cell type in culture.
  • the cells may further be physically separated, e.g., by a membrane or similar device, such that the other cell type may be removed and used separately following the co- culture period.
  • the desired other cells are cultured in contact with a PPDC product, such as conditioned medium, extracellular matrix, and/or a cell fraction of PPDCs.
  • matrices comprising PPDCs or PPDC products are administered to provide trophic support to another cell type.
  • Use of PPDCs or PPDC products to promote expansion and/or differentiation of other cell types may find applicability in research and in clinical/therapeutic areas. For instance, such methods may be utilized to facilitate growth and/or differentiation of cells of a given phenotype in culture (e.g., cells of a soft tissue phenotype) for basic research purposes or for use in drug screening assays. The methods may also be utilized for in vitro expansion of cells of a soft tissue phenotype for later administration for therapeutic purposes.
  • cells may be harvested from an individual, expanded in vitro in co- culture with PPDCs or a PPDC product, then returned to that individual (autologous transfer) or another individual (syngeneic, allogeneic, or xenogeneic transfer).
  • autologous transfer or another individual
  • xenogeneic transfer the population of cells comprising the PPDCs or PPDC products
  • the cultured cell populations may be physically separated in culture, enabling removal of the autologous cells for administration to the patient.
  • the culturing methods are performed in vivo.
  • PPDCs or a PPDC product may be administered to a patient to provide trophic support to another cell type, including but not limited to epithelial cells (e.g., mucosal cells, for example, cells of oral mucosa; gastrointestinal tract; nasal epithelium; respiratory tract epithelium; vaginal epithelium; corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+
  • epithelial cells e
  • PPDCs or PPDC products induce angiogenesis in co- culture with cells such as but not limited to epithelial cells (e.g., mucosal cells, for example, cells of oral mucosa; gastrointestinal tract; nasal epithelium; respiratory tract epithelium; vaginal epithelium; corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), smooth muscle cells, myoblasts, myocytes, stromal cells,
  • epithelial cells
  • angiogenic factors including but not limited to EPO, TIMPl, ANG2, PDGF-bb, TPO, KGF, HGF, FGF, VEGF, and HBEGF, are released by PPDCs.
  • methods of inducing angiogenesis according to the invention include exposing a soft tissue cell or population thereof to a PPDC or PPDC product in vitro or in vivo.
  • endothelial progenitors e.g., CD34+, CD34+/CD117+ cells.
  • PPDCs or PPDC products may be administered to a patient as described herein.
  • a PPDC population or product may be administered to a patient to provide needed angiogenic factors.
  • PPDCs and PPDC products of the invention may be used to produce a vascular network, as demonstrated in Example 14.
  • Methods of producing a vascular network involve exposing (e.g., contacting) a population of soft tissue cells to PPDCs or a PPDC product such as a cell fraction (e.g., lysate or soluble cell fraction thereof), extracellular matrix, or conditioned medium.
  • the population of soft tissue cells preferably contains at least one soft tissue cell of an aortic endothelial cell, coronary artery endothelial cell, pulmonary artery endothelial cell, iliac artery endothelial cell, microvascular endothelial cell, umbilical artery endothelial cell, and umbilical vein endothelial cell.
  • the method of producing a vascular network may be performed in vitro or in vivo. Also included within the scope of the invention are the vascular networks so produced.
  • the vascular networks of the invention may be administered to a patient as a therapeutic regimen.
  • the vascular networks are administered as treatment of a soft tissue condition, for example but not by way of limitation, a vascular condition, such as a vascular disease or injury or improper vascular development.
  • a vascular condition such as a vascular disease or injury or improper vascular development.
  • the vascular network is administered by transplantation to the patient.
  • damaged or diseased vasculature is removed prior to administration of the vascular network of the invention.
  • conditioned media are contemplated for use in in vitro culture of cells, for example, stem or soft tissue progenitor cells, or cells of a soft tissue phenotype, including but not limited to epithelial cells (e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium), bone marrow cells, adipocytes, keratinocytes, vascular endothelial cells ⁇ e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.
  • epithelial cells e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory
  • PPDCs and PPDC products of the invention may be used to treat patients having a soft tissue condition, for example but not limited to patients requiring the repair or replacement of soft tissue resulting from disease or trauma or failure of the tissue to develop normally, or to provide a cosmetic function, such as to augment features of the body.
  • the treatment may comprise at least one of soft tissue repair, reconstruction, bulking, cosmetic treatment, therapeutic treatment, tissue augmentation, and tissue sealing.
  • Provided herein are methods of IPC T/ US ⁇ &V M-B&O B treating soft tissue conditiona in a patient by administering to the patient PPDCs and/or PPDC products of the invention.
  • Therapeutic applications of the PPDCs and PPDC products of the invention include but are not limited to treatment of hernias, congenital defects, damage to the pelvic floor, tear or rupture of a tendon or ligament, a traumatic wound, skin repair and regeneration (e.g., scar revision or the treatment of traumatic wounds, bums, skin ulcers (e.g., decubitus (pressure) ulcers, venous ulcers, and diabetic ulcers), and surgical wounds such as those associated with the excision of skin cancers; treatment of vascular conditions (e.g., vascular disease such as peripheral arterial disease, abdominal aortic aneurysm, carotid disease, and venous disease; vascular injury; improper vascular development); and muscle diseases (e.g., congenital myopathies; myasthenia gravis; inflammatory, neurogenic, and myogenic muscle diseases; and muscular dystrophies such as Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, limb-gir
  • Also provided by the invention are methods of treating a patient in need of angiogenic factors comprising administering to the patient the PPDCs or PPDC products (e.g., conditioned medium) of the invention.
  • the PPDCs or PPDC products e.g., conditioned medium
  • the PPDCs and PPDC products of the invention may be administered alone or as admixtures with other cells.
  • the PPDCs and PPDC products may be administered by way of a matrix.
  • a matrix of the invention may comprise a three-dimensional scaffold. Scaffolds of the invention may be particulate, flat, tubular, single-layered, or multilayered.
  • the PPDCs and PPDC products may be administered with conventional pharmaceutically acceptable carriers. Where PPDCs are to be administered with other cells, the PPDCs may be administered simultaneously or sequentially with the other cells. Where cells are to be administered sequentially with other cell types, the PPDCs may be administered before or after the cells of a second phenotype.
  • Cells which may be administered in conjunction with PPDCs include epithelial cells (e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), myoblasts, myocytes, C TV' ⁇ J S O K ' " 11 A S iEIUII e stromal cells, bladder urothelial cells, smooth
  • the PPDCs and PPDC products may be administered with other beneficial drugs or biological molecules (e.g., growth factors, trophic factors).
  • the pharmaceutical compositions of the invention comprise PPDCs and/or PPDC products and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions comprise PPDCs and/or PPDC products in an effective amount to treat a soft tissue condition.
  • the PPDCs and/or PPDC products may be administered together in a single pharmaceutical composition, or in separate pharmaceutical compositions, simultaneously or sequentially with the other bioactive factor (either before or after administration of the other agents).
  • Bioactive factors which may be co-administered include anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and EL-I inhibitors, pemirolast, tranilast, REMICADE, and NSAIDs (non-steroidal anti-inflammatory drugs; e.g., tepoxalin, tolmetin, suprofen); immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., sirolimus, everolimus); antiproliferatives (e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.,
  • compositions of the invention may comprise, in addition to the PPDC or PPDC product, at least one other cell type.
  • pharmaceutical compositions of the invention may comprise a soft tissue cell.
  • the at least one other cell type to be included in the pharmaceutical compositions of PPDCs and/or PPDC products of the invention include stem cells, epithelial cells, dermal fibroblasts, melanocytes, keratinocytes, and other epithelial progenitor cells, myocytes, myoblasts, and muscle cells (e.g., smooth muscle cells), endothelial cells, and stromal cells.
  • PPDCs are administered as undifferentiated cells, i.e., as cultured in Growth medium.
  • the PPDCs and related products of the invention may be surgically implanted, injected, engrafted, delivered (e.g., by way of a catheter or syringe), or otherwise administered directly or indirectly to the site in need of repair or augmentation.
  • PPDCs and PPDC products may be administered by way of a matrix (e.g., a three-dimensional scaffold), or via injectable viscoelastic supplements such as hyaluronic acid, alginates, self-assembling peptides, hydrogels and collagen.
  • PPDCs and PPDC products may be administered with conventional pharmaceutically acceptable carriers.
  • Routes of administration of PPDCs and PPDC products include intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, oral, and nasal administration.
  • Preferable routes of in vivo administration include transplantation, implantation, injection, delivery via a catheter, microcatheter, suture, stent, microparticle, pump, or any other means known in the art.
  • PPDCs or PPDC products are administered in semi-solid or solid devices, surgical implantation into a precise location in the body is typically a suitable means of administration.
  • Liquid or fluid pharmaceutical compositions may be administered to a more general location (e.g., throughout a diffusely affected area, for example), from which PPDCs or PPDC products migrate to a particular location, e.g., by responding to chemical signals.
  • Dosage forms and regimes for administering PPDCs or PPDC products described herein are developed in accordance with good medical practice, taking into account the condition of the individual patient, e.g., nature and extent of the condition being treated, age, sex, body weight and general medical condition, and other factors known to medical practitioners. Thus, the effective amount of a pharmaceutical composition to be administered to a patient is determined by these considerations as known in the art.
  • PPDCs have been shown not to stimulate allogeneic PBMCs in a mixed lymphocyte reaction. Accordingly, transplantation with allogeneic, or even xenogeneic, PPDCs may be tolerated.
  • PPDCs may be encapsulated in a capsule that is permeable to " £ ' ,'. ⁇ / iJ " ⁇ fi G 1 Ir * ' ' ' ⁇ 1 S •** Ct ' r ⁇ ' ⁇ nutrients " an ' cf oxygen required By the cell and therapeutic factors the cell is yet impermeable to immune humoral factors and cells.
  • the encapsulant is hypoallergenic, is easily and stably situated in a target tissue, and provides added protection to the implanted structure.
  • PPDCs also may be genetically modified to reduce their immunogenicity.
  • Survival of transplanted PPDCs in a living patient can be determined through the use of a variety of scanning techniques, e.g., computerized axial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) or positron emission tomography (PET) scans. Determination of transplant survival can also be done by removing a section of the target tissue and examining it, for example, visually or through a microscope. Alternatively, cells can be treated with stains that are specific for cells of a given lineage.
  • CAT or CT computerized axial tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • Transplanted cells can also be identified by prior incorporation of tracer dyes such as rhodamine- or fluorescein-labeled microspheres, fast blue, bisbenzamide, ferric microparticles, or genetically introduced reporter gene products, such as beta-galactosidase or beta-glucuronidase.
  • tracer dyes such as rhodamine- or fluorescein-labeled microspheres, fast blue, bisbenzamide, ferric microparticles, or genetically introduced reporter gene products, such as beta-galactosidase or beta-glucuronidase.
  • Functional integration of transplanted PPDCs into a subject can be assessed by examining restoration of the function that was damaged or diseased, for example, restoration of joint function, blood flow, muscle contraction, etc., or augmentation of function.
  • compositions and Pharmaceutical compositions
  • compositions of PPDCs and related products are included within the scope of the invention.
  • Compositions of the invention may include one or more bioactive factors, for example but not limited to, a growth factor, a differentiation-inducing factor, a cell survival factor such as caspase inhibitor, an antiinflammatory agent such as p38 kinase inhibitor, or an angiogenic factor such as VEGF or bFGF.
  • bioactive factors include PDGF-bb, EGF, bFGF, IGF-I, and LIF.
  • undifferentiated or differentiation-induced PDPCs are cultured in contact with the bioactive factor.
  • undifferentiated PPDCs remain undifferentiated upon contact with the bioactive factor.
  • the bioactive factor induces differentiation of the PPDCs.
  • compositions of the invention may comprise homogeneous or heterogeneous populations of differentiated and/or undifferentiated PPDCs or PPDC products in a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include organic or inorganic carrier substances suitable which do not deleteriously react with the cells of the invention or related cT/ysois / 'Mi ⁇ aoe products.
  • suitable pharmaceutically acceptable carriers include water, salt solution (such as Ringer's solution), alcohols, oils, gelatins, and carbohydrates, such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, hyaluronic acid, and polyvinyl pyrolidine.
  • Such preparations can be sterilized, and if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring.
  • Pharmaceutical carriers suitable for use in the present invention are known in the art and are described, for example, in Pharmaceutical Sciences (17 th Ed., Mack Pub. Co., Easton, PA) and WO 96/05309, each of which are incorporated by reference herein.
  • compositions may be delivered in the form of a spray, suspension, solution, dry powder, cream, ointment, or gel.
  • the dosage e.g., the number of cells to be administered
  • frequency of administration of the pharmaceutical compositions will depend upon a number of factors, including but not limited to, the nature of the condition to be treated, the extent of the symptoms of the condition, characteristics of the patient (e.g., age, size, gender, health).
  • PPDCs extracellular matrices or cell fractions thereof, conditioned medium, matrices, vascular networks, and compositions produced according to the invention may be used to repair or replace underdeveloped, damaged, or destroyed soft tissue, to augment existing soft tissue, to introduce new or altered tissue, to modify artificial prostheses, or to join biological tissues or structures.
  • some embodiments of the invention include (i) hernia closures with replacement soft tissue constructs grown in three-dimensional cultures; (ii) skin grafts with soft tissue constructs; (iii) prostheses; (iv) blood vessel grafts; and (v) tendon or ligament reconstruction.
  • Examples of such conditions that can be treated according to the methods of the invention include congenital anomalies such as hemifacial microsomia, malar and zygomatic hypoplasia, unilateral mammary hypoplasia, pectus excavatum, pectoralis agenesis (Poland's anomaly) and velopharyngeal incompetence secondary to cleft palate repair or submucous cleft palate (as a retropharyngeal implant); acquired defects (post-traumatic, post-surgical, post-infectious) such as scars, subcutaneous atrophy (e.g., secondary to discoid lupus erythematosus), keratotic lesions, acne pitting of the face, linear scleroderma with subcutaneous atrophy, saddle-nose deformity, Romberg's disease, and unilateral vocal cord paralysis; cosmetic defects such as glabellar frown lines, deep nasolabial creases, circum-oral geographical wrinkles, sunken cheeks and mammary
  • vascular diseases such as peripheral arterial disease, abdominal aortic aneurysm, carotid disease, and venous disease
  • muscle diseases e.g., congenital myopathies; myasthenia gravis; inflammatory, neurogenic, and myogenic muscle diseases
  • muscular dystrophies such as Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, limb-girdle-muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophies, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and Emery-Dreifuss muscular dystrophy
  • connective tissues such as tendons and ligaments.
  • the successful repair or replacement of damaged tissue can be enhanced if the implanted cells and/or tissue can be fixed in place at the site of repair. Post-implantation movement may cause the new cells or tissue to become dislodged from the site if a pro-active fixation technique is not employed.
  • Various methods can be used to fix the new cells and/or tissue in place, including: patches derived from biocompatible tissues, which can be placed over the site; biodegradable sutures, hollow sutures, porous sutures, or other fasteners, e.g., pins, staples, tacks, screws and anchors; non-absorbable fixation devices, e.g., sutures, pins, screws and anchors; adhesives; and the use of interference fit geometries.
  • the PPDCs and PPDC products of the invention may be administered alone, in a pharmaceutically acceptable carrier, through a catheter or microcatheter, via a pump or spray, or on or in a matrix as described herein.
  • the treatment methods of the subject invention involve the implantation of PPDCs, PPDC products, or trans-differentiated cells into individuals in need thereof.
  • PPDCs or PPDC products of the present invention may be delivered to the site of therapeutic need or "home" to the site.
  • the cells of the present invention may be differentiated in vitro prior to implantation in a patient. In vitro differentiation allows for controlled application of bioactive factors. Alternatively, the cells of the present invention may differentiate in situ or provide trophic support to endogenous cells.
  • the appropriate cell implantation dosage in humans can be determined from existing information relating to, e.g., the activity of the cells. From in vitro culture and in vivo animal experiments, the amount of factors produced can be quantitated. This information is also useful in calculating an appropriate dosage of implanted material. Additionally, the patient can be monitored to determine if additional implantation can be made or implanted material reduced accordingly.
  • angiogenic factors such as VEGF, PDGF or bFGF can be added either alone or in combination with endothelial cells or their progenitors, including CD34+, CD34+/CD117+ cells.
  • One or more other components may be co-administered, including selected extracellular matrix components, such as one or more types of collagen known in the art, and/or growth factors, platelet-rich plasma, and drugs.
  • the cells of the invention may be genetically engineered to express and produce growth factors.
  • Bioactive factors which may be usefully incorporated into the cell formulation include anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins, TL-6 and EL-I inhibitors, pemirolast, tranilast, REMICADE, and NSAIDs (non-steroidal anti-inflammatory drugs; e.g., tepoxalin, tolmetin, suprofen); immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., sirolimus, everolimus); antiproliferatives (e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.
  • a formulation comprising PPDCs or PPDC products of the invention is prepared for administration directly to the site where the new soft tissue is desired.
