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WO2007108689A2 - Method for induction of differentiation of stem and progenitor cells - Google Patents

Method for induction of differentiation of stem and progenitor cells Download PDF

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WO2007108689A2
WO2007108689A2 PCT/NL2007/050120 NL2007050120W WO2007108689A2 WO 2007108689 A2 WO2007108689 A2 WO 2007108689A2 NL 2007050120 W NL2007050120 W NL 2007050120W WO 2007108689 A2 WO2007108689 A2 WO 2007108689A2
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cells
differentiation
stem cells
polyamine
progenitor
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WO2007108689A3 (en
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Martinus Nicolaas Helder
Paulus Ignatius Jozef Maria Wuisman
Behrouz Zandieh Doulabi
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Stichting Skeletal Tissue Engineering
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Publication of WO2007108689A3 publication Critical patent/WO2007108689A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells

Definitions

  • the invention relates to propagation and differentiation of stem cells and the chemical stimuli which are used in the environment of those cells to induce differentiation. Further, the invention relates to the prevention of dedifferentiation of already differentiated cells. 5
  • Stem cells are totipotential or pluripotential precursor cells capable of generating a variety of mature cell lineages, and precursor cells are cells capable of generating cells of specific cell 0 lineages. These abilities serve as the basis for the cellular differentiation and specialization necessary for organ and tissue development.
  • stem and progenitor cells have provided new clinical tools to reconstitute and/or supplement bone marrow after myeloablation due to disease, exposure to toxic chemical and/or radiation. 5 Further evidence exists that demonstrates that stem cells can be employed to repopulate many, if not all, tissues and restore physiologic and anatomic functionality. The application of stem cells in tissue engineering, gene therapy delivery and cell therapeutics is also advancing rapidly.
  • stem cells Many different types have 0 been characterized. For example, embryonic stem cells, embryonic germ cells, adult stem cells or committed stem cells or progenitor cells are known. Certain stem cells have not only been isolated and characterized but have also been cultured under conditions to allow differentiation to a limited extent. However, a basic problem remains; that is, it has been difficult to control or regulate the 5 differentiation of stem cells and progenitor cells, such as hematopoietic progenitor cells. Presently, existing methods of modulating the differentiation of these cells are crude and unregulatable, such that the cells differentiate into unwanted cell types, at unwanted times. Moreover, the yield of the product cells is typically low.
  • stem or progenitor cells are typically obtained from embryos or foetal tissue, including abortuses, due, in part, to the limited quantity of stem or progenitor cells found in blood or tissue, and the significant discomfort involved in obtaining bone marrow aspirates.
  • harvesting of stem or progenitor cells from alternative sources in adequate amounts for therapeutic and research purposes is generally laborious, involving, e.g., dissection, harvesting of cells or tissues from a donor subject or patient, culturing and/or propagation of cells in vitro, etc.
  • procurement of these cells from embryos or foetal tissue, including abortuses has raised religious and ethical concerns.
  • Hu et al. discloses human amniotic epithelial cells derived from placenta at delivery that are isolated, cultured, cryopreserved for future use, or induced to differentiate.
  • a placenta is harvested immediately after delivery and the amniotic membrane separated from the chorion, e.g., by dissection.
  • Amniotic epithelial cells are isolated from the amniotic membrane according to standard cell isolation techniques.
  • the disclosed cells can be cultured in various media, expanded in culture, cryopreserved, or induced to differentiate.
  • amniotic epithelial cells are multipotent (and possibly pluripotent), and can differentiate into epithelial tissues such as corneal surface epithelium or vaginal epithelium.
  • the drawback of such methods is that they are labor-intensive and the yield of stem cells is very low.
  • Currently available methods for the ex vivo expansion of cell populations are also labour-intensive.
  • Emerson et al. (Emerson et al., U.S. Pat. No. 6,326,198) discloses media conditions for ex vivo culturing of human stem cell division and/or the optimization of human hematopoietic progenitor stem cells.
  • human stem cells or progenitor cells derived from bone marrow are cultured in a liquid culture medium that is replaced, preferably perfused, either continuously or periodically, at a rate of 1 ml of medium per ml of culture per about 24 to about 48 hour period. Metabolic products are removed and depleted nutrients replenished while maintaining the culture under physiologically acceptable conditions.
  • Kraus et al. discloses that a predetermined target population of cells may be selectively expanded by introducing a starting sample of cells from cord blood or peripheral blood into a growth medium, causing cells of the target cell population to divide, and contacting the cells in the growth medium with a selection element comprising binding molecules with specific affinity (such as a monoclonal antibody for CD34) for a predetermined population of cells (such as CD34 cells), so as to select cells of the predetermined target population from other cells in the growth medium.
  • a selection element comprising binding molecules with specific affinity (such as a monoclonal antibody for CD34) for a predetermined population of cells (such as CD34 cells), so as to select cells of the predetermined target population from other cells in the growth medium.
  • 6,335,195 discloses methods for ex vivo culture of hematopoietic and mesenchymal stem cells and the induction of lineage-specific cell proliferation and differentiation by growth in the presence of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof or All AT2 type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.
  • the stem cells are derived from bone marrow, peripheral blood or umbilical cord blood.
  • Retinoids such as vitamin A and retinoic acid (RA) have been known to affect differentiation of stem cells.
  • retinoic acid has been shown to inhibit proliferation of abnormally committed (chronic myelogenous leukemia) hematopoietic stem cells (Nadkarni et al. 1984, Tumori 70:503-505) and to induce differentiation and loss of self-renewal potential in promyelocytic leukemia cells (Melchner et al., 1985, Blood 66(6): 1469-1472).
  • Retinoic acid has also been shown to induce differentiation of neurons from embryonic stem cells and to repress spontaneous mesodermal differentiation (Slager et al., Dev. Genet.
  • Retinoic acid has further been shown to induce differentiation of transformed germ cell precursors (Damjanov et al., 1993, Labor. Investig. 68(2):220-232), placental cell precursors (Yan et al., 2001, Devel. Biol. 235: 422-432), and endothelial cell precursors (Hatzopoulos et al, 1998, Development 125: 1457-1468).
  • the effect of retinoids on differentiation has yet to be completely understood such that it could be used as a regulatable means of controlling differentiation of stem cells.
