MX2008000001A - Use of adipose tissue-derived stromal stem cells in treating fistula - Google Patents

Abstract

Provided herein are novel methods and compositions utilizing adipose tissue-derived stromal stem cells for treating fistulae.

Description

EMPLOYMENT OF STOMACH CELLS DERIVED FROM ADIPOSE TISSUE IN THE TREATMENT OF THE FISTULA DESCRIPTION Cross-referencing with related applications This application is a continuation in part of U.S. patent application number 11 / 065,461, filed on February 25, 2005, and a partial continuation of U.S. patent application number 11 / 056,241, filed on February 14, 2005, the content of said requests being specifically incorporated in this document by reference in its entirety.

BACKGROUND OF THE INVENTION Generally, a fistula is an abnormal connection or conduit between organs or vessels that are not normally connected. Fistulas can develop in various parts of the body. For example, types of fistulas, named for the areas of the body in which they occur, include anorectal fistula or fistula of the anus or fecal fistula (between the rectum or other anorectal area and the surface of the skin), arteriovenous fistula or AV fistula (between an artery and a vein), biliary fistula (between the bile ducts with the surface of the skin, often caused by surgery of the gallbladder), fistula of the cervix (anomalous opening of the cervix), fistula craniosinus (between the intracranial space and a paranasal sinus), enteroenteric fistula (between two parts of the intestine), enterocutaneous fistula (between the intestine and the surface of the skin, specifically from the duodenum or jejunum or ileus), enterovaginal fistula (between the intestine and the vagina), gastric fistula (between the stomach and the surface of the skin), metroperitoneal fistula (between the uterus and the peritoneal cavity), perilymphatic fistula (a tear between the membranes), anas between the middle and inner ears), pulmonary arteriovenous fistula (between an artery and a vein in the lungs, resulting in a bypass of the blood), rectovaginal fistula (between the rectum and the vagina), umbilical fistula (between the umbilicus and the intestine), tracheoesophageal fistula (between the respiratory and alimentary tracts) and vesicovaginal fistula (between the bladder and the vagina). The causes of fistulas include trauma, complications from medical treatment and disease. Treatment for fistulas varies depending on the cause and degree of the fistula, but usually involves surgery. A variety of surgical procedures are commonly used, most commonly fistulotomy, seton placement (a cord that is passed through the fistula conduit to keep it open for drainage), or an endorectal flap procedure (in which tissue is removed). healthy from the internal side of the fistula to prevent feces or other material from reinfecting the canal). Surgery for anorectal fistulas does not lack side effects, including relapse, reinfection and incontinence. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are the main causes of anorectal, enteroenteric and enterocutaneous fistulas. The reported incidence of fistula in Crohn's disease ranges from 17% to 50%. The treatment of fistulas in patients with Crohn's disease continues to present an extremely complex problem since many of these fistulas do not respond to the available treatments. Such fistulas and their relapse are a very distressing complication that significantly reduces the quality of life of affected patients. Recent improvements in medical treatment (for example, treatment with Infliximab®) and expert surgical treatment have reduced the need for complicated surgery. However, many patients do not heal. The failure in fistula healing is probably due to the inferior quality of the tissues that have been affected by Crohn's disease. In fact, Crohn's disease fistulas provide a model system for wound healing in some of the worst possible conditions. Another major cause of fistulas is trauma, for example for rape, or for injuries experienced during labor, in the tissues of the vagina and bladder and / or rectum that lead to rectovaginal fistula and vesicovaginal fistula. Each year approximately 100,000 in developing countries experience such fistulas (also known as obstetric fistulas) during obstructed labor. During obstructed labor, the pressure of the baby's head against the mother's pelvis cuts the supply of blood to delicate tissues in the area. The dead tissue falls out and the woman is left with a vesicovaginal fistula and sometimes a rectovaginal fistula. This orifice results in permanent incontinence of urine and / or feces. The United Nations Population Fund (UNFPA) estimates the world population of people with obstetric fistula in more than two million. This calculation can be a significant underestimate. The success rates for primary surgical healing range from 88 to 93 percent but decrease with successive attempts. Therefore, a significant percentage of women have obstetric fistulas that can not be cured by surgery. New therapies are needed for fistulas.

SUMMARY OF THE INVENTION The present document provides, inter alia, novel compositions containing stromal stem cells derived from adipose tissue. The compositions containing stromal stem cells derived from adipose tissue described herein have a differentiated phenotype and show a greater phenotype homogeneity than the stromal stem cell compositions derived from adipose tissue previously described, thus making them more suitable for their use in the treatment of fistulas and wounds than the previously described compositions. Compositions containing stromal stem cells derived from adipose tissue can be formulated with solutions or other substances to serve as pharmaceuticals or medical devices, for example, as sutures or adhesives. In addition, novel methods of treating fistulas and wounds are provided using stromal stem cells derived from adipose tissue, as well as kits for the implementation thereof. These embodiments of the present invention, other embodiments, and their features and characteristics will be apparent from the description, drawings and claims that follow.