  • the support for the PPDCs or PPDC products of the invention is biodegradable.
  • PPDCs or PPDC products of the invention may be suspended in a hydrogel solution for injection.
  • suitable hydrogels for use in the invention include self-assembling peptides, such as RAD 16.
  • the hydrogel solution may be allowed to harden, for instance in a mold, to form a matrix having PPDCs or PPDC products dispersed therein prior to implantation.
  • the cell formulations may be cultured so that the cells are mitotically expanded prior to implantation.
  • Hydrogels are organic polymers (natural or P f ' ;. Têt ⁇ • ' y S O B , !i -!HB B O B synthetic) which are cross-linked via covalent, ionic, or hydrogen bonds to create a three- dimensional open-lattice structure which entraps water molecules to form a gel.
  • Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and polyacrylates, which are crosslinked ionically, carboxymethyl cellulose (CMC), oxidized regenerated cellulose (ORC), or block polymers such as polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively.
  • the formulation comprises an in situ polymerizable gel, as described, for example, in U.S. Patent Application Publication 2002/0022676; Anseth et al, J.
  • Other components may also be included in the formulation, including but not limited to any of the following: (1) buffers to provide appropriate pH and isotonicity; (2) lubricants; (3) viscous materials to retain the cells at or near the site of administration, including, for example, alginates, agars and plant gums; and (4) other cell types that may produce a desired effect at the site of administration, such as, for example, enhancement or modification of the formation of tissue or its physicochemical characteristics, or as support for the viability of the cells, or inhibition of inflammation or rejection.
  • the cells may be covered by an appropriate wound covering to prevent cells from leaving the site. Such wound coverings are known to those of skill in the art.
  • Bioactive factors which may be usefully incorporated into the formulations of the invention include anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-I inhibitors, pemirolast, tranilast, REMICADE, and NSAIDs (non-steroidal anti-inflammatory drugs; e.g., tepoxalin, tolmetin, suprofen); immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., sirolimus, everolimus); anti-proliferatives (e.g., azathioprine, mycophenolate mofetil); cortico
  • ⁇ ' H'B 8111 B platelet inhibitors e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-IO, glutathione, L-cysteine, N-acetylcysteine
  • anti-oxidants e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-IO, glutathione, L-cysteine, N-acetylcysteine
  • the cells may be co-administered with scar inhibitory factor as described in U.S. Patent No. 5,827,735, incorporated herein by reference.
  • the cell contacting surface of the well may be coated with a molecule that discourages adhesion of PPDCs to the cell contacting surface.
  • Preferred coating reagents include silicon based reagents i.e., dichlorodimethylsilane or polytetrafluoroethylene based reagents, i.e., TEFLON. Procedures for coating materials with silicon based reagents, specifically dichlorodimethylsilane, are well known in the art. See for example, Sambrook et al. (1989) "Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory Press, the disclosure of which is incorporated by reference herein. It is appreciated that other biocompatible reagents that prevent the attachment of cells to the surface of the well may be useful in the practice of the instant invention.
  • the well may be cast from a pliable or moldable biocompatible material that does not permit attachment of cells per se.
  • Preferred materials that prevent such cell attachment include, but are not limited to, agarose, glass, untreated cell culture plastic and polytetrafluoroethylene, i.e., TEFLON.
  • Untreated cell culture plastics i.e., plastics that have not been treated with or made from materials that have an electrostatic charge are commercially available, and may be purchased, for example, from Falcon Labware, Becton-Dickinson, Lincoln Park, N.J.
  • the aforementioned materials are not meant to be limiting. It is appreciated that any other pliable or moldable biocompatible material that inherently discourages the attachment of PPDCs may be useful in the practice of the instant invention.
  • the size and shape of the well may be determined by the size and shape of the tissue defect to be repaired.
  • the well should be deep enough to contain culture medium overlaying the tissue patch.
  • a tissue patch prepared in accordance with the invention may be "trimmed" or configured to a pre-selected size and/or shape by a surgeon performing V* Ct / U SB S s ' " IhEi 8 O IiI surgical repair of the damaged tissue. Trimming may be performed with the use of a sharp cutting implement, i.e., a scalpel, a pair of scissors or an arthroscopic device fitted with a cutting edge, using procedures well known in the art.
  • a sharp cutting implement i.e., a scalpel, a pair of scissors or an arthroscopic device fitted with a cutting edge
  • the pre-shaped well may be cast in a block of agarose gel under aseptic conditions.
  • Agarose is an economical, biocompatible, pliable and moldable material that can be used to cast pre-shaped wells, quickly and easily.
  • the dimensions of the well may dependent upon the size of the resulting tissue plug that is desired.
  • a pre-shaped well may be prepared by pouring a hot solution of molten LT agarose (BioRad, Richmond, Calif.) into a tissue culture dish containing a cylinder, the cylinder having dimensions that mirror the shape of the well to be formed.
  • the size and shape of the well may be chosen by the artisan and may be dependent upon the shape of the tissue defect to be repaired.
  • the resulting pre-shaped well is suitable for culturing and/or inducing the differentiation of PPDCs. It is appreciated, however, that alternative methods may be used to prepare a pre-shaped well useful in the practice of the invention.
  • PPDCs in suspension may be seeded into and cultured in the pre-shaped well.
  • the PPDCs may be induced to differentiate to a soft tissue phenotype in culture in the well or may have been induced to differentiate prior to seeding in the well.
  • the cells may be diluted by the addition of culture medium to a cell density of about 1 x 10 5 to IxIO 9 PPDCs per milliliter.
  • the cohesive plug of cells may be removed from the well and surgically implanted into the tissue defect. It is anticipated that undifferentiated PPDCs may differentiate in situ thereby to form tissue in vivo.
  • PPDCs are used to generate cell sheets.
  • the sheets may be multilayered, as described in Shimizu, et al., Biomaterials, 24(13):2309-2316 (2003).
  • Soft tissue defects may be identified by any means known in the art, for example, but not limited to computer aided tomography (CAT scanning); X-ray examination; or magnetic resonance imaging (MRI). Defects in soft tissue also are readily identifiable visually during arthroscopic examination or during open surgery. Treatment of the defects can be effected during an orthoscopic or open surgical procedure using the methods and compositions disclosed herein.
  • CAT scanning computer aided tomography
  • X-ray examination or magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • the defect may be treated by
  • tissue patch prepared by the methodologies described herein, and (2) permitting the tissue patch to integrate into pre-determined site.
  • the tissue patch optimally has a size and shape such that when the patch is implanted into the defect, the edges of the implanted tissue contact directly the edges of the defect.
  • the tissue patch may be fixed in place during the surgical procedure. This can be effected by surgically fixing the patch into the defect with biodegradable sutures and/or by applying a bioadhesive to the region interfacing the patch and the defect.
  • diseased, damaged, or underdeveloped tissue may be surgically excised prior to implantation of the patch of synthetic tissue.
  • a synthetic tissue patch is implanted subsequently into the defect by the methods described above.
  • the cells of the invention or co-cultures thereof may be seeded onto a scaffold, such as a three-dimensional scaffold, and implanted in vivo, where the seeded cells will proliferate on or in the framework and form a replacement tissue in vivo in cooperation with the cells of the patient.
  • a scaffold such as a three-dimensional scaffold
  • the scaffolds of the invention can be used to form tubular structures, like those of the gastrointestinal and genitourinary tracts, as well as blood vessels; tissues for hernia repair; tendons and ligaments.
  • PPDCs or co-cultures thereof are inoculated and grown on a three-dimensional framework.
  • the framework may be configured into the shape of the corrective structure desired.
  • the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts found naturally in vivo.
  • Some embodiments of the invention provide a matrix for implantation into a patient.
  • the matrix is seeded with a population of postpartum-derived cells of the invention.
  • the PPDCs may be differentiation-induced or undifferentiated.
  • the PPDC population may be homogeneous or heterogeneous.
  • the matrix may also be inoculated with cells of another desired cell type, for example but not by way of limitation, epithelial cells (e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, -' • ' !H ⁇ ' ⁇ / 3, J '!;:! O ' i ⁇ • ⁇ " •' ! ! - ⁇ ft 1,1 "S)
  • melanocytes dermal fibroblasts
  • vascular endothelial cells e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)
  • myoblasts myocytes, stromal cells, and other soft tissue cells or progenitor cells.
  • the matrix may contain or be pre- treated with one or more bioactive factors including, for example, drugs, anti-inflammatory agents, antiapoptotic agents, and growth factors.
  • the matrix is inoculated with PPDC products of the invention, including for example, extracellular matrix, secreted factors, or cell fractions of the PPDCs.
  • the matrix is biodegradable.
  • the matrix comprises extracellular membrane proteins, for example, MATRIGEL.
  • the matrix comprises natural or synthetic polymers.
  • Matrices of the invention include biocompatible scaffolds, lattices, self- assembling structures and the like, whether biodegradable or not, liquid or solid.
  • matrices are known in the arts of cell-based therapy, surgical repair, tissue engineering, and wound healing.
  • the matrices are pretreated (e.g., seeded, inoculated, contacted with) with PPDCs or PPDC products (e.g., extracellular matrix, conditioned medium, secreted factors, cell fraction, or combination thereof) of the invention.
  • PPDCs or PPDC products are in close association to the matrix or its spaces.
  • the cells or PPDC products adhere to the matrix.
  • the cells or cell products are contained within or bridge interstitial spaces of the matrix.
  • seeded matrices wherein PPDCs or PPDC products are in close association with the matrix and which, when used therapeutically, induce or support ingrowth of the patient's cells and/or proper angiogenesis.
  • the seeded or pre-treated matrices can be introduced into a patient's body in any way known in the art, including but not limited to implantation, injection, surgical attachment, transplantation with other tissue, and the like.
  • the matrices of the invention may be configured for use in vivo, for example, to the shape and/or size of a tissue or organ in vivo.
  • the scaffolds of the invention may be flat or tubular or may comprise sections thereof, as described herein.
  • the scaffolds of the invention may be multilayered.
  • the scaffold may be designed such that the scaffold structure: (1) supports the PPDCs or PPDC products without subsequent degradation; (2) supports the PPDCs or PPDC products from the time of seeding until the scaffold is remodeled by the host tissue; or (3) allows the seeded cells to attach, proliferate, and develop into a tissue structure having sufficient mechanical integrity to support itself in vitro, at which point, the scaffold is degraded.
  • a review of scaffold design is provided by Hutraum, /. Biomat. ScL Polymer Edn., 12(1): 107-124 (2001).
  • Scaffolds of the invention can be administered in combination with any one or more growth factors, cells, drugs, or other components described above that stimulate soft tissue formation or stimulate vascularization or innervation thereof or otherwise enhance or improve the practice of the invention.
  • the cells of the invention can be grown freely in a culture vessel to sub- confluency or confluency, lifted from the culture and inoculated onto a three-dimensional framework. Inoculation of the three-dimensional framework with a high concentration of cells, e.g., approximately 10 6 to 5 x 10 7 cells per milliliter, will result in the establishment of the three- dimensional support in relatively shorter periods of time.
  • PPDCs, co-cultures thereof, or PPDC products may be inoculated onto the framework before or after forming the desired shape, e.g., ropes, tubes, filaments.
  • the framework is preferably incubated in an appropriate growth medium. During the incubation period, the inoculated cells will grow and envelop the framework and will bridge any interstitial spaces therein. It is preferable but not required to grow the cells to an appropriate degree which reflects the in vivo cell density of the tissue being repaired or regenerated.
  • Nonwoven mats may, for example, be formed using fibers comprised of poly(lactic acid-co-glycolic acid) polymer (10/90 PLGA), referred to herein as VNW, available for purchase through Biomedical Structures (Slatersville, Rhode Island)).
  • Foams composed of, for example, poly(epsilon- caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed by processes such as freeze- drying, or lyophilization, as discussed in U.S. Patent No. 6,355,699, are also possible scaffolds.
  • Hydrogels such as self-assembling peptides (e.g., RAD16) may also be used.
  • a scaffold or matrix of the invention comprises collagen/ORC, CMC, or ORC. These materials are frequently used as supports for growth of tissue.
  • the scaffold is lyophilized prior to use.
  • lyophilized scaffolds are rehydrated, with saline for example, prior to use.
  • the framework is a felt, which can be composed of a multifilament yarn made from a bioabsorbable material, e.g., !i>;r;: f / tP S O B / 1 M! B BO 8
  • the yarn is made into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling.
  • PPDCs and PPDC products of the invention are seeded onto foam scaffolds that may be composite structures.
  • the three- dimensional framework may be molded into a useful shape, such as a specific structure in the body to be repaired, replaced, or augmented.
  • the framework may be treated prior to inoculation to enhance attachment of the PPDCs or PPDC products.
  • nylon matrices could be treated with 0.1 molar acetic acid and incubated in polylysine, PBS, and/or collagen to coat the nylon.
  • Polystyrene could be similarly treated using sulfuric acid.
  • the external surfaces of the three-dimensional framework may be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma coating the framework or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellular matrix, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, among others.
  • proteins e.g., collagens, elastic fibers, reticular fibers
  • glycoproteins e.g., glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, der
  • the scaffold is comprised of or is treated with materials that render it non-thrombogenic.
  • These treatments and materials may also promote and sustain endothelial growth, migration, and extracellular matrix deposition.
  • these materials and treatments include but are not limited to natural materials such as basement membrane proteins such as laminin and Type IV collagen, synthetic materials such as ePTFE, and segmented polyurethaneurea silicones, such as PURSPAN (The Polymer Technology Group, Inc., Berkeley, CA). These materials can be further treated to render the scaffold non- thrombogenic.
  • Such treatments include anti -thrombotic agents such as heparin, and treatments which alter the surface charge of the material such as plasma coating.
  • Different proportions of the various types of collagen, for example, deposited on the framework can affect the growth of tissue-specific or other cells which may be later inoculated onto the framework or which may grow onto the structure in vivo.
  • collagen types I and III are preferably deposited in the initial matrix.
  • the framework can be inoculated with a mixture of cells which synthesize the appropriate collagen types desired.
  • the appropriate collagen type to be inoculated on the framework or produced by the cells seeded thereon may be selected.
  • the relative amounts of collagenic and elastic fibers present in the framework can be modulated by controlling the ratio of collagen-producing cells to ⁇ '-err /U SBS .' ' H BBUB elastin-producing cells in the initial inoculum.
  • an arterial scaffold should contain a co-culture of smooth muscle cells which secrete elastin.
  • the seeded or inoculated three-dimensional framework of the invention can be used in a variety of applications. These include but are not limited to transplantation or implantation of either the cultured cells obtained from the matrix or the cultured matrix itself in vivo.
  • the three-dimensional scaffolds may, according to the invention, be used to replace or augment existing tissue, to introduce new or altered tissue, to modify artificial prostheses, or to join together biological tissues or structures.
  • specific embodiments of the invention include but are not limited to, flat structures and tubular three- dimensional tissue implants for repair or regeneration, for example, of the gastrointestinal tract, genitourinary tract, blood vessels, muscles, ligaments, tendons, skin, pelvic floor, fascia, and hernias.
  • PPDCs and PPDC products can be inoculated onto a flat scaffold.
  • the scaffold is preferably incubated in culture medium prior to implantation.
  • Two or more flat frameworks can be laid atop another and sutured together to generate a multilayer framework.
  • the three-dimensional framework can be used to construct single and multi-layer tubular tissues in vitro that can serve as a replacement for damaged or diseased tubular tissue in vivo.
  • a scaffold can be cut into a strip ⁇ e.g., rectangular in shape) of which the width is approximately equal to the inner circumference of the tubular organ into which it will ultimately be inserted.
  • the cells can be inoculated onto the scaffold and incubated by floating or suspending in liquid media.
  • the scaffold can be rolled up into a tube by joining the long edges together. The seam can be closed by suturing the two edges together using fibers of a suitable material of an appropriate diameter.
  • a scaffold can be formed as a tube, inoculated with PPDCs or PPDC products, and suspended in media in an incubation chamber.
  • one of the open ends of the tubular framework can be affixed to a nozzle. Liquid media can be forced through this nozzle from a source chamber connected to the incubation chamber to create a current through the interior of the tubular framework.
  • the other open end can be affixed to an outflow aperture which leads into a collection chamber from which the media can be recirculated through the source chamber.
  • the tube can be detached from the ⁇ ' C T / V S O "H , ⁇ " i 'f.fb S O B nozzle and outflow aperture when incubation is complete. This method is described by
  • two three-dimensional frameworks can be combined into a tube in accordance with the invention using any of the following methods.
  • Two or more flat frameworks can be laid atop another and sutured together. This two-layer sheet can then be rolled up, and, as described above, joined together and secured.
  • One tubular scaffold that is to serve as the inner layer can be inoculated with PPDCs or PPDC products and incubated.
  • a second scaffold can be grown as a flat strip with width slightly larger than the outer circumference of the tubular framework. After appropriate growth is attained, the flat framework can be wrapped around the outside of the tubular scaffold followed by closure of the seam of the two edges of the flat framework and, preferably, securing the flat framework to the inner tube.