  • folic acid analogues such as aminopterin and amethopterin (methotrexate)
  • folic acid analogues are used as chemotherapeutic agents in acute lymphoblastic anemias and other blood proliferation disorders and cancers, and have been shown to effect differentiation of stem cells by killing off certain populations of stem cells (DeLoia et al., 1998, Human Reproduction 13(4):1063-1069), and thus, would not be an effective tool for regulating differentiation of large quantities of stem cells for administration to a patient.
  • cytokines such as IL-I, IL-2, IL-3, IL-6, IL- 7, IL-Il, as well as proteins such as erythropoietin, Kit ligand, M-CSF and GM-CSF have also been shown to direct differentiation of stem cells into specific cell types in the hematopoietic lineage (Dushnik-Levinson et al., 1995, Biol. Neonate 67:77-83), however, these processes are not well understood and still remain too crude and imprecise to allow for a regulatable means of controlling differentiation of stem cells.
  • Murashov, A.K. et al. (FEBS Lett., 2004, 569:165-168) described the use of a poly-L-ornithine/fibronectin coating as support for differentiating embryonic stem cells and they observed an increased differentiation into neuronal cells after application of 17- ⁇ -oestradiol or NGF in comparison with a gelatin coating. However, they ascribed the morphogen inducing neuronal differentiation effect to the use of fibronectin in the coating.
  • Knippenberg, M. et al. (Biochem. Biophys. Res. Comm. 2006, 342:902-908) in one example used spermine in the culture of adipose tissue- derived mesenchymal stem cells, in which example the effects of bone morphogenetic proteins (BMP-2 and BMP- 7) on chondrogenic and/or osteogenic differentiation were investigated. Although these effects were observed for BMP-2 and BMP- 7 no effect of the spermine (alone or in combination with the test proteins) was observed or described. Because control over stem and precursor cell differentiation can produce cell populations that are therapeutically useful, there is a need for the ability to control and regulate the differentiation of those cells.
  • BMP-2 and BMP-7 bone morphogenetic proteins
  • the invention now provides a method for the ex vivo induction of differentiation of stem cells or progenitor cells comprising the steps of: a. providing a progenitor or stem cell suspension in a culture medium; b. adding a polyamine to the culture medium.
  • the polyamines are removed from the culture medium after a period of time; preferably 1 min - 2 hour; more preferably 15 — 60 min.
  • the polyamine compound is preferably selected from the group essentially consisting of ornithine and its derivatives putrescine, spermidine and spermine.
  • differentiation inducing factors may be added in parallel , preferably from the group essentially consisting of steroid hormones, vitamins and/or growth hormones, such as TGFB, BMP, Osf, or LMP-I, and combinations of these.
  • the stem cells are preferably mesenchymal stem cells, preferably from adipose tissue.
  • the method of the invention preferably provides in osteogenic or chondrogenic differentiation.
  • the cells are preferably combined with bioresorbable materials, such as osteoconductive calciumphosphates, bioresorbable polymers such as polylactids or polyglycolids, poly-caprolactones, collagens, minerals, fibrinogens, alginates, hyaluronases, chitosan and/or combinations of these.
  • bioresorbable materials such as osteoconductive calciumphosphates, bioresorbable polymers such as polylactids or polyglycolids, poly-caprolactones, collagens, minerals, fibrinogens, alginates, hyaluronases, chitosan and/or combinations of these.
  • the stem cells and/or bioresorbable materials will either be directly implanted, or further cultured in the proper culture medium prior to implantation.
  • Another embodiment of the invention is the use of a polyamine in a method according to the invention.
  • a further embodiment is a method for preventing dedifferentiation of cells comprising applying a polyamine and/or the use of a polyamine to prevent dedifferentiation of cells.
  • a next embodiment is a method for inducing transdifferentiation of cells comprising applying polyamine and/or use of a polyamine to induce transdifferentiation
  • FIG. 1 Effect of 1,25(OH) 2 D 3 on relative runx-2 gene expression (A), osteopontin gene expression (B), SSAT gene expression (C), and PMF-I gene expression (D) by goat ASCs.
  • 1,25(OH) 2 D 3 significantly up-regulated gene expression of the osteogenic markers runx-2 (A) and osteopontin (B) after 14 days.
  • the gene expression levels for the polyamine-regulated genes SSAT (C) and PMF-I (D) were significantly up-regulated, however, already at 7 days of 1,25(OH) 2 D 3 treatment and not any more at 14 days post-treatment.
  • T/C l,25(OH) 2 D 3 -treated-over-control ratios
  • Data are mean ⁇ SEM of T/C ratios.
  • ASCs adipose tissue stem cells;
  • SSAT spermidine/spermine N-acetyl transferase, PMF-I, polyamine modulated factor-1. * Significant effect of 1,25(OH) 2 D 3 , p ⁇ 0.05.
  • FIG. 1 Effect of spermine on SSAT gene expression in goat-derived AT- MSCs.
  • Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 and 14 days after treatment, SSAT gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ⁇ SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test.
  • SSAT spermidine/spermine N(l) acetyltransferase. * Significant effect of spermine, p ⁇ 0.05.
  • Figure 3 Effect of spermine on runx-2 gene expression in goat-derived AT- MSCs. Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 (A) and 14 (B) days after treatment, runx-2 gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ⁇ SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p ⁇ 0.05.
  • Figure 4 Effect of spermine on osteopontin gene expression in goat-derived AT-MSCs.
  • Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 (A) and 14 (B) days after treatment, osteopontin gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ⁇ SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p ⁇ 0.05.
  • Figure 5. Effect of spermine on BMP-2 and l,25-dihydroxyvitamin-D3 (1,25(OH)2D3) induced osteopontin gene expression in AT-MSCs.
  • Goat-derived AT-MSCs were treated or not for 15 minutes with 10 ⁇ M spermine, followed by 15 minutes treatment with 10 ng/ml BMP-2 or 10 nM 1,25(OH)2D3 combined or not with 10 ⁇ M spermine.
  • Four days after treatment osteopontin gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ⁇ SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p ⁇ 0.05.
  • polyamines spermidine, spermine and their precursors ornithine and putrescine are ubiquitous aliphatic poly cations with multiple cellular functions. Polyamines have been called essential for a number of functions in the cell, including proliferation and apoptosis, however, their explicit role in these cellular processes is mostly unknown.
  • ornithine is formed from L-glutamate in a series of reactions.
  • Ornithine is the starting point for the biosynthesis of proline and arginine and of the here mentioned polyamines.
  • all the polyamines mentioned here are derived from the same building block, ornithine.