Brief description of the figures Figure 1 shows the results of the characterization of cells isolated by the methods of example 1 by immunofluorescence staining. The frequency of immunopositive cells is indicated as follows: -, less than 5%; +/-, 6-15%; +, 16-50%; ++, 51-85%; and +++, 86-100%. P, pass number. Figure 2 represents the characterization by indirect immunofluorescence of stromal stem cells derived from adipose tissue. The cells of patient No. 001 were passed 6 cells after implant no. 6. The blue color indicates cores stained with DAPI. (A) CD90; (B) c-Kit; and (C) vimentin. Figure 3 summarizes the clinical results obtained using certain methods and compositions of the invention. M, woman; H, man; NI, no implant; NA, not analyzed. Figure 4 depicts cell growth curves derived from lipoaspirate at different concentrations of FBS (0.5, 2.5 and 10%, as indicated). Human synovial fibroblasts were cultured in the presence of either 5% or 10% FBS. Cell numbers ± SD are shown in terms of absorbance at 595 nm. The data is from a representative experiment with wells in triplicate. Figure 5 shows the blister in the rectal mucosa after having injected the cells near the internal opening closed with suture. Figure 6 depicts photographs of a fistula before (A) and eight weeks after (B) of the cell injection. Figure 7A depicts fluorescence immunocytometer histograms corresponding to the profile of surface markers (CD3, CD9, CD10, CD11, CD14, CD14, CD15, CD16, CD18, CD19, CD28, CD29, CD31, CD34, CD34, CD36, CD38, CD44, CD45, CD49a, CD49b, CD49c, CD49e, CD49e and CD49f) obtained from cells isolated from liposuction samples of a patient participating in the study, in pass 6. Figure 7B depicts fluorescence immunocytometric histograms corresponding to the profile of surface markers (CD50 CD51, CD54, CD55, CD56, CD58, CD59, CD61E, CD62L, CD62, CD95, CD95, CD104, CD105, CD106, CD133, CD166, glycophorin, β2 microglobulin, HLA I, HLA II and NGFR) obtained from cells isolated from liposuction samples of a patient participating in the study, in passage 6.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions: As used herein, the following terms and phrases will have the meanings set out below. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one skilled in the art to which this invention pertains.

The articles "a" and "an" refer to one or more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" means an element or more than one element. By "adipose tissue" is meant any fatty tissue. Adipose tissue can be brown or white adipose tissue, derived from subcutaneous, omental / visceral, mammary, gonadal or other adipose tissue site. Preferably, the adipose tissue is subcutaneous white adipose tissue. Such cells may comprise a primary cell culture or an immortalized cell line. Adipose tissue can be from any organism that has fatty tissue. Preferably, the adipose tissue is mammalian, most preferably the adipose tissue is human. A convenient source of adipose tissue is surgery by liposuction, however, the source of adipose tissue or the adipose tissue isolation method is not critical to the invention. If stromal cells are desired for autologous transplantation in a subject, adipose tissue will be isolated from that subject. "Stromal stem cells derived from adipose tissue" refers to mesenchymal stem cells that originate from adipose tissue. The term "adhesive" refers to any substance that joins or connects surfaces together; for example, a tail. The term "cell composition" refers to a preparation of cells, which preparation may include, in addition to the cells, non-cellular components such as cell culture medium, for example proteins, amino acids, nucleic acids, nucleotides, coenzymes, antioxidants, metals and similar. In addition, the cell composition may have components that do not affect the growth or viability of the cellular component, but are used to provide the cells in a particular format, for example, as a polymer matrix for encapsulation or a pharmaceutical preparation.

The term "culture" refers to any growth of cells, organisms, multicellular entities or tissue in a medium. The term "cultivar" refers to any method for achieving such growth, and may comprise multiple stages. The term "further culture" refers to cultivating a cell, organism, multicellular entity or tissue up to a certain growth phase, then using another culture method to bring said cell, organism, multicellular entity or tissue to another growth phase. A "cell culture" refers to cell growth in vitro. In a culture of this type, the cells proliferate, but do not organize themselves into a tissue itself. A "tissue culture" refers to the maintenance or growth of tissue, for example, primordial organ explants or an adult organ in vitro so that its architecture and function is preserved. A "monolayer culture" refers to a culture in which the cells multiply in a suitable medium while they are mainly bound together and to a substrate. In addition, a "suspension culture" refers to a culture in which cells multiply while suspended in a suitable medium. Likewise, a "continuous flow culture" refers to the cultivation of cells or explants in a continuous flow of fresh medium to maintain cell growth, for example, viability. The term "conditioned media" refers to the supernatant, for example free of cultured cells / tissue, which results after a period of time in contact with the cultured cells such that the media has been altered to include certain paracrine factors and / or autocrine produced by the cells and secreted in the culture. A "confluent culture" is a cell culture in which all the cells are in contact and therefore the entire surface of the culture vessel is covered, and implies that the cells have also reached their maximum density, although confluence does not necessarily mean that the division will stop happening or the size of the population will not grow.