  • tubular meshes of slightly differing diameters can be grown separately.
  • the framework with the smaller diameter can be inserted inside the larger one and secured.
  • scaffolds can be combined at any stage of growth of the PPDCs, and incubation of the combined scaffolds can be continued when desirable.
  • Scaffolds comprising PPDC products may be layered with scaffolds comprising PPDCs.
  • the lumenal aspect of the tubular construct can be comprised of or treated with materials that render the lumenal surface of the tubular scaffold non-thrombogenic. These treatments and materials may also promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of these materials and treatments include but are not limited to natural materials such as basement membrane proteins such as laminin and Type IV collagen, synthetic materials such as ePTFE, and segmented polyurethaneurea silicones, such as PURSPAN (The Polymer Technology Group, Inc., Berkeley, CA). These materials can be further treated to render the lumenal surface of the tubular scaffold non-thrombogenic. Such treatments include anti-thrombotic agents such as heparin, and treatments which alter the surface charge of the material such as plasma coating.
  • materials that render the lumenal surface of the tubular scaffold non-thrombogenic may also promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of these materials and treatments include but are not limited to natural materials such as basement membrane proteins such as laminin and
  • Advanced bioreactors may be necessary to meet the complex requirements of in vitro engineering of functional skeletal tissues.
  • Bioreactor systems with the ability to apply complex concurrent mechanical strains to three-dimensional matrices, for example, in conjunction with enhanced environmental and fluidic control are provided by Altman et al, J. Biomech. Eng., 124(6):742-749 (2002); U.S. Patent Application Publication No. 2002/0062151.
  • such a bioreactor system may be used in the development of a tissue-engineered tendon or ligament, e.g., anterior cruciate ligament.
  • any suitable method can be employed to shape the three-dimensional culture to assume the conformation of the natural organ or tissue to be simulated.
  • a framework prepared in accordance with the invention may be "trimmed" to a pre-selected size for surgical repair of the damaged tissue. Trimming may be performed with the use of a sharp cutting implement, i.e., a scalpel, a pair of scissors or an arthroscopic device fitted with a cutting edge, using procedures well known in the art.
  • the three-dimensional frameworks can be shaped to assume a conformation which simulates the shape of a natural organ or tissue, such as soft tissue including but not limited to pelvic floor, bladder, fascia, skin, muscle, tendon, ligament, or vasculature (e.g., arteries, veins).
  • a natural organ or tissue such as soft tissue including but not limited to pelvic floor, bladder, fascia, skin, muscle, tendon, ligament, or vasculature (e.g., arteries, veins).
  • These constructions simulate biological structures in vivo and may be readily implanted to repair hernias or to replace damaged or diseased tissues, including hernias, tendons, ligaments, skin, muscle, blood vessels, and components of the gastrointestinal tract, genitourinary tract (e.g., urethra, ureter).
  • PPDCs or PPDC products are seeded on the scaffold in combination (e.g., as a co-culture or as separate layers of cells) with stem cells and /or cells of a soft tissue phenotype.
  • the cells to be co-inoculated with the PPDCs will depend upon the tissue to be simulated.
  • PPDCs may be inoculated onto the scaffold with epithelial cells (e.g., cells of oral mucosa, gastrointestinal tract, nasal epithelium, respiratory tract epithelium, vaginal epithelium, corneal epithelium), bone marrow cells, adipocytes, stem cells, keratinocytes, vascular endothelial cells (e.g., aortic endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells, iliac artery endothelial cells, microvascular endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, and endothelial progenitors (e.g., CD34+, CD34+/CD117+ cells)), bladder urothelial cells, smooth muscle cells, gastrointestinal cells, esophageal cells, larynx cells, mucosal cells, myoblasts, myocytes, my
  • the three-dimensional scaffold of the invention may be used in skin grafting.
  • the scaffold is about 0.5 to 3 millimeter thick and is in the form of a flat sheet.
  • the scaffold is preferably seeded with PPDCs or PPDC products.
  • the scaffolds may be co-inoculated with at least one of stem cells, epithelial cells, dermal fibroblasts, melanocytes, and keratinocytes. In some embodiments, keratinocytes form a layer over the PPDC-seeded framework.
  • the scaffolds of the invention preferably comprise at least one of collagen, elastin, intercellular adhesion molecules, neural cell adhesion molecules, laminin, heparin binding growth factor, fibronectin, proteoglycan, tenascin, E-cahedrin, and fibrillin.
  • the three-dimensional scaffold may be used to generate muscle tissue.
  • the scaffold is preferably seeded with PPDCs or PPDC products.
  • the scaffolds may be co-inoculated with at least one of stem cells, myocytes, and myoblasts.
  • the three-dimensional framework may be modified so that the growth of cells and the production of tissue thereon or therein is enhanced, or so that the risk of rejection of the implant is reduced.
  • one or more biologically active compounds including, but not limited to, antiapoptotic agents, antiinflammatories, angiogenic factors, immunosuppressants or growth factors, may be added to the framework.
  • a subject in need of tissue repair, replacement, or augmentation may benefit from the administration of a PPDC product, such as extracellular matrix (ECM), conditioned medium, or a cell fraction of PPDCs.
  • ECM extracellular matrix
  • coculture of PPDCs with a scaffold deposits ECM onto the framework.
  • ECM Once ECM is secreted onto the framework, the cells may be removed.
  • the ECM may be processed for further use, for example, as an injectable preparation. Scaffolds comprising the ECM may be used therapeutically.
  • ECM may be collected from the scaffold.
  • the collection of the ECM can be accomplished in a variety of ways, depending, for example, on whether the scaffold is biodegradable or non-biodegradable. For example, if the framework is non-biodegradable, the ECM can be removed by subjecting the framework to sonication, high pressure water jets, mechanical scraping, or mild treatment with detergents or enzymes, or any combination of the above.
  • the ECM can be collected, for example, by allowing the framework to degrade or dissolve in solution.
  • the biodegradable framework is composed of a material that can itself be injected along with the ECM, the framework and the ECM product can be processed in toto for subsequent injection.
  • the ECM can be removed from the biodegradable framework by any of the methods described above for collection of ECM from a non-biodegradable framework. All collection processes are preferably designed so as not to denature the ECM produced by the cells of the invention. T / o s o s . ⁇ " H-B s Ci e
  • the ECM may be processed further.
  • the ECM can be homogenized to fine particles using techniques well known in the art such as, for example, by sonication, so that they can pass through a surgical needle.
  • ECM components can be crosslinked, if desired, by gamma irradiation.
  • the ECM can be irradiated between 0.25 to 2 mega rads to sterilize and crosslink the ECM.
  • Cell fractions prepared from the populations of the postpartum-derived cells also have many utilities.
  • whole cell lysates are prepared, e.g., by disrupting cells without subsequent separation of cell fractions.
  • a cell membrane fraction is separated from a soluble fraction of the cells by routine methods known in the art, e.g., centrifugation, filtration, or similar methods.
  • Use of soluble cell fractions or supernatants in vivo allows the beneficial intracellular milieu to be used in a patient without triggering rejection or an adverse response.
  • Methods of lysing cells include various means of freeze-thaw disruption, osmotic disruption, mechanical disruption, ultrasonic disruption, enzymatic disruption (e.g., hyaluronidase, dispase, proteases, and nucleases (for example, deoxyribonuclease and ribonuclease)), or chemical disruption (non-ionic detergents such as, for example, alkylaryl polyether alcohol (TRITON® X-100), octylphenoxy polyethoxy- ethanol (Rohm and Haas Philadelphia, PA), BRIJ-35, a polyethoxyethanol lauryl ether (Atlas Chemical Co., San Diego, CA), polysorbate 20 (TWEEN 20®), a polyethoxyethanol sorbitan monolaureate (Rohm and Haas), polyethylene lauryl ether (Rohm and Haas); and ionic detergents such as, for example, alkylaryl poly
  • Such cell lysates may be prepared from cells directly in their growth medium and thus containing secreted growth factors and the like, or may be prepared from cells washed free of medium in, for example, PBS or other solution. Cells may also be lysed on their growth substrate. Washed cells may be resuspended at concentrations greater than the original population density if preferred. Cell lysates prepared from populations of postpartum-derived cells may be used as is, further concentrated, by for example, ultrafiltration or lyophilization, or even dried, partially purified, combined with pharmaceutically acceptable carriers or diluents as are known in the art, or combined with other compounds such as biologicals, for example pharmaceutically useful protein compositions.
  • cellular membranes are removed from the lysate, for example by centrifugation, or ultracentrifugation, filtration, chromatograph, or sedimentation, to yield a membrane fraction and supernate fraction.
  • the membrane fraction or the supernate may be used according to the methods of the invention.
  • cellular debris is removed by treatment with a mild detergent rinse, such as EDTA, CHAPS or a zwitterionic detergent.
  • Cell lysates may be used in vitro or in vivo, alone or, for example, with cells or on a substrate.
  • the cell lysates, if introduced in vivo may be introduced locally at a site of treatment, or remotely to provide, for example needed cellular growth factors to a patient.
  • the amounts and/or ratios of proteins may be adjusted by mixing the PPDC product of the invention with cells or with ECM or cell fraction of one or more other cell types.
  • biologically active substances such as proteins, growth factors and/or drugs, can be incorporated into the PPDC product formulation.
  • Exemplary biologically active substances include anti-inflammatory agents and growth factors which promote healing and tissue repair.
  • Cells may be co-administered with the PPDC products of the invention.
  • the above described process for preparing PPDC products is preferably earned out under sterile conditions using sterile materials.
  • the processed PPDC product in a pharmaceutically acceptable carrier can be injected intradermally, intraarticularly, or subcutaneously to augment tissue or to repair or correct congenital anomalies, acquired defects or cosmetic defects.
  • Examples of such conditions are congenital anomalies such as hemifacial microsomia, malar and zygomatic hypoplasia, unilateral mammary hypoplasia, pectus excavatum, pectoralis agenesis (Poland's anomaly) and velopharyngeal incompetence secondary to cleft palate repair or submucous cleft palate (as a retropharyngeal implant); acquired defects (post-traumatic, post-surgical, post-infectious) such as scars, subcutaneous atrophy (e.g., secondary to discoid lupus erythematosus), keratotic lesions, acne pitting of the face, linear scleroderma with subcutaneous atrophy, saddle-nose deformity, Romberg's disease, and unilateral vocal cord paralysis; cosmetic defects such as glabellar frown lines, deep nasolabial creases, circum-oral geographical wrinkles, sunken cheeks and mammary hypoplasia; herni
  • the cells and tissues of the invention may be used in vitro to screen for effectiveness as a trophic support or for cytotoxicity of compounds including pharmaceutical agents, growth/regulatory factors, and anti-inflammatory agents.
  • the cells of the invention are maintained in vitro and exposed to the compound to be tested.
  • the activity of a cytotoxic compound can be measured by its ability to damage or kill cells in culture. This may readily be assessed by vital staining techniques.
  • the effect of trophic factors may be assessed by analyzing the number of living cells in vitro, e.g., by total cell counts, and differential cell counts or by detecting a marker of differentiation.
  • the cells and tissues of the invention may be used as model systems for the study of soft tissue conditions.
  • the cells and tissues of the invention may also be used to study the mechanism of action of cytokines, growth factors and inflammatory mediators, e.g., EL-I, TNF and prostaglandins.
  • cytotoxic and/or pharmaceutical agents can be screened for those that are most efficacious for a particular patient. Agents that prove to be efficacious in vitro could then be used to treat the patient therapeutically.
  • the cells of the invention can be cultured in vitro to produce biological products in high yield.
  • such cells which either naturally produce a particular biological product of interest (e.g., a growth factor, regulatory factor, or peptide hormone), or have been genetically engineered to produce a biological product, could be clonally expanded using, for example, the three-dimensional culture system described above.
  • the cells excrete the biological product into the nutrient medium, the product can be readily isolated from the spent or conditioned medium using standard separation techniques, e.g., such as differential protein precipitation, ion-exchange chromatography, gel filtration chromatography, electrophoresis, and high performance liquid chromatography.
  • a "bioreactor” PCT/ O SOEf '" " 1 MMSe 01:1 may be used to take advantage of the flow method for feeding, for example, a three-dimensional culture in vitro.
  • the biological product is washed out of the culture and may then be isolated from the outflow, as above.
  • a biological product of interest may remain within the cell and, thus, its collection may require that the cells are lysed.
  • the biological product may then be purified using any one or more of the above-listed techniques.
  • the PPDCs and PPDC products can conveniently be employed as part of a kit, for example, for culture or in vivo administration.
  • the invention provides a kit including the PPDCs and/or PPDC products and additional components, such as a matrix (e.g., a scaffold), hydrating agents (e.g., physiologically-compatible saline solutions, prepared cell culture media), cell culture substrates (e.g., culture dishes, plates, vials, etc.), cell culture media (whether in liquid or powdered form), antibiotic compounds, hormones, a bioactive factor, a second cell type, a differentiation-inducing agent, cell culture media, and the like.
  • the kit can include any such components, preferably it includes all ingredients necessary for its intended use. If desired, the kit also can include cells (typically cryopreserved), which can be seeded into the lattice as described herein.
  • kits that utilize the PPDCs, PPDC populations, products of PPDCs in various methods for augmentation, regeneration, and repair as described above.
  • the kits may include one or more cell populations, including at least PPDCs and a pharmaceutically acceptable carrier (liquid, semi-solid or solid).
  • the kits also optionally may include a means of administering the cells, for example by injection.
  • the kits further may include instructions for use of the cells.
  • Kits prepared for field hospital use, such as for military use may include full-procedure supplies including tissue scaffolds, surgical sutures, and the like, where the cells are to be used in conjunction with repair of acute injuries.
  • Kits for assays and in vitro methods as described herein may contain one or more of (1) PPDCs or products of PPDCs, (2) reagents for practicing the in vitro method, (3) other cells or cell populations, as appropriate, and (4) instructions for conducting the in vitro method.
  • PPDCs of the invention can be cryopreserved and maintained or stored in a "cell bank". Cryopreservation of cells of the invention may be carried out according to known methods. For example, but not by way of limitation, cells may be suspended in a "freeze medium" such as, for example, culture medium further comprising 0 to 95 percent FBS and 0 to 10 percent dimethylsulfoxide (DMSO), with or without 5 to 10 percent glycerol, at a density, for example, of about 0.5 to 10 x 10 6 cells per milliliter.
  • the cryopreservation medium may comprise cryopreservation agents including but not limited to methylcellulose.
  • the cells are dispensed into glass or plastic ampoules that are then sealed and transferred to the freezing chamber of a controlled rate freezer.
  • the optimal rate of freezing may be determined empirically.
  • a programmable rate freezer for example, can give a change in temperature of -1 to -10°C per minute.
  • the preferred cryopreservation temperature is about -8O 0 C to about -180°C, more preferably is about -90°C to about -160°C, and most preferably is about -125 to about -140 0 C.
  • Cryopreserved cells preferably are transferred to liquid nitrogen prior to thawing for use. In some embodiments, for example, once the ampoules have reached about -9O 0 C, they are transferred to a liquid nitrogen storage area. Cryopreserved cells can be stored for a period of years. Alternatively, cells could be freeze-dried using agents such as but not limited to trehalose, sucrose, maltose, and sorbitol.
  • the cryopreserved cells of the invention constitute a bank of cells, portions of which can be "withdrawn” by thawing and then used as needed. Thawing should generally be carried out rapidly, for example, by transferring an ampoule from liquid nitrogen to a 37 °C water bath. The thawed contents of the ampoule should be immediately transferred under sterile conditions to a culture vessel containing an appropriate medium such as DMEM conditioned with 10 percent FBS.
  • the invention provides for banking of tissues, cells, PPDC derivatives, and cell populations in freeze-dried form.
  • a trehalose pre-incubation step is necessary to achieve this.
  • sucrose or other additives might be used. This will allow the generation of room temperature stable products with long shelf lives.
  • the objective of this study was to derive populations of cells from placental and umbilicus tissues. Postpartum umbilicus and placenta were obtained upon birth of either a full term or pre-term pregnancy. Cells were harvested from 5 separate donors of umbilicus and placental tissue. Different methods of cell isolation were tested for their ability to yield cells with: 1) the potential to differentiate into cells with different phenotypes, or 2) the potential to provide critical trophic factors useful for other cells and tissues.
  • Umbilicus cell derivation Umbilical cords were obtained from National Disease Research Interchange (NDRI , Philadelphia, PA). The tissues were obtained following normal deliveries. The cell isolation protocol was performed aseptically in a laminar flow hood. To remove blood and debris, the umbilicus was washed in phosphate buffered saline (PBS; Invitrogen, Carlsbad, CA) in the presence of antimycotic and antibiotic (100 Units/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B) (Invitrogen Carlsbad, CA)).
  • PBS phosphate buffered saline
  • antibiotic 100 Units/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B
  • the tissues were then mechanically dissociated in 150 cm 2 tissue culture plates in the presence of 50 milliliters of medium (DMEM-Low glucose or DMEM-High glucose; Invitrogen) until the tissue was minced into a fine pulp.