  • ODC ornithine decarboxylase
  • the catabolic pathway is controlled predominantly by the action of spermine/spermidine Nl-acetyltransferase (SSAT).
  • SSAT spermine/spermidine Nl-acetyltransferase
  • the concentration of the polyamine(s) should be in the range of 1 nM tot 100 ⁇ M, preferably in the range of about 100 nM to about 10 ⁇ M. It has also appeared that the presence of polyamine(s) in the medium can be limited to a certain time of minimal 5 minutes, but preferably more than 15 minutes. Further, to prevent pleiotropic or even toxic effects of the added polyamine(s) it is preferable to remove the polyamine(s) from the medium after the induction. However, this is not strictly necessary and the polyamine(s) may stay in the medium even if the stem cells have differentiated. In such a case, the polyamine(s) might prevent the differentiated cells from dedifferentiation.
  • Dulbecco's Modified Eagles Medium (DMEM) (obtainable as Cat# 11965-084 from GibcoBRL) with the addition of foetal bovine serum (FBS) (Cat# 10437-028, Gibco-BRL) has appeared to be a suitable medium. If desired antibiotics and/or antimycotics may be added, and also ascorbate-2-phosphate and ⁇ -glycerophosphate to ensure proper collagen processing and mineralization] can be added. After inducing differentiation with polyamines, as discussed above, the culturing of the cells can continue in this medium without the polyamines for continued differentiation and proliferation.
  • DMEM Dulbecco's Modified Eagles Medium
  • FBS foetal bovine serum
  • polyamines can be co- introduced with compounds which are known to induce differentiation wherein the polyamines function to enhance or boost the inducing effects of these compounds.
  • Other differentiation factors which can be used in this respect are known to the person skilled in the art and can be compounds such as steroid hormones, vitamins (such as vitamin A, retinoic acid and 1,25- dihydroxyvitamin D3), cytokines (such as IL-I, IL-2, IL-3, IL-6, IL-7, IL-I), proteins (such as erythropoietin, Kit ligand, M-CSF and GM-CSF) and growth factors (such as TGFB, BMP, Osf and LMP-I)
  • vitamins such as vitamin A, retinoic acid and 1,25- dihydroxyvitamin D3
  • cytokines such as IL-I, IL-2, IL-3, IL-6, IL-7, IL-I
  • proteins such as erythropoietin, Kit ligand, M
  • the stem cells which can be used and induced according to this invention can be any stem cells, such as umbilical stem cells and foetal stem cells.
  • progenitor cells or mesenchymal stem cells are used, which can be derived from adipose tissue.
  • Mesenchymal stem cells (MSC) from human bone marrow are known to be able to differentiate into chondrocytes, adipocytes, myeloblasts and osteoblasts.
  • Stromal cells of human adipose tissue have been shown to have similar characteristics in vitro (Zuk et al, 2001 Tissue Eng 7:211-228) .
  • Adipose stem cells can be obtained relatively easy, without long purification procedures and with high yield through resection and from liposuction aspirates (either in a conventional way or through ultrasonic mediated liposuction). 300 cc Aspirate gives a yield of about 2 to 6 x 10 8 cells. This takes away the need for in vitro expansion. Preferably stem cells derived from resection or conventional liposuction are used.
  • the adipose stem cells can be maintained fro prolonged periods in vitro without apparent loss of multipotency and they are, being autologous stem cells, immunocompatible.
  • the aspiration of adipose tissue can be accomplished using any known method, e.g. through procedures that have been proven repeatedly, wherein also has been pointed at procedures for obtaining stem cells from these aspirates (Halvorsen et al., 2001, Tissue Eng 7:729-41; Mizuno et al., 2002, Plast Reconstr. Surg. 109:199-209; Zuk et al., 2001 Tissue Eng7:211-228).
  • the aspirate can be obtained from various parts of the body, e.g. buttock, thigh or abdomen.
  • Aspirates can be processed directly or can be stored for a short period. The person skilled in the art will be able to choose the storage conditions to retrieve sufficient amount and quality after storage. Preferably, the aspirate is processed directly.
  • a special embodiment of this invention is to direct the stem cells to osteogenic or chondrogenic differentiation routes. This can be done by adding growth factors which are able to specifically direct differentiation in the osteoblast-forming or chondroblast forming pathways.
  • growth factors which are able to specifically direct differentiation in the osteoblast-forming or chondroblast forming pathways.
  • inducers for osteoblast formation are: BMP-2, 1,25-dihydroxyvitaniin D3, dexamethasone, while examples of inducers for chondroblast formation are: TGF- ⁇ i-3 and BMP-7 .
  • polyamines can be used to boost the effect of such inducers.
  • stem cells can be obtained (e.g. by aspiration of adipose tissue), can be induced ex vivo, and can then be replaced in the body for tissue outgrowth (e.g. in constructive bone surgery).
  • the stem cells can be reintroduced in the body as such, but they can also be combined with filler and/or carrier material.
  • the carrier material can be comprised of, optionally processed, autologous or homologous bone tissue or a combination thereof.
  • synthetic materials can be applied, such as osteoconductive calciumphosphates, bioresorbable polymers, like polylactides and polyglycolides, collagens, fibrinogens, alginates, hyaluronic acid derivatives, chitins and/or a combination of these materials.
  • Such filler or carrier material can take any form (porous, amorphous, granules, fibrous, powder) as long as it, temporarily, can be a mixable, fluid or pliable substance, which has the ability to function as attachment substrate for bone, cartilage, vertebral disc or connective tissue forming cells or tissues.
  • the substance should be injectable, but it could also be used as a pliable or even solid substance in open surgery.
  • the carrier material is preferably applied as a scaffold, which is defined for the present invention, as an optionally porous, physiological support for individual cells or tissues.
  • These cells are preferably the stem cells or the differentiated stem cells according to the invention.
  • This scaffold preferably has an architecture which enhances the migration, maintenance and/or proliferation and differentiation of the cells according to the present invention or the tissues which develop from these cells.
  • the term 'porous' means, for the present invention, having holes (pores) of sufficient size to be occupied by a cell and being able of being penetrated by polyamines alone or in combination with other inducing factors as discussed above, and/or by a cell suspension.
  • Another embodiment of the present invention is a method for preventing cells, e.g. derived from cartilage, intervertebral disc, bone, and connective tissue, to dedifferentiate by the application of a polyamine, preferably selected from the group consisting of ornithine, putrescine, spermidine and spermine.