The term "culture medium" or "medium" is recognized in the art, and generally refers to any substance or preparation used for the cultivation of living cells. The term "medium", as used with reference to a cell culture, includes the components of the environment surrounding the cells. The media can be solid, liquid, gaseous or a mixture of phases and materials. The media includes liquid growth media as well as liquid media that do not maintain cell growth. The media also includes gelatinous media such as agar, agarose, gelatin and collagen matrices. Exemplary gaseous media include the gaseous phase to which cells growing in a petri dish or other solid or semi-solid support are exposed. The term "medium" also refers to the material intended for use in a cell culture, even if it has not yet been brought into contact with the cells. In other words, a liquid rich in nutrients prepared for bacterial culture is a means. Similarly, a powder mixture that when mixed with water or other liquid becomes suitable for cell culture can be termed "powder medium". The "defined medium" refers to media that are composed of chemically defined (usually purified) components. The "defined media" does not contain poorly characterized biological extracts such as yeast extract and calf broth. The "rich medium" includes means that are designed to support the growth of most or all viable forms of a particular species. Rich media often include complex biological extracts. A "suitable medium for the growth of a high density culture" is any means that allows a cell culture to reach an OD600 of 3 or more when other conditions (such as temperature and oxygen transfer rate) allow such growth. The term "basal medium" refers to a medium that favors the growth of many types of microorganisms that do not require any special nutrient supplement. Most of the basal media are generally composed of four basic chemical groups: amino acids, carbohydrates, inorganic salts and vitamins. A basal medium generally serves as a base for a more complex medium, to which supplements such as serum, buffers, growth factors, lipids and the like are added. Examples of basal media include, but are not limited to, Eagle's basal medium, minimum essential medium, Dulbecco's modified Eagle medium, Medium 199, Ham's F-10 nutrient mixes and Ham's F-12, 5A Me's Coy , Dulbecco MEM / FI 2, RPMI 1640 and Dulbecco's medium modified by Iscove (IMDM). The terms "comprises" and "comprising" are used in the open, inclusive sense, which means that additional elements may be included. The term "differentiation" refers to the formation of cells that express markers known to be associated with cells that are more specialized and closer to becoming terminally differentiated cells incapable of further differentiation or division. For example, in a pancreatic context, differentiation may be observed in the production of islet-type cell clusters that contain an increased proportion of beta epithelial cells that produce increased amounts of insulin. The terms "additional" or "major" differentiation refer to cells that are more specialized and closer to becoming terminally differentiated cells incapable of further differentiation or division than the cells from which they were cultured. The term "final differentiation" refers to cells that have become terminally differentiated cells incapable of further differentiation or division. The term "fistula" refers to any abnormal connection or communication or connection, usually between two internal organs or that leads from an internal organ to the surface of the body. Examples of fistulas include, but are not limited to, anorectal fistula or fistula of the anus or fecal fistula, arteriovenous fistula or AV fistula, biliary fistula, fistula of the cervix, fistula craniosinus, enteroenteric fistula, enterocutaneous fistula, enterovaginal fistula, gastric fistula, Meterperitoneal fistula, peri-lymphatic fistula, pulmonary arteriovenous fistula, rectovaginal fistula, umbilical fistula, tracheoesophageal fistula, and vesicovaginal fistula. The term "including" is used in the present document to refer to "including but not limited to". "Including" or "including but not limited to" are used interchangeably. "Marker" refers to a biological molecule whose presence, concentration, activity or state of phosphorylation can be detected and used to identify the phenotype of a cell. A "patch" is a dressing or covering applied to cover and protect a wound or other sore. A "patient", "subject" or "guest" that is to be treated by the object method can mean either a human or a non-human animal. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, according to a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent that encapsulates the material, which participates in carrying or transporting the compound object from an organ, or part of the body, to another organ or part of the body. Each vehicle must be "acceptable" in the sense of being compatible with the other components of the formulation and not causing injury to the patient. The term "phenotype" refers to the observable characteristics of a cell, such as size, morphology, protein expression, etc. The term "progenitor cell" refers to a cell that has the ability to create a progeny that is more differentiated than itself. For example, the term can refer to an undifferentiated cell or a differentiated cell in a small degree of final differentiation that can proliferate and give rise to more progenitor cells that have the capacity to generate a large number of stem cells which in turn can result in differentiated or differentiable daughter cells. In a preferred embodiment, the term progenitor cell refers to a generalized stem cell whose descendants (progeny) are specialized, often in different directions, by differentiation, for example, by acquiring completely individual characters, such as occurs in the progressive diversification of tissues and embryonic cells. Cell differentiation is a complex process that normally occurs through many cell divisions. A differentiated cell can be derived from a multipotent cell that itself derives from a multipotent cell, and so on. Although each of these multipotent cells can be considered a stem cell, the range of cell types to which each one can give rise can vary considerably. Some differentiated cells may also have the ability to give rise to cells with greater development potential. Such capacity can be natural or can be induced artificially with the treatment with various factors. By this definition, the stem cells can also be progenitor cells, as well as the more immediate precursors of terminally differentiated cells.