  • the chopped tissues were transferred to 50 milliliter conical tubes (approximately 5 grams of tissue per tube).
  • the tissue was then digested in either DMEM-Low glucose medium or DMEM-High glucose medium, each containing antimycotic and antibiotic (100 Units/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B (Invitrogen)) and digestion enzymes.
  • C:D collagenase (Sigma, St Louis, MO), 500 Units/milliliter; and dispase (Invitrogen), 50 Units/milliliter in DMEM-Low glucose medium).
  • C:D:H collagenase, dispase and hyaluronidase
  • the conical tubes containing the tissue, medium and digestion enzymes were incubated at 37 0 C in an orbital shaker (Environ, Brooklyn, NY) at 225 rpm for 2 hrs.
  • the filtrate was resuspended in Growth medium (total volume 50 milliliters) and centrifuged at 150 x g for 5 minutes. The supernatant was aspirated, and the cells were resuspended in 50 milliliters of fresh Growth medium. This process was repeated twice more.
  • the cells isolated from umbilicus were seeded at 5,000 cells/cm 2 onto gelatin- coated T-75 cm 2 flasks (Corning Inc., Corning, NY) in Growth medium (DMEM-Low glucose (Invitrogen), 15 percent (v/v) defined bovine serum (Hyclone, Logan, UT; Lot#AND18475), 0.001 percent (v/v) 2-mercaptoethanol (Sigma), 100 Units/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B (Invitrogen)). After about 2-4 days, spent medium was aspirated from the flasks.
  • Growth medium DMEM-Low glucose (Invitrogen), 15 percent (v/v) defined bovine serum (Hyclone, Logan, UT; Lot#AND18475), 0.001 percent (v/v) 2-mercaptoethanol (Sigma), 100 Units/milliliter penicillin, 100 micrograms/milli
  • Cells were washed with PBS three times to remove debris and blood-derived cells. Cells were then replenished with Growth medium and allowed to grow to confluence (about 10 days from passage 0 to passage 1). On subsequent passages (from passage 1 to 2, etc.), cells reached sub-confluence (75-85 percent confluence) in 4-5 days. For these subsequent passages, cells were seeded at 5000 cells/cm 2 . Cells were grown in a humidified incubator with 5 percent carbon dioxide and 20 percent oxygen at 37 0 C.
  • Placental Cell Isolation Placental tissue was obtained from NDRI (Philadelphia, PA). The tissues were from a pregnancy and were obtained at the time of a normal surgical delivery. Placental cells were isolated as described for umbilicus cell isolation.
  • the cell isolation protocol was performed aseptically in a laminar flow hood.
  • the placental tissue was washed in phosphate buffered saline (PBS; Invitrogen, Carlsbad, CA) in the presence of antimycotic and antibiotic (100 Units/milliliter penicillin, 100 microgram/milliliter streptomycin, 0.25 microgram/milliliter amphotericin B; Invitrogen) to f / USD*.? / " 1 I GBOB remove blood and debris.
  • the placental tissue was then dissected into three sections: top-line
  • mid-line mixed cell isolation neonatal and maternal or villous region
  • bottom line bottom line
  • the separated sections were individually washed several times in PBS with antibiotic/antimycotic to further remove blood and debris. Each section was then mechanically dissociated in 150 cm 2 tissue culture plates in the presence of 50 milliliters of DMEM-Low glucose (Invitrogen) to a fine pulp. The pulp was transferred to 50 milliliter conical tubes. Each tube contained approximately 5 grams of tissue. The tissue was digested in either DMEM-Low glucose or DMEM-High glucose medium containing antimycotic and antibiotic (100 Units/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B (Invitrogen)) of PBS and digestion enzymes.
  • DMEM-Low glucose Invitrogen
  • C:D collagenase and dispase
  • collagenase Sigma, St Louis, MO
  • dispase Invitrogen
  • C:D:H hyaluronidase
  • the conical tubes containing the tissue, medium, and digestion enzymes were incubated for 2 h at 37°C in an orbital shaker (Environ, Brooklyn, NY) at 225 rpm.
  • the cell suspension was filtered through a 70 micrometer nylon cell strainer (BD Biosciences), chased by a rinse with an additional 5 milliliters of Growth medium.
  • the total cell suspension was passed through a 40 micrometer nylon cell strainer (BD Biosciences) followed with an additional 5 milliliters of Growth medium as a rinse.
  • the filtrate was resuspended in Growth medium (total volume 50 milliliters) and centrifuged at 150 x g for 5 minutes. The supernatant was aspirated and the cell pellet was resuspended in 50 milliliters of fresh Growth medium. This process was repeated twice more. After the final centrifugation, supernatant was aspirated and the cell pellet was resuspended in 5 milliliters of fresh Growth medium. A cell count was determined using the Trypan Blue Exclusion test. Cells were then cultured at standard conditions. [0219] LIBERASE (Boehringer Mannheim Corp., Indianapolis, IN) Cell Isolation.
  • the lysate was centrifuged (10 minutes at 200 x g).
  • the cell pellet was resuspended in complete minimal essential medium (Gibco, Carlsbad CA) containing 10 percent fetal bovine serum (Hyclone, Logan UT), 4 millimolar glutamine (Mediatech Herndon, VA ), 100 Units penicillin per 100 milliliters and 100 micrograms streptomycin per 100 milliliters (Gibco, Carlsbad, CA).
  • the resuspended cells were centrifuged (10 minutes at 200 x g), the supernatant was aspirated, and the cell pellet was washed in complete medium. Cells were seeded directly into either T75 flasks (Corning, NY),
  • T75 laminin-coated flasks, or T175 fibronectin-coated flasks both Becton Dickinson, Bedford, MA.
  • Postpartum-derived cells were isolated from residual blood in the cords but not from cord blood. The presence of cells in blood clots washed from the tissue that adhere and grow under the conditions used may be due to cells being released during the dissection process.
  • Table 1-2 Isolation and culture expansion of postpartum-derived cells under varying conditions:
  • Placenta-derived cells at passage 8 were seeded at 1 x 10 3 cells/well in 96 well plates in Growth medium (DMEM-low glucose (Gibco, Carlsbad CA), 15% (v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan, UT), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, MO), 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco)).
  • Growth medium DMEM-low glucose (Gibco, Carlsbad CA)
  • fetal bovine serum Cat. #SH30070.03; Hyclone, Logan, UT
  • betamercaptoethanol Sigma, St. Louis, MO
  • 50 Units/milliliter penicillin 50 micrograms/milliliter streptomycin (Gibco)).
  • the medium was changed as described in Table 2-1, and cells were incubated in normal (20%, v/v) or low (5%, v/v) oxygen at 37 0 C, 5% CO 2 for 48 hours.
  • MTS was added to the culture medium (CELLTITER 96 AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI) for 3 hours and the absorbance measured at 490 nanometers (Molecular Devices, Sunnyvale CA).
  • Standard curves for the MTS assay established a linear correlation between an increase in absorbance and an increase in cell number.
  • the absorbance values obtained were converted into estimated cell numbers and the change (%) relative to the initial seeding was calculated.
  • Postpartum-derived cells may be grown in a variety of culture media in normal or low oxygen. Short-term growth of placenta-derived cells was determined in 12 basal media with 0, 2, and 10% (v/v) serum in 5% or 20% O 2 . In general placenta-derived cells did not grow in serum-free conditions with the exceptions of Ham's FlO and Cellgro-free, which are also protein-free. Growth in these serum-free media was approximately 25-33% of the maximal growth observed with Growth medium containing 15% serum.
  • placenta-derived cells may be grown in serum-free conditions and that Growth medium is one of several media (10% serum in Iscove's, RPMI or Ham's F12 media) that can be used to grow placenta-derived cells.
  • CELLGRO-FREE a serum and protein-free medium without hormones or growth factors, which is designed for the growth of mammalian cells in vitro (Mediatech product information).
  • Complete-serum free medium also developed for serum-free culture was not as effective in supporting growth of the placenta-derived cells.
  • Complete-serum free was developed by Mediatech, based on a 50/50 mix of DMEM/F12 with smaller percentages of RPMI 1640 and McCoy's 5 A.
  • This medium also contains selected trace elements and high molecular weight carbohydrates, extra vitamins, a non-animal protein source, and a small amount of BSA (1 gram/liter). It does not contain any insulin, transferrin, cholesterol, or growth or attachment factors. It is bicarbonate buffered for use with 5% CO 2 . Originally designed for hybridomas and suspension cell lines, it may be suitable for some anchorage dependent cell lines.
  • Placenta-derived cells (P3), fibroblasts (P9), and umbilicus-derived cells (P5) were seeded at 5 x 10 3 cells/cm 2 in gelatin-coated T75 flasks (Corning, Corning, NY). After 24 P C IV tJ S O S ,.• ' '4-la IB U 'B hours the medium was removed and the cells were washed with phosphate buffered saline (PBS)
  • PBS phosphate buffered saline
  • DMEM Modified Growth medium
  • D-valine special order Gibco
  • fetal bovine serum Hyclone, Logan, UT
  • betamercaptoethanol Sigma
  • 50 Units/milliliter penicillin 50 micrograms/milliliter streptomycin (Gibco)
  • Placenta-derived, umbilicus-derived, and fibroblast cells seeded in the D-valine- containing medium did not proliferate, unlike cells seeded in Growth medium containing dialyzed serum. Fibroblasts changed morphologically, increasing in size and changing shape. All of the cells died and eventually detached from the flask surface after 4 weeks.
  • Postpaitum-derived cells require L-valine for cell growth and for long-term viability. L-valine is preferably not removed from the growth medium for postpartum- derived cells.
  • the objective of this study was to determine a suitable cryopreservation medium for the cryopreservation of postpartum-derived cells.
  • Placenta-derived cells grown in Growth medium DMEM-low glucose (Gibco, Carlsbad CA), 15% (v/v) fetal bovine serum (Cat. #SH30070.03, Hyclone, Logan, UT), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, MO), 50 Units/milliliter penicillin, 50 microgram/milliliter streptomycin (Gibco)), in a gelatin-coated T75 flask were washed with phosphate buffered saline (PBS; Gibco) and trypsinized using 1 milliliter Trypsin/EDTA (Gibco). The trypsinization was stopped by adding 10 milliliters Growth medium.
  • PBS phosphate buffered saline
  • the cells were s ⁇ C T/ O S 1 Ds ""• ⁇ ⁇ - ⁇ " ; Mf- B B O H centrifuged at 150 x g, supernatant removed, and the cell pellet was resuspended in 1 milliliter
  • the cells were cooled at approximately l°C/min overnight in a -8O 0 C freezer using a "Mr Frosty" freezing container according to the manufacturer's instructions (Nalgene, Rochester, NY). Vials of cells were transferred into liquid nitrogen for 2 days before thawing rapidly in a 37 0 C water bath. The cells were added to 10 milliliters Growth medium and centrifuged before the cell number and viability was estimated as before. Cells were seeded onto gelatin-coated flasks at 5,000 cells/cm 2 to determine whether the cells would attach and proliferate.
  • cryopreservation of cells is one procedure available for preparation of a cell bank or a cell product.
  • Four cryopreservation mixtures were compared for their ability to protect human placenta-derived cells from freezing damage.
  • Dulbecco's modified Eagle's medium (DMEM) and 10% (v/v) dimethylsulfoxide (DMSO) is the preferred medium of those compared for cryopreservation of placenta-derived cells.
  • DMEM Dulbecco's modified Eagle's medium
  • DMSO dimethylsulfoxide
  • Tissue culture plastic flasks were coated by adding 20 milliliters 2% (w/v) porcine gelatin (Type B: 225 Bloom; Sigma, St Louis, MO) to a T75 flask (Corning, Corning, NY) for 20 minutes at room temperature. After removing the gelatin solution, 10 milliliters phosphate-buffered saline (PBS) (Invitrogen, Carlsbad, CA) were added and then aspirated.
  • PBS phosphate-buffered saline
  • Cells were initially seeded at 5,000 cells/cm 2 on gelatin-coated T75 flasks in DMEM-Low glucose growth medium ((Invitrogen, Carlsbad, CA), with 15% (v/v) defined bovine serum (Hyclone, Logan, UT; Lot#AND 18475), 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis, MO), 100 Units/milliliter penicillin, 100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliter amphotericin B; Invitrogen, Carlsbad, CA). For subsequent passages, cell cultures were treated as follows. After trypsinization, viable cells were counted after Trypan Blue staining. Cell suspension (50 microliters) was combined with Trypan Blue (50 microliters, Sigma, St. Louis MO). Viable cell numbers were estimated using a hemocytometer.
  • the cell yield, population doubling (In (cell final/cell initial)/ln 2), and doubling time (time in culture (h)/population doubling) were calculated for each passage.
  • Cloning may be used in order to expand a population of neonatal or maternal cells successfully from placental tissue. Following isolation of three different cell populations from the placenta (neonatal aspect, maternal aspect, and villous region), these cell populations are expanded under standard growth conditions and then karyotyped to reveal the identity of the isolated cell populations. By isolating the cells from a mother who delivers a boy, it is possible to distinguish between the male and female chromosomes by performing metaphase spreads.
  • top-line cells are karyotype positive for neonatal phenotype
  • mid-line cells are karyotype positive for both neonatal and maternal phenotypes
  • bottom-line cells are karyotype positive for maternal cells.
  • a clonal neonatal or maternal cell population can be expanded from placenta-derived cells isolated from the neonatal aspect or the maternal aspect, respectively, of the placenta.
  • Cells are serially diluted and then seeded onto gelatin-coated plates in Growth medium for expansion at 1 cell/well in 96-well gelatin-coated plates. From this initial cloning, expansive clones are identified, trypsinized, and reseeded in 12-well gelatin-coated plates in Growth medium and then subsequently passaged into T25 gelatin-coated flasks at 5,000 cells/cm 2 in Growth medium.
  • Subcloning is performed to ensure that a clonal population of cells has been identified.
  • cells are trypsinized and reseeded at 0.5 cells/well.
  • the subclones that grow well are expanded in gelatin-coated T25 flasks at 5,000 cells cm 2 /flask .
  • Cells are passaged at 5,000 cells cm 2 /T75 flask.
  • the growth characteristics of a clone may be plotted to demonstrate cell expansion.
  • Karyotyping analysis can confirm that the clone is either neonatal or maternal.
  • Postpartum-derived cells can be expanded in culture for such purposes. Comparisons were made of the growth of postpartum-derived cells in culture to that of other cell populations including mesenchymal stem cells. The data demonstrated that postpartum-derived cell lines as developed herein can expand for greater than 40 doublings to provide sufficient cell numbers, for example, for pre-clinical banks. Furthermore, these postpartum-derived cell populations can be expanded well at low or high density. This study has demonstrated that mesenchymal stem cells, in contrast, cannot be expanded to obtain large quantities of cells.
  • Table 5-1 Growth characteristics for different cell populations grown to senescence
  • Table 5-2 Growth characteristics for different cell populations using low density growth expansion from passage 10 to senescence
  • Cell lines used in cell therapy are preferably homogeneous and free from any contaminating cell type. Human cells used in cell therapy should have a normal chromosome number (46) and structure. To identify postpartum-derived placental and umbilicus cell lines PCf / l! . S S O !5 / ' H H B U B that are homogeneous and free from cells of non-postpartum tissue origin, karyotypes of cell samples were analyzed.
  • PPDCs from postpartum tissue of a male neonate were cultured in Growth medium (DMEM-low glucose (Gibco Carlsbad, CA), 15% (v/v) fetal bovine serum (FBS) (Hyclone, Logan, UT), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, MO), and 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco, Carlsbad, CA)).
  • Growth medium DMEM-low glucose (Gibco Carlsbad, CA)
  • FBS fetal bovine serum
  • betamercaptoethanol Sigma, St. Louis, MO
  • Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco, Carlsbad, CA)
  • Postpartum tissue from a male neonate (X, Y) was selected to allow distinction between neonatal- derived cells and maternal-derived cells (X 5 X).
  • Cells were seeded at 5,000 cells per square centimeter in Growth medium in a T25 flask (Corning, Corning, NY) and expanded to about 80% confluence. A T25 flask containing cells was filled to the neck with Growth medium. Samples were delivered to a clinical cytogenetics lab by courier (estimated lab to lab transport time is one hour). Chromosome analysis was performed by the Center for Human & Molecular Genetics at the New Jersey Medical School, Newark, NJ. Cells were analyzed during metaphase when the chromosomes are best visualized. Of twenty cells in metaphase counted, five were analyzed for normal homogeneous karyotype number (two) .
  • a cell sample was characterized as homogeneous if two karyotypes were observed.
  • a cell sample was characterized as heterogeneous if more than two karyotypes were observed. Additional metaphase cells were counted and analyzed when a heterogeneous karyotype number (four) was identified.
  • N- Neonatal side V- villous region
  • M- maternal side C- clone
  • Chromosome analysis identified placenta- and umbilicus-derived PPDCs whose karyotypes appear normal as interpreted by a clinical cytogenetic laboratory.