  • a polyamine preferably selected from the group consisting of ornithine, putrescine, spermidine and spermine.
  • the polyamines can be used in combination with other factors, such as vitamins, growth factors, hormones, and the like.
  • polyamines are added to the culture medium intermittently, i.e. with some intervals. This would allow for maintaining tissue specificity while continuous cell expansion or proliferation is maintained.
  • a further embodiment of the present invention is a method to provide transdifferentiation from cells of one tissue type to cells of another tissue type. It is envisaged that e.g. skin fibroblasts can be turned into e.g. chondrocytes by application of polyamines, optionally in combination with one of the above mentioned differentiation factors. Such a transdifferentiation would allow for both in vivo and in vitro regeneration of tissues from adjacent cells.
  • Polyamine levels in tissues are modulated by a number of polyamine-regulated genes, including polyamine-modulated transcription factor- 1 (PMF-I) and spermidine/spermine N-acetyl transferase (SSAT (14-16).
  • PMF-I polyamine-modulated transcription factor- 1
  • SSAT spermidine/spermine N-acetyl transferase
  • example 1 the regulation of polyamine-related genes after growth factor- induced differentiation was illustrated.
  • polyamine spermine can modulate polyamine-regulated genes (example: SSAT) as well as osteogenic differentiation markers (runx-2, osteopontin, ALP activity) in AT-MSCs.
  • SSAT polyamine-regulated genes
  • osteogenic differentiation markers runx-2, osteopontin, ALP activity
  • runx-2 As well as osteopontin gene expression were significantly upregulated, However, 14 days after treatment, although runx-2 and osteopontin gene expression both showed increased expression levels in particular with the higher spermine dosages, only runx-2 reached significance with the 10 ⁇ M dosage.
  • polyamines i.e. spermine in this study
  • BMP-2 Bone Morphogenic Protein-2
  • 1,25- dihydroxyvitamin-D3 1,25(OH)2D3
  • BMP-2 Bone Morphogenic Protein-2
  • 1,25(OH)2D3 1,25(OH)2D3-induced osteopontin gene expression.
  • Cells were either treated or not for 15 minutes with 10 ⁇ M spermine, followed by 15 minutes treatment with spermine combined with either BMP-2 (10 ng/ml) or 1,25(OH)2D3 (10 ⁇ M), or with BMP-2 or 1,25(OH)2D3 alone.
  • BMP-2 ng/ml
  • 1,25(OH)2D3 1,25(OH)2D3
  • treatment with neither spermine, nor BMP-2 or 1,25(OH)2D3 significantly affected osteopontin gene expression.
  • treatment with the combination of spermine and either BMP-2 or 1,25(OH)2D3 synergistically increased osteopontin gene expression.

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Abstract

The invention relates to propagation and differentiation of stem cells and the chemical stimuli which are used in the environment of those cells to induce differentiation. More in particular, the invention provides a method for the ex vivo induction of differentiation of mesenchymal stem cells, preferably derived from adipose tissue, or progenitor cells by adding a polyamine to the cells for a short time (1 min - 2 hours) and subsequent removal. Other differentiation inducing factors may be added in parallel, preferably from the group essentially consisting of steroid hormones, vitamins and/or growth hormones, LMP-I, and combinations of these. The method described preferably provides osteogenic or chondrogenic differentiation, and cells are therefore preferably combined with bioresorbable materials, such as osteoconductive calciumphosphates, bioresorbable polymers such as polylactids or polyglycolids, poly-caprolactones, collagens, minerals, fibrinogens, alginates, hyaluronases, chitosan and/or combinations of these. The stem cells and/or bioresorbable materials will either be directly implanted, or further cultured in the proper culture medium prior to implantation. In addition, polyamine use is described as a method for preventing dedifferentiation of cells and/or inducing transdifferentiation of cells.

Description

Title: New method for induction of differentiation of stem and progenitor cells
The invention relates to propagation and differentiation of stem cells and the chemical stimuli which are used in the environment of those cells to induce differentiation. Further, the invention relates to the prevention of dedifferentiation of already differentiated cells. 5
There is considerable interest in the identification, isolation and generation of human stem and progenitor cells. Stem cells are totipotential or pluripotential precursor cells capable of generating a variety of mature cell lineages, and precursor cells are cells capable of generating cells of specific cell 0 lineages. These abilities serve as the basis for the cellular differentiation and specialization necessary for organ and tissue development.
Recent success at transplanting stem and progenitor cells have provided new clinical tools to reconstitute and/or supplement bone marrow after myeloablation due to disease, exposure to toxic chemical and/or radiation. 5 Further evidence exists that demonstrates that stem cells can be employed to repopulate many, if not all, tissues and restore physiologic and anatomic functionality. The application of stem cells in tissue engineering, gene therapy delivery and cell therapeutics is also advancing rapidly.
Many different types of mammalian and progenitor stem cells have 0 been characterized. For example, embryonic stem cells, embryonic germ cells, adult stem cells or committed stem cells or progenitor cells are known. Certain stem cells have not only been isolated and characterized but have also been cultured under conditions to allow differentiation to a limited extent. However, a basic problem remains; that is, it has been difficult to control or regulate the 5 differentiation of stem cells and progenitor cells, such as hematopoietic progenitor cells. Presently, existing methods of modulating the differentiation of these cells are crude and unregulatable, such that the cells differentiate into unwanted cell types, at unwanted times. Moreover, the yield of the product cells is typically low.
Furthermore, obtaining sufficient numbers of human stem cells for therapeutic or research purposes is problematic. Isolation of normally occurring populations of stem or progenitor cells in adult tissues has been technically difficult and costly, due, in part, to the limited quantity of stem or progenitor cells found in blood or tissue, and the significant discomfort involved in obtaining bone marrow aspirates. In general, harvesting of stem or progenitor cells from alternative sources in adequate amounts for therapeutic and research purposes is generally laborious, involving, e.g., dissection, harvesting of cells or tissues from a donor subject or patient, culturing and/or propagation of cells in vitro, etc. With respect to stem cells in particular, procurement of these cells from embryos or foetal tissue, including abortuses, has raised religious and ethical concerns. The widely held belief that the human embryo and foetus constitute independent life has prompted governmental restrictions on the use of such sources for all purposes, including medical research. Alternative sources that do not require the use of cells procured from embryonic or foetal tissue are therefore desired for further progress in the use of stem cells clinically. There are, however, few viable alternative sources of stem or progenitor cells, particularly human stem or progenitor cells, and thus the supply is limited.
Hu et al. (WO 00/73421) discloses human amniotic epithelial cells derived from placenta at delivery that are isolated, cultured, cryopreserved for future use, or induced to differentiate. According to Hu et al., a placenta is harvested immediately after delivery and the amniotic membrane separated from the chorion, e.g., by dissection. Amniotic epithelial cells are isolated from the amniotic membrane according to standard cell isolation techniques. The disclosed cells can be cultured in various media, expanded in culture, cryopreserved, or induced to differentiate. Hu et al. discloses that amniotic epithelial cells are multipotent (and possibly pluripotent), and can differentiate into epithelial tissues such as corneal surface epithelium or vaginal epithelium. The drawback of such methods, however, is that they are labor-intensive and the yield of stem cells is very low. Currently available methods for the ex vivo expansion of cell populations are also labour-intensive. For example, Emerson et al. (Emerson et al., U.S. Pat. No. 6,326,198) discloses media conditions for ex vivo culturing of human stem cell division and/or the optimization of human hematopoietic progenitor stem cells. According to the disclosed methods, human stem cells or progenitor cells derived from bone marrow are cultured in a liquid culture medium that is replaced, preferably perfused, either continuously or periodically, at a rate of 1 ml of medium per ml of culture per about 24 to about 48 hour period. Metabolic products are removed and depleted nutrients replenished while maintaining the culture under physiologically acceptable conditions.
Kraus et al. (U.S. Pat. No. 6,338,942,) discloses that a predetermined target population of cells may be selectively expanded by introducing a starting sample of cells from cord blood or peripheral blood into a growth medium, causing cells of the target cell population to divide, and contacting the cells in the growth medium with a selection element comprising binding molecules with specific affinity (such as a monoclonal antibody for CD34) for a predetermined population of cells (such as CD34 cells), so as to select cells of the predetermined target population from other cells in the growth medium. Rodgers et al. (U.S. Pat. No. 6,335,195) discloses methods for ex vivo culture of hematopoietic and mesenchymal stem cells and the induction of lineage-specific cell proliferation and differentiation by growth in the presence of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof or All AT2 type 2 receptor agonists, either alone or in combination with other growth factors and cytokines. The stem cells are derived from bone marrow, peripheral blood or umbilical cord blood. The drawback of such methods, however, is that such ex vivo methods for inducing proliferation and differentiation of stem cells are time-consuming, as discussed above, and also result in low yields of stem cells.
Retinoids, such as vitamin A and retinoic acid (RA), have been known to affect differentiation of stem cells. For example, retinoic acid has been shown to inhibit proliferation of abnormally committed (chronic myelogenous leukemia) hematopoietic stem cells (Nadkarni et al. 1984, Tumori 70:503-505) and to induce differentiation and loss of self-renewal potential in promyelocytic leukemia cells (Melchner et al., 1985, Blood 66(6): 1469-1472). Retinoic acid has also been shown to induce differentiation of neurons from embryonic stem cells and to repress spontaneous mesodermal differentiation (Slager et al., Dev. Genet. 1993;14(3):212-24, Ray et al., 1997, J. Biol. Chem. 272(30): 18702-18708). Retinoic acid has further been shown to induce differentiation of transformed germ cell precursors (Damjanov et al., 1993, Labor. Investig. 68(2):220-232), placental cell precursors (Yan et al., 2001, Devel. Biol. 235: 422-432), and endothelial cell precursors (Hatzopoulos et al, 1998, Development 125: 1457-1468). The effect of retinoids on differentiation, however, has yet to be completely understood such that it could be used as a regulatable means of controlling differentiation of stem cells.
The effects of folic acid analogues, such as aminopterin and amethopterin (methotrexate), on the differentiation of hematopoietic stem cells has been studied. Folic acid analogues are used as chemotherapeutic agents in acute lymphoblastic anemias and other blood proliferation disorders and cancers, and have been shown to effect differentiation of stem cells by killing off certain populations of stem cells (DeLoia et al., 1998, Human Reproduction 13(4):1063-1069), and thus, would not be an effective tool for regulating differentiation of large quantities of stem cells for administration to a patient. Several cytokines, such as IL-I, IL-2, IL-3, IL-6, IL- 7, IL-Il, as well as proteins such as erythropoietin, Kit ligand, M-CSF and GM-CSF have also been shown to direct differentiation of stem cells into specific cell types in the hematopoietic lineage (Dushnik-Levinson et al., 1995, Biol. Neonate 67:77-83), however, these processes are not well understood and still remain too crude and imprecise to allow for a regulatable means of controlling differentiation of stem cells.
To date, no one has described the use of compounds, such as the polyamine compounds discussed below, for the induction of differentiation of stem cells or precursor cells. In particular, no one has demonstrated the use of such compounds to modulate the differentiation of progenitor cells, such as mesenchymal progenitor cells. Likewise, no one has described the use of the compounds described herein to expand the progenitor cell populations so as to produce a osteogenic or chondrogenic composition containing such cells. Such expanded progenitor cell cultures would be especially useful in the treatment of deossification and cartilage repair, in general surgery where induction of the formation of bone and cartilage tissue is indicated. Trauma, inflammation, tumour, degeneration, release of prostheses of joints, release of fixation implants, cut or torn ligaments, tendons, muscles or nerves can lead to a loss of tissue and/or tissue-function in the skeleton and accompanying support- and movement structures in many patients. If non-operative therapies fail, a surgical intervention for conservation, repair and/or regeneration of the animal musculoskeletal apparatus is the general accepted therapy. In these procedures often stem cells and differentiated cells are used to induce regeneration of bone and cartilage tissues.
Murashov, A.K. et al. (FEBS Lett., 2004, 569:165-168) described the use of a poly-L-ornithine/fibronectin coating as support for differentiating embryonic stem cells and they observed an increased differentiation into neuronal cells after application of 17-β-oestradiol or NGF in comparison with a gelatin coating. However, they ascribed the morphogen inducing neuronal differentiation effect to the use of fibronectin in the coating.