  • Karyotype analysis also identified cell lines free from maternal cells, as determined by homogeneous karyotype.
  • Characterization of cell surface proteins or "markers" by flow cytometry can be used to determine a cell line's identity. The consistency of expression can be determined from multiple donors and in cells exposed to different processing and culturing conditions. Postpartum-derived cell lines isolated from the placenta and umbilicus were characterized by flow cytometry, thereby providing a profile for the identification of the cells of the invention.
  • Placenta- and Umbilicus-Derived Cell Comparison Placenta-derived cells were compared to umbilicus-derived cells at passage 8.
  • Donor to Donor Comparison To compare differences among donors, placenta-derived cells from different donors were compared to each other, and umbilicus-derived cells from different donors were compared to each other.
  • Placental Layer Comparison Cells isolated from the maternal aspect of placental tissue were compared to cells isolated from the villous region of placental tissue and cells isolated from the neonatal fetal aspect of placenta.
  • Placenta-derived cells were compared to Umbilicus-derived cells. Placenta- and umbilicus-derived cells analyzed by flow cytometry showed positive for production of CDlO, CD 13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, indicated by the increased values of fluorescence relative to the IgG control. These cells were negative for detectable production of CD31, CD34, CD45, (ZDl 17, CD141, and HLA-DR, DP, DQ, indicated by fluorescence values comparable to the IgG control. Variations in fluorescence values of positive curves was accounted. The mean (i.e., CD13) and range (i.e., CD90) of the positive curves showed some variation, but the curves appeared normal, confirming a homogeneous population. Both curves individually exhibited values greater than the IgG control.
  • Placenta-derived cells at passages 8, 15, and 20 analyzed by flow cytometry all were positive for production of CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, as reflected in the increased value of fluorescence relative to the IgG control. The cells were negative for production of T s ' V H O S ' •' l! 4-61;10 E
  • CD31, CD34, CD45, CDl 17, CD141, and HLA-DR, DP, DQ having fluorescence values consistent with the IgG control.
  • Umbilicus-derived cells at passage 8, 15, and 20 analyzed by flow cytometry all expressed CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, indicated by increased fluorescence relative to the IgG control. These cells were negative for CD31, CD34, CD45, CDl 17, CD 141, and HLA-DR, DP, DQ, indicated by fluorescence values consistent with the IgG control.
  • Placenta-derived cells isolated from separate donors analyzed by flow cytometry each expressed CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, with increased values of fluorescence relative to the IgG control.
  • the cells were negative for production of CD31, CD34, CD45, CDl 17, CD 141, and HLA-DR, DP, DQ as indicated by fluorescence value consistent with the IgG control.
  • Umbilicus-derived cells isolated from separate donors analyzed by flow cytometry each showed positive for production of CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values of fluorescence relative to the IgG control. These cells were negative for production of CD31, CD34, CD45, CDl 17, CD141, and HLA-DR, DP, DQ with fluorescence values consistent with the IgG control.
  • Placenta-derived cells expanded on either gelatin-coated or uncoated flasks analyzed by flow cytometry all expressed CDlO, CD 13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values of fluorescence relative to the IgG control. These cells were negative for production of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ indicated by fluorescence values consistent with the IgG control.
  • Umbilicus-derived cells expanded on gelatin and uncoated flasks analyzed by flow cytometry all were positive for production of CDlO, CD 13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, with increased values of fluorescence relative to the IgG control. These cells were negative for production of CD31, CD34, CD45, CDl 17, CD141, and HLA-DR, DP, DQ, with fluorescence values consistent with the IgG control.
  • CD73, CD90, PDGFr-alpha and HLA-A, B, C as indicated by the increased values of fluorescence relative to the IgG control.
  • These cells were negative for production of CD31, CD34, CD45, CDl 17, CD141, and HLA-DR, DP, DQ as indicated by fluorescence values consistent with the IgG control.
  • Placental Layer Comparison Cells derived from the maternal, villous, and neonatal layers of the placenta, respectively, analyzed by flow cytometry showed positive for production of CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, as indicated by the increased value of fluorescence relative to the IgG control. These cells were negative for production of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence values consistent with the IgG control.
  • Placenta- and umbilicus-derived postpartum cells are positive for CDlO, CD 13, CD44, CD73, CD90, PDGFr-alpha, HLA-A 5 B, C and negative for CD31, CD34, CD45, CDl 17, CD141and HLA-DR, DP, DQ. This identity was consistent between variations in variables including the donor, passage, culture vessel surface coating, digestion enzymes, and placental layer.
  • Affymetrix GeneChip® arrays were used to compare gene expression profiles of umbilicus- and placenta-derived cells with fibroblasts, human mesenchymal stem cells, and another cell line derived from human bone marrow. This analysis provided a characterization of the postpartum-derived cells and identified unique molecular markers for these cells.
  • tissue-derived cells Human umbilical cords and placenta were obtained from National Disease Research Interchange (NDRI, Philadelphia, PA) from normal full term deliveries with patient consent. The tissues were received and cells were isolated as described in Example 1. Cells were cultured in Growth medium (Dulbecco's Modified Essential Media (DMEM-low glucose; Invitrogen, Carlsbad, CA) with 15% (v/v) fetal bovine serum (Hyclone, Logan UT), 100 Units/milliliter penicillin, 100 micrograms/milliliter streptomycin (Invitrogen, Carlsbad, CA), and 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis MO)) on gelatin-coated tissue culture plastic flasks. The cultures were incubated at 37°C in standard atmosphere.
  • Growth medium Dulbecco's Modified Essential Media (DMEM-low glucose; Invitrogen, Carlsbad, CA) with 15% (v/v) fetal bovine serum (Hyclone,
  • Fibroblasts Human dermal fibroblasts were purchased from Cambrex Incorporated (Walkersville, MD; Lot number 9F0844) and were obtained from ATCC CRL-1501 (CCD39SK). Both lines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, CA) with 10% (v/v) fetal bovine serum (Hyclone) and 100 Units/milliliter penicillin, 100 micrograms/milliliter streptomycin (Invitrogen). The cells were grown on standard tissue-treated plastic.
  • hMSC Human Mesenchymal Stem Cells
  • ICBM Human Ileac Crest Bone Marrow Cells
  • Human ileac crest bone marrow was received from NDRI with patient consent.
  • the marrow was processed according to the method outlined by Ho et al. (WO03/025149).
  • the marrow was mixed with lysis buffer (155 micromolar NH 4 Cl, 10 micromolar KHCO 3 , and 0.1 micromolar EDTA, pH 7.2) at a ratio of 1 part bone marrow to 20 parts lysis buffer.
  • the cell suspension was vortexed, incubated for 2 minutes at ambient temperature, and centrifuged for 10 minutes at 500 x g.
  • the supernatant was discarded and the cell pellet was resuspended in Minimal Essential Medium-alpha (Invitrogen) supplemented with 10 % (v/v) fetal bovine serum and 4 micromolar glutamine.
  • the cells were centrifuged again and the cell pellet was resuspended in fresh medium.
  • the viable mononuclear cells were counted using trypan-blue exclusion (Sigma, St. Louis, MO).
  • the mononuclear cells were seeded in tissue-cultured plastic flasks at 5 x 10 4 cells/cm 2 .
  • the cells were incubated at 37°C with 5% CO 2 at either standard atmospheric O 2 or at 5% O 2 .
  • Cells were cultured for 5 days without a media change. Media and non-adherent cells were removed after 5 days of culture. The adherent cells were maintained in culture.
  • Table 8-1 Cells analyzed by the microarray study. The cell lines are listed by their identification code along with passage at the time of analysis, cell growth substrate, and Growth medium.
  • Table 8-2 The Euclidean Distances for the Cell Pairs.
  • the Euclidean distance was calculated for the cell types using the 290 genes that were differentially expressed between the cell types. Similarity between the cells is inversely proportional to the Euclidean distance.
  • Tables 8-3, 8-4, and 8-5 show the expression of genes increased in placenta- derived cells (Table 8-3), increased in umbilicus-derived cells (Table 8-4), and reduced in umbilicus- and placenta-derived cells (Table 8-5).
  • the column entitled “Probe Set ID” refers to the manufacturer's identification code for the sets of several oligonucleotide probes located on a particular site on the chip, which hybridize to the named gene (column "Gene Name”), comprising a sequence that can be found within the NCBI (GenBank) database at the specified accession number (column "NCBI Accession Number").
  • Tables 8-6, 8-7, and 8-8 show the expression of genes increased in human fibroblasts (Table 8-6), ICBM cells (Table 8-7), and MSCs (Table 8-8).
  • Homo sapiens cDNA FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic, intermediate polypeptide 1 ankyrin 3, node of Ranvier (ankyrin G) inhibin, beta A (activin A, activin AB alpha polypeptide) ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function)
  • LIM protein (similar to rat protein kinase C-binding enigma) inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein hypothetical protein FLJ22004
  • cytokine receptor CRL2 precursor [Homo sapiens] transforming growth factor, beta 2 hypothetical protein MGC29643 antigen identified by monoclonal antibody MRC OX-2
  • Jagged 1 (Alagille syndrome) proteoglycan 1, secretory granule
  • B-cell CLL/lymphoma 6 zinc finger protein 51
  • zinc finger protein 36 zinc finger protein 36
  • C3H type homolog (mouse)
  • the GENECHIP analysis was performed to provide a molecular characterization of the postpartum cells derived from umbilicus and placenta. This analysis included cells derived from three different umbilical cords and three different placentas. The study also included two different lines of dermal fibroblasts, three lines of mesenchymal stem cells, and three lines of ileac crest bone marrow cells. The mRNA that was expressed by these cells was analyzed by AffyMetrix GENECHIP that contained oligonucleotide probes for 22,000 genes.
  • Results showed that 290 genes are differentially expressed in these five different cell types. These genes include ten genes that are specifically increased in the placenta-derived cells and seven genes specifically increased in the umbilicus-derived cells. Fifty-four genes were found to have specifically lower expression levels in placenta and umbilical cord.
  • Placenta-derived cells three isolates, including one isolate predominately neonatal as identified by karyotyping analysis), umbilicus-derived cells (four isolates), and Normal Human Dermal Fibroblasts (NHDF; neonatal and adult) were grown in Growth medium (DMEM-low glucose (Gibco, Carlsbad, CA), 15% (v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan, UT), 0.001% (v/v) beta-mercaptoethanol (Sigma, St.
  • Growth medium DMEM-low glucose (Gibco, Carlsbad, CA)
  • fetal bovine serum Cat. #SH30070.03; Hyclone, Logan, UT
  • beta-mercaptoethanol Sigma, St.
  • MSCs Mesenchymal Stem Cells
  • MSCGM Mesenchymal Stem Cell Growth Medium Bullet kit
  • IL-8 secretion experiment cells were thawed from liquid nitrogen and plated in gelatin-coated flasks at 5,000 cells/cm 2 , grown for 48 hours in Growth medium, and then grown for an additional 8 hours in 10 milliliters of serum starvation medium (DMEM-I ow glucose (Gibco, Carlsbad, CA), 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco, Carlsbad, CA), and 0.1% (w/v) Bovine Serum Albumin (BSA; Sigma, St. Louis, MO)). After this treatment, RNA was extracted and the supernatants were centrifuged at 150 x g for 5 minutes to remove cellular debris. Supernatants were then frozen at -80°C for ELISA analysis.
  • serum starvation medium DMEM-I ow glucose (Gibco, Carlsbad, CA), 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gi
  • ELISA assay The amount of IL-8 secreted by the cells into serum starvation medium was analyzed using ELISA assays (R&D Systems, Minneapolis, MN). All assays were tested according to the instructions provided by the manufacturer.
  • RNA isolation was extracted from confluent postpartum-derived cells and fibroblasts. RNA was extracted from cells treated as described above for IL-8 expression analysis. Cells were lysed with 350 microliters buffer RLT containing beta- mercaptoethanol (Sigma, St. Louis, MO) according to the manufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, CA). RNA was extracted according to the manufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, CA) and subjected to DNase treatment (2.7 U/sample) (Sigma St. Louis, MO). RNA was eluted with 50 microliters DEPC-treated water and stored at -80 0 C.
  • Genes identified by cDNA microarray as uniquely regulated in postpartum- derived cells were further investigated using real-time and conventional PCR.
  • PCR was performed on cDNA samples using ASSAYS-ON- DEMAND gene expression products: oxidized LDL receptor (Hs00234028); renin (Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2 (Hs00605742); IL-8 (Hs00174103); and GAPDH were mixed with cDNA and TaqMan Universal PCR master mix according to the manufacturer's instructions (Applied Biosystems, Foster City, CA) using a 7000 sequence detection system with ABI Prism 7000 SDS software (Applied Biosystems, Foster City, CA).
  • Thermal cycle conditions were initially 50 0 C for 2 minutes and 95°C for 10 min, followed by 40 cycles of 95 0 C for 15 seconds and 6O 0 C for 1 minute.
  • PCR data was analyzed according to manufacturer's specifications (User Bulletin #2 from Applied Biosystems for ABI Prism 7700 Sequence Detection System).
  • Amplification was optimized for each primer set: for IL-8, CXC ligand 3, and reticulon (94 0 C for 15 seconds, 55°C for 15 seconds and 72 0 C for 30 seconds for 30 cycles); for renin (94 0 C for 15 seconds, 53 0 C for 15 seconds and 72 0 C for 30 seconds for 38 cycles); for oxidized LDL receptor and GAPDH (94°C for 15 seconds, 55°C for 15 seconds and 72°C for 30 seconds for 33 cycles). Primers used for amplification are listed in Table 1. Primer concentration in the final PCR reaction was 1 & f ⁇ /- ? 5 *V ill 1 ⁇ •'" !l W"b 1 K H "1 !
  • GAPDH K micromolar except for GAPDH which was 0.5 micromolar.
  • GAPDH primers were the same as real-time PCR, except that the manufacturer's TaqMan probe was not added to the final PCR reaction.
  • Samples were run on 2% (w/v) agarose gel and stained with ethidium bromide (Sigma, St. Louis, MO). Images were captured using a 667 Universal Twinpack film (VWR International, South Plainfield, NJ) using a focal-length POLAROID camera (VWR International, South Plainfield, NJ).
  • Oxidized LDL receptor S 5 ' -GAGAAATCCAAAGAGCAAATGG-S ' ( SEQ ID NO : 1 )
  • Renin S 5 ' -TCTTCGATGCTTCGGATTCC- B ' ( SEQ ID NO : 3 )
  • Interleukin-8 S 5 ' - TCTGCAGCTCTGTGTGAAGG-3 ' ( SEQ ID NO : 7 )
  • anti-human GROalpha - PE (1:100; Becton Dickinson, Franklin Lakes, NJ)
  • anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, CA)
  • anti-human oxidized LDL receptor 1 ox-LDL Rl; 1:100; Santa Cruz Biotech
  • anti-human NOGA-A (1:100; Santa Cruz, Biotech).
  • fluorescence was visualized using an appropriate fluorescence filter on an Olympus inverted epi-fluorescent microscope (Olympus, Melville, NY). In all cases, positive staining represented fluorescence signal above control staining where the entire procedure outlined above was followed with the exception of application of a primary antibody solution (no 1° control). Representative images were captured using a digital color videocamera and ImagePro software (Media Cybernetics, Carlsbad, CA). For triple-stained samples, each image was taken using only one emission filter at a time. Layered montages were then prepared using Adobe Photoshop software (Adobe, San Jose, CA).
  • Antibody was added to aliquots as per manufacturer's specifications, and the cells were incubated in the dark for 30 minutes at 4 0 C. After incubation, cells were washed with PBS and centrifuged to remove excess antibody. Cells requiring a secondary antibody were resuspended in 100 microliters of 3% FBS. Secondary antibody was added as per manufacturer's specification, and the cells were incubated in the dark for 30 minutes at 4°C. After incubation, cells were washed with PBS and centrifuged to remove excess secondary antibody. Washed cells were resuspended in 0.5 milliliter PBS and analyzed by flow cytometry.
  • results of real-time PCR for selected "signature" genes performed on cDNA from cells derived from human placentas, adult and neonatal fibroblasts, and Mesenchymal Stem Cells (MSCs) indicate that both oxidized LDL receptor and renin were expressed at higher level in the placenta-derived cells as compared to other cells.
  • the data obtained from real-time PCR were analyzed by the ⁇ CT method and expressed on a logarithmic scale. Levels of reticulon and oxidized LDL receptor expression were higher in umbilicus-derived cells as compared to other cells. No significant difference in the expression levels of CXC ligand 3 and GCP-2 were found between postpartum-derived cells and controls (data not shown).
  • CXC-ligand 3 was expressed at very low levels. GCP-2 was expressed at levels comparable to human adult and neonatal fibroblasts. The results of real-time PCR were confirmed by conventional PCR. Sequencing of PCR products further validated these observations. No significant difference in the expression level of CXC ligand 3 was found between postpartum-derived cells and controls using conventional PCR CXC ligand 3 primers listed in Table 9-1.