Knippenberg, M. et al. (Biochem. Biophys. Res. Comm. 2006, 342:902-908) in one example used spermine in the culture of adipose tissue- derived mesenchymal stem cells, in which example the effects of bone morphogenetic proteins (BMP-2 and BMP- 7) on chondrogenic and/or osteogenic differentiation were investigated. Although these effects were observed for BMP-2 and BMP- 7 no effect of the spermine (alone or in combination with the test proteins) was observed or described. Because control over stem and precursor cell differentiation can produce cell populations that are therapeutically useful, there is a need for the ability to control and regulate the differentiation of those cells.
SUMMARY OF THE INVENTION
The invention now provides a method for the ex vivo induction of differentiation of stem cells or progenitor cells comprising the steps of: a. providing a progenitor or stem cell suspension in a culture medium; b. adding a polyamine to the culture medium. Preferably in such a method the polyamines are removed from the culture medium after a period of time; preferably 1 min - 2 hour; more preferably 15 — 60 min. The polyamine compound is preferably selected from the group essentially consisting of ornithine and its derivatives putrescine, spermidine and spermine. In a method according to the invention other differentiation inducing factors may be added in parallel , preferably from the group essentially consisting of steroid hormones, vitamins and/or growth hormones, such as TGFB, BMP, Osf, or LMP-I, and combinations of these. The stem cells are preferably mesenchymal stem cells, preferably from adipose tissue. The method of the invention preferably provides in osteogenic or chondrogenic differentiation. Therefore the cells are preferably combined with bioresorbable materials, such as osteoconductive calciumphosphates, bioresorbable polymers such as polylactids or polyglycolids, poly-caprolactones, collagens, minerals, fibrinogens, alginates, hyaluronases, chitosan and/or combinations of these. The stem cells and/or bioresorbable materials will either be directly implanted, or further cultured in the proper culture medium prior to implantation.
Another embodiment of the invention is the use of a polyamine in a method according to the invention.
A further embodiment is a method for preventing dedifferentiation of cells comprising applying a polyamine and/or the use of a polyamine to prevent dedifferentiation of cells.
A next embodiment is a method for inducing transdifferentiation of cells comprising applying polyamine and/or use of a polyamine to induce transdifferentiation
LEGENDS TO THE FIGURES
Figure 1. Effect of 1,25(OH)2D3 on relative runx-2 gene expression (A), osteopontin gene expression (B), SSAT gene expression (C), and PMF-I gene expression (D) by goat ASCs. 1,25(OH)2D3 significantly up-regulated gene expression of the osteogenic markers runx-2 (A) and osteopontin (B) after 14 days. Also the gene expression levels for the polyamine-regulated genes SSAT (C) and PMF-I (D) were significantly up-regulated, however, already at 7 days of 1,25(OH)2D3 treatment and not any more at 14 days post-treatment.
Data are expressed as l,25(OH)2D3-treated-over-control ratios (T/C). Data are mean ± SEM of T/C ratios. ASCs, adipose tissue stem cells; SSAT, spermidine/spermine N-acetyl transferase, PMF-I, polyamine modulated factor-1. * Significant effect of 1,25(OH)2D3, p<0.05.
Figure 2. Effect of spermine on SSAT gene expression in goat-derived AT- MSCs. Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 and 14 days after treatment, SSAT gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ± SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. SSAT, spermidine/spermine N(l) acetyltransferase. * Significant effect of spermine, p<0.05.
Figure 3. Effect of spermine on runx-2 gene expression in goat-derived AT- MSCs. Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 (A) and 14 (B) days after treatment, runx-2 gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ± SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p<0.05.
Figure 4. Effect of spermine on osteopontin gene expression in goat-derived AT-MSCs. Cells were treated for 30 minutes with or without various concentrations of spermine, and 4 (A) and 14 (B) days after treatment, osteopontin gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ± SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p<0.05. Figure 5. Effect of spermine on BMP-2 and l,25-dihydroxyvitamin-D3 (1,25(OH)2D3) induced osteopontin gene expression in AT-MSCs. Goat-derived AT-MSCs were treated or not for 15 minutes with 10 μM spermine, followed by 15 minutes treatment with 10 ng/ml BMP-2 or 10 nM 1,25(OH)2D3 combined or not with 10 μM spermine. Four days after treatment, osteopontin gene expression was determined by real time PCR. Data are normalized to 18S gene expression, and presented as mean ± SEM of treatment-over-control (T/C) ratio's. Statistical analysis was performed using the Student t test. * Significant effect of spermine, p<0.05.
DETAILED DESCRIPTION OF THE INVENTION
The polyamines spermidine, spermine and their precursors ornithine and putrescine are ubiquitous aliphatic poly cations with multiple cellular functions. Polyamines have been called essential for a number of functions in the cell, including proliferation and apoptosis, however, their explicit role in these cellular processes is mostly unknown.
Under most circumstances the major sources for cellular polyamines are synthesis from amino acid precursors and transport across the plasma membrane. In the biosynthesis pathway ornithine is formed from L-glutamate in a series of reactions. Ornithine is the starting point for the biosynthesis of proline and arginine and of the here mentioned polyamines. In other words, all the polyamines mentioned here are derived from the same building block, ornithine. To form putrescine ornithine is decarboxylated by the action of ornithine decarboxylase (ODC). Next an aminopropyl group generated by the action of S-adenosylmethionine decarboxylase on S-adenosylmethionine is attached to putrescine and spermidine to form spermidine and spermine, respectively. Both enzymes are highly regulated and subjected to feedback control by cellular polyamines, which is in concordance with a critical role of polyamines in cellular processes.
The catabolic pathway is controlled predominantly by the action of spermine/spermidine Nl-acetyltransferase (SSAT). Recently, several studies on the effects of blocking or enhancing the action of ODC and/or SSAT have been conducted, which have verified the crucial role of polyamines in various cell functions. It also appears that high concentrations of intracellular polyamines have toxic effects, which also indicates that it is essential for a cell to tightly regulate the biosynthesis of these compounds. Surprisingly, it has now been found that polyamines can be used in the proliferation and differentiation of stem cells. To this end polyamines, i.e. ornithine, putrescine, spermidine or spermine, are added to a medium in which stem cells are maintained or grown. The concentration of the polyamine(s) should be in the range of 1 nM tot 100 μM, preferably in the range of about 100 nM to about 10 μM. It has also appeared that the presence of polyamine(s) in the medium can be limited to a certain time of minimal 5 minutes, but preferably more than 15 minutes. Further, to prevent pleiotropic or even toxic effects of the added polyamine(s) it is preferable to remove the polyamine(s) from the medium after the induction. However, this is not strictly necessary and the polyamine(s) may stay in the medium even if the stem cells have differentiated. In such a case, the polyamine(s) might prevent the differentiated cells from dedifferentiation. While many culture media can be applied, Dulbecco's Modified Eagles Medium (DMEM) (obtainable as Cat# 11965-084 from GibcoBRL) with the addition of foetal bovine serum (FBS) (Cat# 10437-028, Gibco-BRL) has appeared to be a suitable medium. If desired antibiotics and/or antimycotics may be added, and also ascorbate-2-phosphate and β-glycerophosphate to ensure proper collagen processing and mineralization] can be added. After inducing differentiation with polyamines, as discussed above, the culturing of the cells can continue in this medium without the polyamines for continued differentiation and proliferation.