  • Table 9-2 IL-8 protein expression measured by ELISA
  • Placenta-derived cells were also examined for the expression of oxidized LDL receptor, GCP-2, and GROalpha by FACS analysis. Cells tested positive for GCP-2. Oxidized LDL receptor and GRO were not detected by this method.
  • Placenta-derived cells were also tested for the expression of selected proteins by immunocytochemical analysis. Immediately after isolation (passage 0), cells derived from the human placenta were fixed with 4% paraformaldehyde and exposed to antibodies for six proteins: von Willebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscle actin, and vimentin. Cells stained positive for both alpha-smooth muscle actin and vimentin. This pattern was preserved through passage 11. Only a few cells ( ⁇ 5%) at passage 0 stained positive for cytokeratin 18.
  • Placenta-derived cells at passage 11 were also investigated by immunocytochemistry for the expression of GROalpha and GCP-2. Placenta-derived cells were GCP-2 positive, but GROalpha expression was not detected by this method.
  • the complete mRNA data at least partially verifies the data obtained from the microarray experiments.
  • anti-human GROalpha - PE (1:100; Becton Dickinson, Franklin Lakes, NJ)
  • anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, CA)
  • anti-human oxidized LDL receptor 1 ox -LDL Rl; 1:100; Santa Cruz Biotech
  • anti -human NOGO-A (1:100; Santa Cruz Biotech).
  • Fixed specimens were trimmed with a scalpel and placed within OCT embedding compound (Tissue-Tek OCT; Sakura, Torrance, CA) on a dry ice bath containing ethanol. Frozen blocks were then sectioned (10 micron thick) using a standard cryostat (Leica Microsystems) and mounted onto glass slides for staining.
  • Table 10-1 Summary of Primary Antibodies Used Antibody Concentration Vendor
  • Immunohistochemistry was performed similar to previous studies (e.g., Messina, et al. (2003) Exper. Neurol. 184: 816-829). Tissue sections were washed with phosphate-buffered saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula, CA), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 1 hour to access intracellular antigens.
  • PBS phosphate-buffered saline
  • Triton Triton X-100
  • fluorescence was visualized using the appropriate fluorescence filter on an Olympus inverted epi -fluorescent microscope (Olympus, Melville, NY). Positive staining was represented by fluorescence signal above control staining. Representative images were captured using a digital color videocamera and ImagePro software (Media
  • Vimentin, desmin, SMA, CKl 8, vWF, and CD34 markers were expressed in a subset of the cells found within umbilical cord (data not shown). In particular, vWF and CD34 expression were restricted to blood vessels contained within the cord. CD34+ cells were on the innermost layer (lumen side). Vimentin expression was found throughout the matrix and blood vessels of the cord. SMA was limited to the matrix and outer walls of the artery and vein but was not contained within the vessels themselves. CKl 8 and desmin were observed within the vessels only, desmin being restricted to the middle and outer layers.
  • GROalpha, GCP-2, ox-LDL Rl, and NOGO-A Tissue Expression None of these markers were observed within umbilical cord or placental tissue (data not shown).
  • Postpartum-derived cell lines were evaluated in vitro for their immunological characteristics in an effort to predict the immunological response, if any, these cells would elicit upon in vivo transplantation. Postpartum-derived cell lines were assayed by flow cytometry for the expression of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed by antigen-presenting cells (APC) and are required for the direct stimulation of na ⁇ ve CD4 + T cells (Abbas & Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171).
  • APC antigen-presenting cells
  • HLA-G Abbas & Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171
  • CD 178 Cells, et.al, (1999) Journal of K - C T ,. " U S O '!; “ ;. , ⁇ " H I Ii B O B Immunological Methods 224, 185-196)
  • PD-L2 Abbas & Lichtman, CELLULAR AND
  • Cell culture Cells were cultured in Growth medium (DMEM-low glucose (Gibco, Carlsbad, CA), 15% (v/v) fetal bovine serum (FBS); (Hyclone, Logan, UT), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, MO), 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco, Carlsbad, CA)) until confluent in T75 flasks (Corning, Corning, NY) coated with 2% gelatin (Sigma, St. Louis, MO).
  • Growth medium DMEM-low glucose (Gibco, Carlsbad, CA), 15% (v/v) fetal bovine serum (FBS); (Hyclone, Logan, UT), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, MO), 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco, Carls
  • the stimulation index for the allogeneic donor was calculated as the mean proliferation of the receiver plus mitomycin C-treated allogeneic donor divided by the baseline proliferation of the receiver.
  • the stimulation index of the postpartum-derived cells was calculated as the mean proliferation of the receiver plus mitomycin C-treated postpartum-derived cell line divided by the baseline proliferation of the receiver.
  • Table 11-5 Average stimulation index of umbilicus-derived cells and an allogeneic donor in a mixed lymphocyte reaction with five individual allogeneic receivers.
  • Histograms of umbilicus-derived cells analyzed by flow cytometry show positive expression of PD-L2, as noted by the increased value of fluorescence relative to the IgG control, and negative expression of CD178 and HLA-G, as noted by fluorescence value consistent with the IgG control.
  • Placenta- and umbilicus-derived cell lines were negative for the expression of immuno-modulating proteins HLA-G and CD178 and positive for the expression of PD-L2, as measured by flow cytometry.
  • Allogeneic donor PBMCs contain antigen-presenting cells expressing HLA-DP, DR, DQ, CD80, CD86, and B7-H2, thereby allowing for the stimulation of allogeneic PBMCs (e.g., na ⁇ ve CD4 + T cells).
  • HGF hepatocyte growth factor
  • MCP-I monocyte chemotactic protein 1
  • IL-8 interleukin-8
  • keratinocyte growth factor KGF
  • basic fibroblast growth factor bFGF
  • VEGF vascular endothelial growth factor
  • TPO tissue inhibitor of matrix metalloproteinase 1
  • ANG2 angiopoietin 2
  • PDGF-bb platelet derived growth factor
  • TPO thrombopoietin
  • HB-EGF stromal-derived factor Ia
  • SDF-Ia neurotrophic/neuroprotective activity
  • BDNF brain-derived neurotrophic factor
  • IL-6 interleukin-6
  • GCP-2 granulocyte chemotactic protein-2
  • TGFbeta2 transforming growth factor beta2
  • chemokine activity microphage inflammatory protein Ia (MIPIa), macrophage inflammatory protein lbeta (MIPIb), monocyte chemoattractant-1 (MCP-I), Rantes (regulated on activation, normal T cell expressed and secreted), 1309, thymus and activation-regulated chemokine (TARC), Eotaxin, macrophage- derived chemokine (MDC), IL-8).
  • MIPIa interleukin-6
  • GCP-2 granulocyte chemotactic protein-2
  • TGFbeta2 transforming growth factor beta2
  • chemokine activity microphage inflammatory protein Ia (MIPIa), macrophage inflammatory protein lbeta (MIPIb), monocyte chemoattractant-1 (MCP-I), Rantes (regulated on activation, normal T cell expressed
  • PPDCs derived from placenta and umbilicus as well as human fibroblasts derived from human neonatal foreskin were cultured in Growth medium (DMEM-low glucose (Gibco, Carlsbad, CA), 15% (v/v) fetal bovine serum (SH30070.03; Hyclone, Logan, UT), 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco)) on gelatin- coated T75 flasks. Cells were cryopreserved at passage 11 and stored in liquid nitrogen.
  • Growth medium DMEM-low glucose (Gibco, Carlsbad, CA)
  • fetal bovine serum SH30070.03; Hyclone, Logan, UT
  • Cells were cryopreserved at passage 11 and stored in liquid nitrogen.
  • the medium was changed to a serum-free medium (DMEM- low glucose (Gibco), 0.1% (w/v) bovine serum albumin (Sigma), 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco)) for 8 hours.
  • Conditioned serum-free media was collected at the end of incubation by centrifugation at 14,000 x g for 5 minutes and stored at - 80°C.
  • PBS phosphate-buffered saline
  • trypsin/EDTA Gibco
  • Trypsin activity was inhibited by addition of 8 milliliters Growth medium.
  • Cells were centrifuged at 150 x g for 5 minutes. Supernatant was removed, and cells were
  • the arrays are produced by spotting a 2 x 2, 3 x 3, or 4 x 4 pattern of four to 16 different capture antibodies into each well of a 96- well plate. Following a sandwich ELISA procedure, the entire plate is imaged to capture chemiluminescent signal generated at each spot within each well of the plate. The amount of signal generated in each spot is proportional to the amount of target protein in the original standard or sample.
  • MCP-I and EL-6 were secreted by placenta- and umbilicus- derived PPDCs and dermal fibroblasts (Table 12-1). Umbilicus-derived cells secreted at least 10-fold higher amounts of MCP-I and IL6 than other cell populations. GCP-2 and EL-8 were highly expressed by umbilicus-derived PPDCs. TGF-beta2 was not detectable. VEGF was detected in fibroblast medium.
  • HGF, FGF, and BDNF secreted from umbilicus-derived cells were noticeably higher than fibroblasts and placenta-derived cells (Tables 12-2 and 12-3).
  • TEVIPl, TPO, HBEGF, MCP-I, TARC, and IL-8 were higher in umbilicus-derived cells than other cell populations (Table 12-3). No ANG2 or PDGF-bb were detected.
  • hFB human fibroblasts
  • Pl placenta-derived PPDC (042303)
  • Ul umbilicus-derived PPDC (022803)
  • P3 placenta-derived PPDC (071003)
  • U3 umbilicus-derived PPDC (071003)).
  • ND Not Detected.
  • hFB human fibroblasts
  • Pl placenta-derived PPDC (042303)
  • Ul umbilicus-derived PPDC (022803)
  • P3 placenta-derived PPDC (071003)
  • U3 umbilicus-derived PPDC (071003)).
  • ND Not Detected.
  • Umbilicus derived-cells secreted significantly higher amount of trophic factors than placenta-derived cells and fibroblasts. Some of these trophic factors, such as HGF, bFGF, MCP-I and EL-8, play important roles in angiogenesis. Other trophic factors, such as BDNF and IL-6, have important roles in neural regeneration. Under these conditions, the expression of some factors was confined to umbilicus-derived cells, such as MIPIb, Rantes, 1309, and FGF. References
  • Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain. Dev. Biol. 258;319-33.
  • Tissue factor a membrane-bound procoagulant glycoprotein
  • Tissue factor also plays an important role in embryonic vessel formation, for example, in the formation of the primitive vascular wall (Brodsky et al. (2002) Exp. Nephrol. 10:299-306).
  • PPDCs a membrane-bound procoagulant glycoprotein
  • Human Tissue factor Human tissue factor SIMPLASTIN (Organon Tekailca Corporation, Durham, NC) was reconstituted with 20 milliliters distilled water. The stock solution was serially diluted (1:2) in eight tubes. Normal human plasma (George King Bio- Medical, Overland Park, KS) was thawed at 37 0 C in a water bath and then stored in ice before use. To each well of a 96-well plate was added 100 microliters phosphate buffered saline (PBS), 10 microliters diluted Simplastin® (except a blank well), 30 microliters 0.1 molar calcium chloride, and 100 microliters of normal human plasma. The plate was immediately placed in a temperature-controlled microplate reader and absorbance measured at 405 nanometers at 40 second intervals for 30 minutes.
  • PBS phosphate buffered saline
  • Simplastin® except a blank well
  • J-82 and postpartum-derived cells were grown in Iscove's modified Dulbecco's medium (IMDM; Gibco, Carlsbad, CA) containing 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan UT), 1 millimolar sodium pyruvate (Sigma Chemical, St. Louis, MO), 2 millimolar L-Glutamin (Mediatech Herndon, VA), 1 x non-essential amino acids (Mediatech Herndon, VA). At 70% confluence, cells were transferred to wells of a 96-well plate at 100,000, 50,000, and 25,000 cells/well.
  • IMDM Iscove's modified Dulbecco's medium
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • L-Glutamin Mediatech Herndon, VA
  • VA non-essential amino acids
  • Postpartum-derived cells derived from placenta and umbilicus were cultured in Growth Medium (DMEM-low glucose (Gibco), 15% (v/v) FBS, 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco), and 0.001% betamercaptoethanol (Sigma)) in gelatin-coated T75 flasks (Corning, Corning, NY). Placenta- derived cells at passage 5 and umbilicus-derived cells at passages 5 and 11 were transferred to wells at 50,000 cells/well. Culture medium was removed from each well after centrifugation at 150 x g for 5 minutes. Cells were suspended in PBS without calcium and magnesium.
  • Growth Medium DMEM-low glucose (Gibco), 15% (v/v) FBS, 50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin (Gibco), and 0.001% betamercaptoethanol (Sigma)
  • CNTO 859 (Centocor, Malvern, PA) for 30 minutes.
  • Calcium chloride (30 microliter) was added to each well. The plate was immediately placed in a temperature-controlled microplate reader and absorbance measured at 405 nanometers at 40 second intervals for 30 minutes.
  • Cells were re-suspended in 100 microliter of 3% FBS and secondary antibody added as per the manufacturer's instructions. Cells were incubated in the dark for 30 minutes at 4°C. After incubation, cells were washed with PBS and centrifuged to remove unbound secondary antibody. Washed cells were re-suspended in 500 microliters of PBS and analyzed by flow cytometry.
  • Flow cytometry analysis revealed that both placenta- and umbilicus-derived postpartum-derived cells express tissue factor.
  • a plasma clotting assay demonstrated that tissue factor was active. Both placenta- and umbilicus-derived cells increased the clotting rate as indicated by the time to half maximal absorbance (T Vi to max; Table 13-1). Clotting was observed with both early (P5) and late (P18) cells. The T Vi to max is inversely proportional to the number of J82 cells.
  • Table 13-1 The effect of human tissue factor (SIMPLASTIN), placenta-derived cells (PIa), and umbilicus-derived cells (Umb) on plasma clotting was evaluated.
  • the time to half maximal absorbance (T 1/2 to max) at the plateau in seconds was used as a measurement unit.
  • tissue factor which can induce clotting.
  • the addition of an antibody to tissue factor can inhibit tissue factor.
  • Tissue factor is normally found on cells in a conformation that is inactive but is activated by mechanical or chemical (e.g., LPS) stress (Sakariassen et al. (2001) Thromb. Res. 104:149-74; Engstad et al. (2002) Int. Immunopharmacol. 2:1585-97).
  • LPS low-Sus-induced Thromb. Res. 104:149-74
  • Engstad et al. (2002) Int. Immunopharmacol. 2:1585-97 Thus, minimization of stress during the preparation process of PPDCs may prevent activation of tissue factor.
  • tissue factor has been associated with angiogenic activity.
  • tissue factor activity may be beneficial when umbilicus- or placenta-derived PPDCs are transplanted in tissue but should be inhibited when PPDCs are injected intravenously.
  • Angiogenesis or the formation of new vasculature, is necessary for the growth of new tissue. Induction of angiogenesis is an important therapeutic goal in many pathological conditions.
  • the present study was aimed at identifying potential angiogenic activity of the postpartum-derived cells in in vitro assays. The study followed a well-established method of seeding endothelial cells onto a culture plate coated with MATRIGEL (BD Discovery Labware, Bedford, MA), a basement membrane extract (Nicosia and Ottinetti (1990) In Vitro Cell Dev. Biol. 26(2): 119-28).
  • MATRIGEL BD Discovery Labware, Bedford, MA
  • MATRIGEL basement membrane extract
  • Treating endothelial cells on MATRIGEL (BD Discovery Labware, Bedford, MA) with angiogenic factors will stimulate the cells to form a network that is similar to capillaries. This is a common in vitro assay for testing stimulators and inhibitors of blood vessel formation (Ito et al. (1996) Int. J. Cancer 67(1): 148-52).
  • the present studies made use of a co- culture system with the postpartum-derived cells seeded onto culture well inserts. These permeable inserts allow for the passive exchange of media components between the endothelial and the postpartum-derived cell culture media.
  • hMSC Human mesenchymal stem cells
  • Actively growing MSCs were trypsinized and counted and seeded onto COSTAR TRANSWELL 6.5 millimeter diameter tissue culture inserts (Corning, Corning, NY) at 15,000 cells per insert. Cells were cultured on the inserts for 48-72 hours in Growth medium under standard growth conditions.
  • HUVEC Human umbilical vein endothelial cells
  • HCAEC Human coronary artery endothelial cells
  • MATRIGEL Endothelial Network Formation assays. Culture plates were coated with MATRIGEL (BD Discovery Labware, Bedford, MA) according to manufacturer's specifications. Briefly, MATRIGELTM (BD Discovery Labware, Bedford, MA) was thawed at 4°C and approximately 250 microliters were aliquoted and distributed evenly onto each well of a chilled 24-well culture plate (Corning). The plate was then incubated at 37 °C for 30 minutes to allow the material to solidify. Actively growing endothelial cell cultures were trypsinized and counted. Cells were washed twice in Growth medium with 2% FBS by centrifugation, resuspension, and aspiration of the supernatant. Cells were seeded onto the coated wells 20,000 cells per well in approximately 0.5 milliliter Growth medium with 2% (v/v) FBS. Cells were then incubated for approximately 30 minutes to allow cells to settle.