Further, it has surprisingly appeared that polyamines can be co- introduced with compounds which are known to induce differentiation wherein the polyamines function to enhance or boost the inducing effects of these compounds. Other differentiation factors which can be used in this respect are known to the person skilled in the art and can be compounds such as steroid hormones, vitamins (such as vitamin A, retinoic acid and 1,25- dihydroxyvitamin D3), cytokines (such as IL-I, IL-2, IL-3, IL-6, IL-7, IL-I), proteins (such as erythropoietin, Kit ligand, M-CSF and GM-CSF) and growth factors (such as TGFB, BMP, Osf and LMP-I) The polyamine may be added together with the inducing compound or before or after the inducing compound. Because of the strong boosting effect of the polyamines it has appeared possible to use much lower concentrations of differentiation inducers. This also means a strong reduction in the costs of culturing and differentiating stem cells, since the currently used differentiation inducers are relatively expensive, certainly in comparison with the costs of the polyamines. The stem cells which can be used and induced according to this invention can be any stem cells, such as umbilical stem cells and foetal stem cells. Preferably progenitor cells or mesenchymal stem cells are used, which can be derived from adipose tissue. Mesenchymal stem cells (MSC) from human bone marrow are known to be able to differentiate into chondrocytes, adipocytes, myeloblasts and osteoblasts. Stromal cells of human adipose tissue have been shown to have similar characteristics in vitro (Zuk et al, 2001 Tissue Eng 7:211-228) .
Adipose stem cells can be obtained relatively easy, without long purification procedures and with high yield through resection and from liposuction aspirates (either in a conventional way or through ultrasonic mediated liposuction). 300 cc Aspirate gives a yield of about 2 to 6 x 108 cells. This takes away the need for in vitro expansion. Preferably stem cells derived from resection or conventional liposuction are used.
The adipose stem cells can be maintained fro prolonged periods in vitro without apparent loss of multipotency and they are, being autologous stem cells, immunocompatible.
The aspiration of adipose tissue (liposuction) can be accomplished using any known method, e.g. through procedures that have been proven repeatedly, wherein also has been pointed at procedures for obtaining stem cells from these aspirates (Halvorsen et al., 2001, Tissue Eng 7:729-41; Mizuno et al., 2002, Plast Reconstr. Surg. 109:199-209; Zuk et al., 2001 Tissue Eng7:211-228). The aspirate can be obtained from various parts of the body, e.g. buttock, thigh or abdomen. It is known to a person skilled in the art that the amount and quality of the harvested stem cells depends on the site of liposuction and the depth of aspiration of the adipose tissue. Aspirates can be processed directly or can be stored for a short period. The person skilled in the art will be able to choose the storage conditions to retrieve sufficient amount and quality after storage. Preferably, the aspirate is processed directly.
A special embodiment of this invention is to direct the stem cells to osteogenic or chondrogenic differentiation routes. This can be done by adding growth factors which are able to specifically direct differentiation in the osteoblast-forming or chondroblast forming pathways. Examples for inducers for osteoblast formation are: BMP-2, 1,25-dihydroxyvitaniin D3, dexamethasone, while examples of inducers for chondroblast formation are: TGF-βi-3 and BMP-7 . As indicated before, polyamines can be used to boost the effect of such inducers.
However, it has appeared that also if polyamine are given alone differentiation towards the osteogenic or chondrogenic pathways is caused. Furthermore, it appeared that spermine (and to a lesser extent spermidine) stimulate differentiation into osteoblasts, whereas putrescine (and to a lesser extent) ornithine may stimulate a more chondrogenic phenotype .
Since induction of differentiation is effected with very low doses of inducers and in very short time periods, a specific use of the present invention is found in surgical procedures, wherein during one surgical treatment stem cells can be obtained (e.g. by aspiration of adipose tissue), can be induced ex vivo, and can then be replaced in the body for tissue outgrowth (e.g. in constructive bone surgery). The stem cells can be reintroduced in the body as such, but they can also be combined with filler and/or carrier material. The carrier material can be comprised of, optionally processed, autologous or homologous bone tissue or a combination thereof. Also synthetic materials can be applied, such as osteoconductive calciumphosphates, bioresorbable polymers, like polylactides and polyglycolides, collagens, fibrinogens, alginates, hyaluronic acid derivatives, chitins and/or a combination of these materials. Such filler or carrier material can take any form (porous, amorphous, granules, fibrous, powder) as long as it, temporarily, can be a mixable, fluid or pliable substance, which has the ability to function as attachment substrate for bone, cartilage, vertebral disc or connective tissue forming cells or tissues. Preferably, the substance should be injectable, but it could also be used as a pliable or even solid substance in open surgery.
According to the invention the carrier material is preferably applied as a scaffold, which is defined for the present invention, as an optionally porous, physiological support for individual cells or tissues. These cells are preferably the stem cells or the differentiated stem cells according to the invention. This scaffold preferably has an architecture which enhances the migration, maintenance and/or proliferation and differentiation of the cells according to the present invention or the tissues which develop from these cells. The term 'porous' means, for the present invention, having holes (pores) of sufficient size to be occupied by a cell and being able of being penetrated by polyamines alone or in combination with other inducing factors as discussed above, and/or by a cell suspension.
An advantage of the application of the above mentioned autologous and homologous tissue transplants and bioresorbable materials as scaffold is, that they can be used as release system for controlled release of polyamines, whether or not in combination with other inducing factors.
Another embodiment of the present invention is a method for preventing cells, e.g. derived from cartilage, intervertebral disc, bone, and connective tissue, to dedifferentiate by the application of a polyamine, preferably selected from the group consisting of ornithine, putrescine, spermidine and spermine. As for the induction of differentiation, the polyamines can be used in combination with other factors, such as vitamins, growth factors, hormones, and the like. By inhibiting dedifferentiation loss of phenotype is prevented.