  • MATRIGEL Endothelial Network Formation
  • Endothelial cell cultures were then treated with either 10 nanomolar human bFGF (Peprotech, Rocky Hill, NJ) or 10 nanomolar human VEGF (Peprotech, Rocky Hill, NJ) to serve as a positive control for endothelial cell response.
  • Transwell inserts seeded with postpartum-derived cells were added to appropriate wells with Growth medium with 2% FBS in the insert chamber. Cultures were incubated at 37 0 C with 5% CO 2 for approximately 24 hours. The well plate was removed from the incubator, and images of the endothelial cell cultures were collected with an Olympus inverted microscope (Olympus, Melville, NY).
  • HUVEC form cell networks (data not shown). HUVEC cells form limited cell networks in co-culture experiments with hMSC and with 10 nanomolar bFGF (data not shown). HUVEC cells without any treatment showed very little or no network formation (data not shown).
  • CAECs form cell networks (data not shown).
  • Table 14-1 shows levels of known angiogenic factors released by the postpartum-derived cells in Growth medium.
  • Postpartum-derived cells were seeded onto inserts as described above. The cells were cultured at 37 °C in atmospheric oxygen for 48 hours on the inserts and then switched to a 2% FBS media and returned at 37 0 C for 24 hours. Media was removed, immediately frozen and stored at -80 0 C, and analyzed by the Searchlight Multiplex ELISA assay (Pierce Chemical Company, Rockford, IL). Results shown are the averages of duplicate measurements.
  • the cells do release measurable quantities of tissue inhibitor of metallinoprotease-1 (TIMP-I), angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF).
  • TMP-I tissue inhibitor of metallinoprotease-1
  • ANG2 angiopoietin 2
  • TPO thrombopoietin
  • KGF keratinocyte growth factor
  • HGF hepatocyte growth factor
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • Plac placenta derived cells
  • Umb cord Umbilicus derived cells
  • Table 14-2 shows levels of known angiogenic factors released by the postpartum-derived cells.
  • Postpartum-derived cells were seeded onto inserts as described above. The cells were cultured in Growth medium at 5% oxygen for 48 hours on the inserts and then switched to a 2% FBS medium and returned to 5% O 2 incubation for 24 hours. Media was removed, immediately frozen, and stored at -80 0 C, and analyzed by the Searchlight Multiplex ELISA assay (Pierce Chemical Company, Rockford, IL). Results shown are the averages of duplicate measurements. The results show that the postpartum-derived cells do not release detectable levels of platelet-derived growth factor-bb (PDGF-BB) or heparin-binding epidermal growth factor (HBEGF).
  • PDGF-BB platelet-derived growth factor-bb
  • HEGF heparin-binding epidermal growth factor
  • the cells do release measurable quantities of tissue inhibitor of metallinoprotease-1 (TIMP-I), angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF).
  • TPO tissue inhibitor of metallinoprotease-1
  • KGF keratinocyte growth factor
  • HGF hepatocyte growth factor
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • Table 14-2 Potential angiogenic factors released from postpartum-derived cells. Postpartum-derived cells were cultured in 24 hours in media with 2% FBS in 5% oxygen. Media was removed and assayed by the SearchLight multiplex ELISA assay (Pierce). Results are the means of a duplicate analysis. Values are concentrations in the media reported in picograms per milliter of culture media.
  • Plac placenta derived cells
  • Umb cord Umbilicus derived cells
  • Cells derived from the postpartum umbilical cord and placenta are useful for regenerative therapies.
  • the tissue produced by postpartum-derived cells transplanted into SCID mice with a biodegradable material was evaluated.
  • the materials evaluated were non-woven comprised of poly(lactic acid-co-glycolic acid) polymer (10/90 PLGA), 35/65 PCL/PGA foam, and RAD16 self-assembling peptide hydrogel.
  • a nonwoven scaffold was prepared using a traditional needle punching technique as described below.
  • Fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (10/90 PLGA), were obtained from Ethicon, Inc. (Somerville, NJ). The fibers were filaments of approximately 20 microns in diameter. The fibers were then cut and crimped into uniform 2-inch lengths to form 2-inch staple fiber.
  • a dry lay needle-punched nonwoven matrix (VNW) was then prepared utilizing the 10/90 PLGA staple fibers. The staple fibers were opened and carded on standard nonwoven machinery. The resulting mat was in the form of webbed staple fibers. The webbed staple fibers were needle- punched to form the dry lay needle-punched nonwoven scaffold.
  • the nonwoven scaffold was rinsed in water followed by another incubation in ethanol to remove any residual chemicals or processing aids used during the manufacturing process.
  • Foams composed of 35/65 poly(epsilon-caprolactone)/poly(glycolic acid) (35/65 PCL/PGA) copolymer, were formed by the process of lyophilization, as discussed in U.S. Patent No. 6,355,699.
  • mice Musculus/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc., Indianapolis, Indiana), 5 weeks of age. All handling of the SCID mice took place under a hood. The mice were individually weighed and anesthetized with an intraperitoneal injection of a mixture of 60 milligram/kilogram KETASET (ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa) and 10 milligram/kilogram ROMPUN (xylazine, Mobay Corp., Shawnee, Kansas) and saline.
  • KETASET ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa
  • ROMPUN milligram/kilogram
  • Subcutaneous Implantation Technique Four skin incisions, each approximately 1.0 cm in length, were made on the dorsum of the mice. Two cranial sites were located transversely over the dorsal lateral thoracic region, about 5-mm caudal to the palpated inferior edge of the scapula, with one to the left and one to the right of the vertebral column. Another two were placed transversely over the gluteal muscle area at the caudal sacro-lumbar level, about 5-mm caudal to the palpated iliac crest, with one on either side of the midline. Implants were randomly placed in these sites.
  • the skin was separated from the underlying connective tissue to make a small pocket and the implant placed (or injected for RAD16) about 1-cm caudal to the incision.
  • the appropriate test material was implanted into the subcutaneous space. The skin incision was closed with metal clips.
  • mice were individually housed in microisolator cages throughout the course of the study within a temperature range of 64 0 F - 79°F and relative humidity of 30% to 70% and maintained on an approximate 12 hour light/12 hour dark cycle. The temperature and relative humidity were maintained within the stated ranges to the greatest extent possible. Diet consisted of Irradiated Pico Mouse Chow 5058 (Purina Co.) and water fed ad libitum.
  • mice were euthanized at their designated intervals by carbon dioxide inhalation.
  • the subcutaneous implantation sites with their overlying skin were excised and frozen for histology.
  • Non-woven scaffolds seeded with umbilicus- or placenta-derived cells showed increased matrix deposition and mature blood vessels (data not shown).
  • Cells were pelleted in growth media and then resuspended in a total volume of 40 milliliters of PBS. The cells were washed three times in PBS to remove residual FBS from the growth media. This was done by centrifuging the cells for 5 minutes at 1.5 RPM and then resuspending the cells in 40 milliliters of PBS until the three washes were complete.
  • the cells were equally divided into two tubes with PBS for the freeze/thaw procedure.
  • the lysates were prepared by repeated freeze/thaw cycles.
  • To freeze the cells the tubes were placed in a slurry of dry ice and isopropanol for 10 minutes. After 10 minutes, the tubes were placed in a 37°C water bath for 10 minutes.
  • the cell suspensions were transferred to ten sterile siliconized microcentrifuge tubes, to prevent protein adsorption, and centrifuged at 13,000xg for 10 minutes at 4°C to separate the cell membranes from the cytosolic components.
  • the tubes were then placed on ice and the supernatant was very gently mixed by tapping the centrifuge tube to ensure uniformity.
  • the supernatant was transferred to new siliconized tubes and placed on ice.
  • SEARCHLIGHT Multiplexed ELISA assay Chemokines, BDNF and angiogenic factors were measured using SEARCHLIGHT Proteome Arrays (Pierce Biotechnology Inc.).
  • the proteome arrays are multiplexed sandwich ELISAs for the quantitative measurement of two to 16 proteins per well.
  • the arrays are produced by spotting a 2 x 2, 3 x 3, or 4 x 4 pattern of four to 16 different capture antibodies into each well of a 96-well plate. Following a typical sandwich ELISA procedure, the entire plate is imaged to capture chemiluminescent signal generated at each spot within each well of the plate. The amount of signal generated in each spot is proportional to the amount of target protein in the original standard or sample.
  • Table 16-1 SEARCHLIGHT Multiplexed ELISA results. Average for duplicate adjusted for dilution.
  • UDC lysate contains significant levels of beneficial factors including pro-angiogenic as well as factors that can stimulate cell proliferation and extracellular matrix production (KGF, PDGF-BB, HGF, TGFa) and neurotrophic factors (BDNF, IL-6). These factors might have beneficial effects on local environment by inducing cell proliferation, differentiation and survival. In addition, pro-angiogenic factors might induce new blood vessel formation in the wound environment and stimulate extracellular matrix formation. Furthermore, the high level of TIMPs might be extremely beneficial in the chronic wound environment, since chronic wounds are known to be associated with high levels of MMPs, known to mediate extracellular matrix degradation.
  • Umbilicus-derived cells produce and secrete various growth factors involved in tissue regeneration, including basic Fibroblast Growth Factor (bFGF), hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF), and keratinocyte growth factor (KGF).
  • bFGF basic Fibroblast Growth Factor
  • HGF hepatocyte growth factor
  • BDNF brain-derived neurotrophic factor
  • KGF keratinocyte growth factor
  • the purpose of the present study was to provide methods for the production of lyophilized UDC lysate.
  • the method consistently allowed the harvest of proteins from lysed UDCs.
  • the growth factor bFGF was present in six separate production lots of lyophilized UDC lysate averaging 3.09 +/- 1.06 picograms per microgram of total protein.
  • SDS-PAGE analysis of UDC lysate showed the banding pattern of protein was consistent between separate production lots, pre- and post- lyophilization, and lyophilization into a synthetic biomaterial.
  • the current method allowed reproducible production of lyophilized material containing growth factors for application in tissue regeneration.
  • UDCs were seeded at 5,000 cells per cm 2 in gelatin-coated flasks with growth media (Dulbecco's Modified Eagles Media (DMEM)-low glucose, 15% fetal bovine serum (FBS), penicillin/streptomycin (P/S), Betamercaptoethanol (BME) and expanded for 3 to 4 days (25,000 cells per cm 2 target harvest density).
  • growth media Dulbecco's Modified Eagles Media (DMEM)-low glucose, 15% fetal bovine serum (FBS), penicillin/streptomycin (P/S), Betamercaptoethanol (BME) and expanded for 3 to 4 days (25,000 cells per cm 2 target harvest density).
  • DMEM Dulbecco's Modified Eagles Media
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • BME Betamercaptoethanol
  • Protein Assay To determine total protein content, 10 microliters of lysate supernatant fluid was diluted into 990 microliters PBS, and the dilution was analyzed by Bradford assay (standard range 1.25- 25 micrograms). This value was used to calculate the total protein per cell, the main metric used to ensure the consistency of the process.
  • Lysate Lyophilization Multiple 1.5 milliliter sterile labeled cryo vials were loaded into a sterile heat transfer block. Aliquots of lysate supernatant fluid at defined total protein concentration were loaded into the cryovials. The heat block containing uncapped cryovials was aseptically loaded into an autoclaved pouch with tube openings facing the paper side of the pouch. The pouch was sealed before removal from the laminar flow hood. The pouch was loaded into the lyophilizer. [0429] Pre-cut materials (i.e., 90/10 PGA/PLA non-woven) were aseptically placed into the wells of 24- or 48-well sterile, ultra low cluster cell culture dishes (Corning Inc., Corning NY).
  • Lysate supernatant fluid was delivered at a defined total protein concentration onto the material. For example, a material measuring 6 mm in diameter and 0.5 mm in thickness received 2 microliters of a 15 microgram/microliter total protein solution to create a 30 microgram lysate protein material. The lid of the dish was replaced and secured with tape. The dish with materials was loaded into the lyophilizer
  • Test materials with applied lysate were loaded into a FTS Systems Dura-Stop MP Stoppering Tray Dryer and lyophilized using the following ramping program. All steps had a ramping rate of 2.5°C/minute and a 100-mT vacuum.
  • bFGF Enyme Linked Immunosorbent Assay Analysis. Vials from six separate production lots of lyophilized lysate powder were reconstituted in PBS and analyzed for total protein content by Bradford assay. The samples were then further diluted to achieve a 20 microgram/milliliter solution. Solutions further serially diluted in PBS and analyzed by ELISA using a Quantikine human bFGF kit (R&D Systems cat. no. DFB50).
  • Table 17-4 Summary of bFGF (picograms) per given quantity of total lysate protein as measured by ELISA assay
  • Equation slope and Y-intercept are derived from the average slope and Y-intercept values obtained from regression analysis of six production lots.
  • the purpose of this study was to evaluate the ability of human umbilicus cell- derived cell lysate when delivered in a scaffold to induce cellular infiltration and tissue formation. Since this is a xenogeneic source of cells, an early time-point was chosen to evaluate the inflammatory response the cell lysate might exhibit.
  • Two types of scaffolds VNW and 35/65 PCL/PGA foam) were tested to determine their potential to act as carriers for lysate delivery.
  • the cellular components of human umbilicus-derived cells (UDCs) loaded on two different scaffold types, were tested to evaluate the cell infiltration and inflammatory response elicited in a subcutaneous rat implantation study.
  • the excised tissue was placed in 10% neutral buffered formalin for histological processing (paraffin sections) and stained with hematoxylin and eosin and trichrome. Tissue sections were histologically analyzed for the percentage of ingrowth into the scaffold, the quality of ingrowth into the scaffold, the encapsulation of the scaffold, and the inflammatory response within the scaffold.
  • Treatment Groups The VNW was purchased from Biomedical Structures (Slatersville, Rhode Island). The scaffolds were placed in desiccant paper pillows that were then packaged in T-vent aluminum pouches and sterilized via ethylene oxide sterilization (nominal B cycle). The scaffolds were stored at room temperature prior to use. The following treatment groups were included in the study:
  • VNW and human umbilicus-derived cells UTC
  • VNW 3551-14-HD
  • Fibroblasts human adult; passage 10): IFl 853
  • Caprolactone/Glycolide molar composition of high purity grade (99+%) 1,4-Dioxane was prepared by dissolving five parts polymer to ninety- five parts of solvent at 60°C for 4 hours. The polymer solution was filtered through an extra coarse thimble prior to making the foam scaffolds. This polymer solution was diluted with dioxane to make a 3% w/w solution. A pre-determined amount of polymer solution was poured into a pre-cooled aluminum mold and lyophilized to remove the solvent from the frozen structure by phase separation resulting in the interconnecting pore structure.
  • VNWs were scoured to remove residual processing oils. The material was scoured twice. The VNW was agitated in isopropanol (IPA) in the BRANSONIC Ultrasonic Cleaner (BUC) for at least 30 minutes. The IPA was drained, and the VNW was washed with deionized water three times. The VNW was then agitated in deionized water in the BUC for an additional 30 minutes. The VNW was dried under vacuum overnight or until dry to the touch.
  • IPA isopropanol
  • BUC BRANSONIC Ultrasonic Cleaner
  • the cell groups (UDC and HF) were created from the original suspension. 100 microliters was added to each scaffold. The cell-loaded scaffolds were placed in a shaker for 20 minutes to encourage incorporation of the cells into the scaffolds. The cell-loaded scaffolds were then lyophilized, in tissue culture plates, prior to implantation in the rat.
  • the lysate groups were prepared by freezing and thawing cells for three cycles (-80 0 C for 10 min/37°C) and then 100 microliters was added to each scaffold.
  • the lysate-loaded scaffolds were placed in a shaker for 20 minutes to encourage incorporation of the lysate into the scaffolds.
  • the lysate loaded scaffolds were then lyophilized, in tissue culture plates, prior to implantation in the rat.
  • the supernatant groups were prepared by freezing and thawing cells for three cycles (-80 0 C for 10 min/37°C) and then centrifuged at 13,000 x g for 10 minutes at 4°C. The supernatant was collected and 100 microliters was added to each scaffold. The supernatant- loaded scaffolds were placed in a shaker for 20 minutes to encourage incorporation of supernatant into the scaffolds. The supernatant-loaded scaffolds were then lyophilized, in tissue culture plates, prior to implantation in the rat.
  • the Growth medium control group was prepared by adding 100 microliters of Growth medium (containing 10% fetal bovine serum) to each scaffold; these scaffolds were washed with PBS three times after the addition of growth medium. These scaffolds were then lyophilized, in tissue culture plates, prior to implantation in the rat.
  • Growth medium containing 10% fetal bovine serum
  • the scaffold alone groups were prepared by adding 100 microliters of PBS to each scaffold. The scaffolds were then lyophilized, in tissue culture plates, prior to implantation in the rat.
  • 35 mole% and 63.3 mole% were determined for epsilon-caprolactone and glycolide, respectively, and 0.11 mole% of CAP and 0.58 mole% of GLY.