Preferably polyamines are added to the culture medium intermittently, i.e. with some intervals. This would allow for maintaining tissue specificity while continuous cell expansion or proliferation is maintained. A further embodiment of the present invention is a method to provide transdifferentiation from cells of one tissue type to cells of another tissue type. It is envisaged that e.g. skin fibroblasts can be turned into e.g. chondrocytes by application of polyamines, optionally in combination with one of the above mentioned differentiation factors. Such a transdifferentiation would allow for both in vivo and in vitro regeneration of tissues from adjacent cells. EXAMPLES
EXAMPLE 1. EXPRESSION OF POLYAMINE-RELATED GENES UPON OSTEOGENIC DIFFERENTIATION
Polyamine levels in tissues are modulated by a number of polyamine-regulated genes, including polyamine-modulated transcription factor- 1 (PMF-I) and spermidine/spermine N-acetyl transferase (SSAT (14-16). We investigated whether these genes are modulated during l,25-dihydroxyvitamin-D3 (vit.D) induced osteogenic differentiation of goat adipose stem cells (ASCs). Treatment with osteogenic medium containing 10 nM of vit. D showed induction of the osteogenic markers runx-2 and osteopontin after 14 days (2.6- fold and 2.3-fold respectively) (Figure IA, IB). Furthermore, 7 days of treatment up-regulated PMF-I and SSAT RNA expression levels by 6.4-fold and 3.4-fold respectively, but no differences were observed after 14 days (Figure 1C, ID). Thus, induction of PMF-I and SSAT expression occurred prior to upregulation of runx-2 and osteopontin.
These data show that polyamine-related genes are upregulated in early osteogenic differentiation in ASCs, suggesting a concomitant role for polyamines in early osteogenic events.
EXAMPLE 2. POLYAMINES REGULATE RUNX-2 AND OSTEOPONTIN GENE EXPRESSION AND ALKALINE PHOSPHATASE ACTIVITIY IN ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS
In example 1, the regulation of polyamine-related genes after growth factor- induced differentiation was illustrated. Here, it was investigated whether the polyamine spermine can modulate polyamine-regulated genes (example: SSAT) as well as osteogenic differentiation markers (runx-2, osteopontin, ALP activity) in AT-MSCs.
Goat-derived AT-MSCs, harvested as described above, were treated for 30 minutes with various dosages of the polyamine spermine, and the effect on expression patterns was determined after 4 and 14 days post-culture in normal (plain) culture medium. It was shown that, compared to control cultures, SSAT expression was significantly upregulated after 4 days when spermine dosages of 3, 10 and 30 μM were applied (Figure 2A; t/cl d4 SSAT). After 14 days, the patterns were similar but differences did not reach significance any more (Figure 2B; t/c2 dl4 SSAT). These data verify the inducing capacity of spermine on a known responsive gene. Next, osteogenic markers were tested for their differential expression patterns upon spermine stimulation. It was found that 4 days after spermine treatment, runx-2 as well as osteopontin gene expression were significantly upregulated, However, 14 days after treatment, although runx-2 and osteopontin gene expression both showed increased expression levels in particular with the higher spermine dosages, only runx-2 reached significance with the 10 μM dosage.
From this example, it can be concluded that polyamines (i.e. spermine in this study) induce differentiation of ASCs along the osteogenic lineage.
EXAMPLE 3. ENHANCEMENT OF GROWTH FACTOR ACTION BY POLYAMINES
The growth factor Bone Morphogenic Protein-2 (BMP-2) and the hormone 1,25- dihydroxyvitamin-D3 (1,25(OH)2D3) have been implicated in osteogenic differentiation. Therefore, we studied the effect of spermine on either BMP-2 or l,25(OH)2D3-induced osteopontin gene expression. Cells were either treated or not for 15 minutes with 10 μM spermine, followed by 15 minutes treatment with spermine combined with either BMP-2 (10 ng/ml) or 1,25(OH)2D3 (10 μM), or with BMP-2 or 1,25(OH)2D3 alone. At the concentrations used, treatment with neither spermine, nor BMP-2 or 1,25(OH)2D3 significantly affected osteopontin gene expression. However, treatment with the combination of spermine and either BMP-2 or 1,25(OH)2D3 synergistically increased osteopontin gene expression.

Claims

Claims
1. A method for the ex vivo induction of differentiation of progenitor or stem cells comprising the steps of: a. providing a progenitor or stem cell suspension in a culture medium; b. adding a polyamine to the culture medium.
2. A method according to claim 1, further comprising removing the polyamines from the culture medium after a period of time, preferably wherein said period of time is 1 min — 2 hour, more preferably wherein said period of time is 15 — 60 min.
3. A method according to claim 1 or 2, wherein the polyamine is selected from the group essentially consisting of ornithine, putrescine, spermidine and spermine.
4. A method according to any of claims 1-3, wherein other differentiation inducing factors are used, preferably from the group essentially consisting of steroid hormones, vitamins and/or growth hormones, such as TGFB, BMP, Osf, or LMP-I, and combinations of these.
5. A method according to any of claims 1-4, wherein the stem cells are mesenchymal stem cells, preferably from adipose tissue.
6. A method according to any of claims 1-5, wherein the stem cells show osteogenic differentiation.
7. A method according to any of claims 1-5, wherein the stem cells show chondrogenic differentiation.
8. A method according to any of claims 1-7, wherein the culture medium also contains bioresorbable materials, such as osteoconductive calciumphosphates, bioresorbable polymers such as polylactids, poly- caprolactones, or polyglycolids, collagens, minerals, fibrinogens, alginates, hyaluronases, chitosan and/or combinations of these.
9. A method according to any of claims 1-8, wherein the polyamines are also present in the medium during the proliferation phase of the stem cells.
10. A method according to any of claims 1-9, wherein the progenitor or stem cells, either or not in combination with bioresorbable materials, will be ready-to-use for implantation in a subject.
11. Use of a polyamine in a method for the ex vivo induction of differentiation of progenitor or stem cells.
12. Method for preventing dedifferentiation of cells comprising applying a polyamine.
13. Use of a polyamine to prevent dedifferentiation of cells.
14. Method for inducing transdifferentiation of cells comprising applying polyamine.
15. Use of a polyamine to induce transdifferentiation of cells.
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