  • the morphology of pores was analyzed by SEM method.
  • VNW scaffold used in this study was 2.06 mm thick.
  • the density, as determined by Biomechanical Structures, was 108.49 mg/cc.
  • the percent porosity was calculated to be 92.8%.
  • the VNW was tested in triplicate for residual IPA and residual ethylene oxide (EtO).
  • the samples all demonstrated less than 1 ppm residual IPA.
  • the residual EtO levels in the VNW were 8, 9, and 10 ppm.
  • the residual EtO in each sample was much less than the 250 ppm limit.
  • Tissue sections were histologically analyzed for the percentage of ingrowth into the scaffold, the quality of ingrowth into the scaffold, the encapsulation of the scaffold, and inflammatory response within the scaffold.
  • Samples tested at day 3 included UDC lysate, UDC supernatant, HF, and VNW scaffold. All treatments were incorporated into the VNW scaffold. The purpose of a three day time-point was to determine if there was an immediate immune response to the implanted cellular components.
  • the treatments incorporated into the foam scaffold demonstrated a wide range of ingrowth.
  • the average amount of ingrowth ranged from about 31% to 90%. In general, there was more ingrowth seen in the cranial sites.
  • VNW scaffolds only The treatments incorporated into the VNW demonstrated a narrower range of ingrowth into the scaffold. The average amount of ingrowth ranged from about 69% to 100%. All cranial sites with VNW demonstrated the same or more ingrowth than the caudal sites. Cellularity of Ingrowth (VNW scaffolds only):
  • the grade 'minimal' was given for reactions mainly concentrated at the surface of the material with no significant extension into the scaffold or outwards from the surface.
  • 'Slight' indicated partial cellular infiltration of the scaffold as noted above, but with no significant cellular response outwards from the surface.
  • Foams were given a 'moderate' score when there was total or almost total infiltration of the cell types noted above, but there was no significant extension of the reaction beyond the surface of the scaffold.
  • a 'pronounced' score indicated that, in addition to the 'moderate' score, there was a pronounced degree of inflammatory cell infiltration surrounding the scaffold or the primary response to the material was neutrophilic.
  • VNW the grade 'minimal' indicated only small collections of cells around individual fibers or fiber bundles and these infiltrates did not tend to coalesce.
  • 'Slight' reactions for VNWs were given when there were greater concentrations of the cell types noted above (plus possibly other mononuclear cells).
  • VNWs were given a 'moderate' score when there was total or almost total infiltration of the cell types noted above, but there was no significant extension of the reaction beyond the surface of the scaffold.
  • the Growth medium control group (negative control) appeared to impact the scaffolds in a negative way.
  • the amount of ingrowth into the VNW was very similar across the treatment groups, both in the cranial and caudal positions.
  • the UDC Supernatant demonstrated significantly increased cellularity of ingrowth (fibroblasts and capillaries within the VNW scaffold) as compared to the GM Control and the VNW alone.
  • the UDC Supernatant demonstrated greater cellularity of ingrowth at both the cranial and caudal sites compared to all other treatments.
  • Collagen deposition was statistically greater for the cranial sites compared to GM Control and Scaffold Control. Additionally, the caudal sites demonstrated statistically greater collagen deposition than the Scaffold Control.
  • VNW was purchased from Biomedical Structures (Slatersville, Rhode Island). The scaffolds were placed in desiccant paper pillows that were then packaged in T- vent aluminum pouches and sterilized via EtO sterilization (nominal B cycle). The scaffolds were stored at room temperature prior to use. The following treatment groups were included in the study:
  • Fibroblasts human adult; passage 10): IFl 853 UDC (passage 11): 040604B Growth medium (Hayflick media): 1192731 Test Article Preparation
  • VNWs were scoured to remove residual processing oils. The material was scoured twice. The VNW was agitated in isopropanol (IPA) in the BRANSON ULTRASONIC CLEANER (BUC) for at least 30 minutes. The PA was drained, and the VNW was washed with deionized water three times. The VNW was then agitated in deionized water in the BUC for an additional 30 minutes. The VNW was dried under vacuum overnight or until dry to the touch.
  • IPA isopropanol
  • BUC BRANSON ULTRASONIC CLEANER
  • Cells were pelleted in Growth media, combined into one pellet and then resuspended in a total volume of 20 milliliters of PBS. The cells were washed three times in PBS to remove residual FBS from the Growth media. This was done by centrifuging the cells for 5 minutes at 1.5 RPM and then resuspending the cells in 20 ml of PBS until the three washes were complete.
  • the lysates were prepared by repeated freeze/thaw cycles.
  • the cell pellets (UDC and HF) were resuspended in 425 microliters of PBS.
  • the tubes were placed in a slurry of dry ice and isopropanol for 10 minutes. After 10 minutes, the tubes were placed in a 37°C water bath for 10 minutes. This procedure was repeated for a total of three cycles of freezing and thawing.
  • the cell suspensions were transferred to sterile siliconized microcentrifuge tubes, to prevent protein adsorption, and centrifuged at 13,000xg for 10 minutes at 4 0 C to separate the cell membranes from the cytosolic components. After removal of the cell membranes, the supernatant was gently mixed by tapping the centrifuge tube to ensure uniformity. The supernatant was transferred to new siliconized tubes and placed on ice. Approximately 425 microliters of UDC supernatant was collected. To ensure that there was enough UDC supernatant to be loaded onto the scaffolds 50 microliters of PBS was added to make a final volume of 475 microliters.
  • UDC Supernatant and HF Supernatant Sterile VNW scaffolds (6 mm punches) were aseptically transferred to sterile multi-well plates. 25 microliters of cell lysate supernatant (UDC or HF) were placed on the VNW; the drop of cell lysate sank into the scaffold. The dish was covered with a sterile lid and taped to ensure the lid would stay in place during the lyophilization process. The dishes were then immediately placed on dry ice until lyophilization.
  • UDC or HF cell lysate supernatant
  • GM Control Sterile VNW scaffolds (6 mm punches) were aseptically transferred to sterile multi-well plates. 30 microliters of Hayflick media was placed in the same " manner onto each of the scaffolds as described above. The scaffolds were washed three times in PBS to remove residual media. The dish was covered with a sterile lid and taped to ensure the lid would stay in place during the lyophilization process. The dishes were then immediately placed on dry ice until lyophilization.
  • VNW Control Sterile VNW scaffolds (6 mm punches) were aseptically transferred to sterile multi-well plates. The dish was covered with a sterile lid and taped to ensure the lid would stay in place during the lyophilization process. The dishes were then immediately placed on dry ice until lyophilization.
  • the treatments were lyophilized using a 48-hour lyophilization cycle. After the lyophilization was complete, the dishes were wrapped in parafilm and stored at -80 0 C until the day of surgery. On the day of surgery, the treatments were removed from the freezer and placed into a foil-covered ice bucket to prevent proteolytic activity.
  • VNW scaffold (Lot 355-73-1) was purchased from Biomedical Structures (Slatersville, Rhode Island). The VNW scaffold used in this study was 2.06 mm thick. The density, as determined by Biomedical Structures, was 108.49 mg/cc. The percent porosity was calculated to be 92.8%. The sample demonstrated less than 1 ppm residual IPA. Residual ethylene oxide (EtO) levels were tested four times. The residual EtO levels in the VNW were 130, 132, 133 and 137 ppm. The residual EtO in the sample was much less than the 250 ppm limit.
  • EtO residual ethylene oxide
  • the scaffolds were placed in desiccant paper pillows that were then packaged in T-vent aluminum pouches and sterilized via EtO sterilization (nominal B cycle). The scaffolds were stored at room temperature prior to use. The cell isolates were co-lyophilized with the scaffolds in an aseptic manner.
  • Each rat was anesthetized via Isoflurane inhalant anesthesia. After induction of anesthesia, the entire back of the animal from the dorsal cervical area to the dorsal lumbrosacral area was clipped free of hair using electric animal clippers. The area was then scrubbed with Chlorhexidine diacetate, rinsed with alcohol, dried and painted with an aqueous iodophor solution of 1% available iodine. The anesthetized and surgically prepared animal was placed in the desired recumbent position.
  • UDC Supernatant demonstrated statistically significant more cellularity than either the GM Control or Scaffold Control groups (p ⁇ 0.05, Tukey-Kramer). When the data was separated into cranial and caudal sites, there were no statistical differences demonstrated. See Table 19-2.
  • Table 19-2 Mean and (SEM) of the score for cellularity of ingrowth
  • VNWs the grade 'minimal' indicated only small collections of cells (macrophages and macrophage giant cells) around individual fibers or fiber bundles and these infiltrates did not tend to coalesce. 'Slight' reactions for VNWs were given when there were greater concentrations of the cell types (macrophages and macrophage giant cells plus possibly other mononuclear cells). VNWs were given a 'moderate' score when there was total or almost total infiltration of the cell types noted above, but there was no significant extension of the reaction beyond the surface of the scaffold. A 'pronounced' score indicated that, in addition to the 'moderate' score, there was a pronounced degree of inflammatory cell infiltration surrounding the scaffold, or if the primary response to the material was neutrophilic.
  • the amount of ingrowth into the VNW was very similar across the treatment groups, both in the cranial and caudal positions.
  • the UDC Supernatant demonstrated significantly increased cellularity of ingrowth (fibroblasts and capillaries within the VNW scaffold) as compared to the GM Control and the VNW alone.
  • Collagen deposition was statistically greater for the cranial sites compared to GM Control and Scaffold Control. Additionally, the caudal sites demonstrated statistically greater collagen deposition than the Scaffold Control.
  • the GM Control demonstrated reduced amounts of cellularity of ingrowth and greater collagen deposition than the Scaffold Control.
  • UDCs produce various growth factors involved in tissue regeneration, including basic Fibroblast Growth Factor (bFGF), hepatocyte growth factor (HGF), brain derived neurotrophic factor (BDNF), and keratinocyte growth factor (KGF).
  • bFGF basic Fibroblast Growth Factor
  • HGF hepatocyte growth factor
  • BDNF brain derived neurotrophic factor
  • KGF keratinocyte growth factor
  • the present study evaluated the ability of UDC lysate, lyophilized onto a material, to increase mouse NIF1/3T3 fibroblast proliferation when co-cultured in a transwell system.
  • Collagen/oxidized regenerated cellulose (ORC) containing lyophilized UDC lysate was placed in the upper portion of a transwell system and co-cultured with mouse NIH/3T3 fibroblasts plated at low density in the lower portion of the system. After three days the cells were harvested and counted. The transwells containing materials were transferred to new transwell systems and again co-cultured with mouse NIH/3T3 fibroblasts plated at low density in the lower portion of the system. After an additional three days (six days total material time in culture), the cells were harvested and counted. This second culture timepoint was performed to assess the release kinetics of the UDC lysate from the biomaterial.
  • Test materials with applied lysate were loaded into a FTS Systems Dura-Stop MP Stoppering Tray Dryer and lyophilized using the ramping program set forth in Example 17. All steps had a ramping rate of 2.5°C/minute and a 100-mT vacuum.
  • Mouse Fibroblasts Mouse NIH/3T3 fibroblasts (ATCC CRL-1658) were expanded in growth media (DMEM high glucose with 10% fetal calf serum and penicillin/streptomycin). All treatments were in triplicate. 10% FCS (empty transwell) 1% FCS (empty transwell)
  • Transwell Assay The mouse NIH/3T3 fibroblasts were plated into the lower portion of a 6-well transwell plate (Corning cat. no. 3412) at 5,000 cells per cm 2 and cultured overnight. The media was removed by aspiration and the appropriate media (2.5 ml per well, 1.5ml per transwell), transwells, and treatments were added. On day 3, transwell containing materials were removed and transferred to new 6 well plates that were seeded with NIH/3T3 at 5,000 cells per cm 2 and the prior day. Cells in transwells were harvested by trypsinization and counted using a Guava PCA instrument (Guava Technologies, Hayward CA)

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Abstract

L'invention concerne des cellules dérivées de tissus du post-partum et des produits de celles-ci ayant le potentiel de supporter des cellules et/ou de différentier en cellules de lignées de tissus mous et des procédés de préparation et d'utilisation de ces cellules dérivées de tissus du post-partum. L'invention concerne également des procédés d'utilisation de telles cellules dérivées du post-partum et des produits relatifs à celles-ci dans des traitements d'états de tissus mous.
PCT/US2005/046808 2004-12-23 2005-12-22 Reparation et regeneration de tissus mous au moyen de cellules derivees du post-partum et produits cellulaires WO2006071777A2 (fr)

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US7875273B2 (en) 2004-12-23 2011-01-25 Ethicon, Incorporated Treatment of Parkinson's disease and related disorders using postpartum derived cells
US7875272B2 (en) 2003-06-27 2011-01-25 Ethicon, Incorporated Treatment of stroke and other acute neuraldegenerative disorders using postpartum derived cells
US8273526B2 (en) 2007-06-18 2012-09-25 Children's Hospital & Research Center At Oakland Method of isolating stem and progenitor cells from placenta
WO2012158952A1 (fr) * 2011-05-19 2012-11-22 Advanced Technologies And Regenerative Medicine, Llc Traitement de la dégénérescence du disque intervertébral en utilisant des cellules humaines dérivées de tissu de cordon ombilical
US8574899B2 (en) 2010-12-22 2013-11-05 Vladimir B Serikov Methods for augmentation collection of placental hematopoietic stem cells and uses thereof
US8771677B2 (en) 2008-12-29 2014-07-08 Vladimir B Serikov Colony-forming unit cell of human chorion and method to obtain and use thereof
US9175261B2 (en) 2005-12-16 2015-11-03 DePuy Synthes Products, Inc. Human umbilical cord tissue cells for inhibiting adverse immune response in histocompatibility-mismatched transplantation
US9611513B2 (en) 2011-12-23 2017-04-04 DePuy Synthes Products, Inc. Detection of human umbilical cord tissue derived cells
JP2017070761A (ja) * 2006-10-06 2017-04-13 アントフロゲネシス コーポレーション ヒト胎盤コラーゲン組成物、並びにそれらの製造方法及び使用方法
US9943552B2 (en) 2009-03-26 2018-04-17 DePuy Synthes Products, Inc. hUTC as therapy for Alzheimer's disease
US10179900B2 (en) 2008-12-19 2019-01-15 DePuy Synthes Products, Inc. Conditioned media and methods of making a conditioned media
US10557116B2 (en) 2008-12-19 2020-02-11 DePuy Synthes Products, Inc. Treatment of lung and pulmonary diseases and disorders
CN110988352A (zh) * 2019-11-11 2020-04-10 余波澜 辅助检测胎盘植入的试剂盒及其应用
US10744164B2 (en) 2003-06-27 2020-08-18 DePuy Synthes Products, Inc. Repair and regeneration of ocular tissue using postpartum-derived cells
US12215352B2 (en) 2015-03-04 2025-02-04 Mesoblast International Sarl Cell culture method for mesenchymal stem cells

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US9592258B2 (en) 2003-06-27 2017-03-14 DePuy Synthes Products, Inc. Treatment of neurological injury by administration of human umbilical cord tissue-derived cells
US9572840B2 (en) 2003-06-27 2017-02-21 DePuy Synthes Products, Inc. Regeneration and repair of neural tissue using postpartum-derived cells
US9125906B2 (en) 2005-12-28 2015-09-08 DePuy Synthes Products, Inc. Treatment of peripheral vascular disease using umbilical cord tissue-derived cells

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US7413734B2 (en) 2003-06-27 2008-08-19 Ethicon, Incorporated Treatment of retinitis pigmentosa with human umbilical cord cells
US9717763B2 (en) 2003-06-27 2017-08-01 DePuy Synthes Products, Inc. Postpartum cells derived from umbilical cord tissue, and methods of making and using the same
US7875273B2 (en) 2004-12-23 2011-01-25 Ethicon, Incorporated Treatment of Parkinson's disease and related disorders using postpartum derived cells
US9175261B2 (en) 2005-12-16 2015-11-03 DePuy Synthes Products, Inc. Human umbilical cord tissue cells for inhibiting adverse immune response in histocompatibility-mismatched transplantation
JP2017070761A (ja) * 2006-10-06 2017-04-13 アントフロゲネシス コーポレーション ヒト胎盤コラーゲン組成物、並びにそれらの製造方法及び使用方法
US8273526B2 (en) 2007-06-18 2012-09-25 Children's Hospital & Research Center At Oakland Method of isolating stem and progenitor cells from placenta
US10179900B2 (en) 2008-12-19 2019-01-15 DePuy Synthes Products, Inc. Conditioned media and methods of making a conditioned media
US10557116B2 (en) 2008-12-19 2020-02-11 DePuy Synthes Products, Inc. Treatment of lung and pulmonary diseases and disorders
US8771677B2 (en) 2008-12-29 2014-07-08 Vladimir B Serikov Colony-forming unit cell of human chorion and method to obtain and use thereof
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US8574899B2 (en) 2010-12-22 2013-11-05 Vladimir B Serikov Methods for augmentation collection of placental hematopoietic stem cells and uses thereof
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