"Proliferation" refers to an increase in the number of cells. "Proliferating" and "proliferation" refers to cells that experience mitosis. As used herein, the term "solution" includes a pharmaceutically acceptable carrier or diluent in which the cells of the invention are still viable. The term "substantially pure", with respect to stem cell populations derived from adipose tissue, refers to a population of stem cells derived from adipose tissue that is pure in at least about 75%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%, with respect to stromal stem cells derived from adipose tissue that constitute a total cell population. By restructuring, the term "substantially pure" refers to a population of stromal stem cells derived from adipose tissue of the present invention containing less than about 20%, more preferably less than about 10%, most preferably less than about 5% of cells directed to a lineage in the original population isolated and without amplification before the subsequent culture and amplification.

"Support" as used herein refers to any device or material that can serve as a base or matrix for the growth of stromal stem cells derived from adipose tissue.

The term "suture" refers to thread or fiber or other fastening material that can be used to sew a wound. The term "treating" as used herein refers to repairing a fistula or wound, as well as preventing a fistula or wound from worsening or relapse. "Therapeutic agent" or "therapeutic compound" refers to an agent that may have a desired biological effect on a host. Chemotherapeutic and genotoxic agents are examples of therapeutic agents that are generally known to be of chemical origin, as opposed to biological, or that elicit a therapeutic effect by a particular mechanism of action, respectively. Examples of therapeutic agents of biological origin include growth factors, hormones and cytokines. A variety of therapeutic agents are known in the art and can be identified by their effects. Certain therapeutic agents can regulate cell proliferation and differentiation. Examples include nucleotides, drugs, hormones, non-specific proteins (non-antibodies), oligonucleotides (e.g., antisense oligonucleotides that bind to a target nucleic acid sequence (e.g., mRNA sequence)), chemotherapeutic peptides and peptidomimetics. A "wound" is an injury or damage to the tissue, caused by physical means, which causes the alteration of the normal continuity of the tissue. 2. Novel compositions containing stromal stem cells derived from adipose tissue In one aspect, the invention relates to compositions containing stromal stem cells derived from adipose tissue with certain characteristics, such as a particular phenotype. For example, stromal stem cells derived from adipose tissue in a cell composition of the invention can be characterized by expression of cell surface marker, size, glucose consumption, lactate production and cell viability / performance. Yet another aspect of the present invention relates to compositions containing stromal stem cells derived from adipose tissue that include, as a cellular component, substantially pure preparations of stromal stem cells derived from adipose tissue having a particular phenotype, or the progeny of the same. The compositions containing stromal stem cells derived from adipose tissue of the present invention include not only substantially pure populations of the progenitor cells, but may also include cell culture components., for example, culture media including amino acids, metals, coenzyme factors, as well as small populations of other stromal cells, for example, some of which may arise by further differentiation of the cells of the invention. In addition, other non-cellular components may include those that make the cellular component suitable for support in particular circumstances, for example, implantation, for example, continuous culture, or suitable for use as a biomaterial or pharmaceutical composition. In certain embodiments, the compositions containing stromal stem cells derived from adipose tissue are produced by the culture methods described in section 4 and the examples. In one embodiment, a composition containing stromal stem cells derived from adipose tissue is provided, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or preferably at least about 96%, 97%, 98% or 99% of the stem cells express the markers CD9, CD10, CD13 , CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and / or CD105. In certain embodiments of the compositions containing stromal stem cells derived from adipose tissue, less than about 15%, about 10%, about 5%, and preferably about 4%, 3%, 2% or 1%. % of the stem cells express the markers CD34, CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and / or CD133.

In another embodiment, a composition containing stromal stem cells derived from adipose tissue is provided, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at less about 85%, at least about 90%, at least about 95% or preferably at least about 96%, 97%, 98% or 99% of the stem cells express the c-Kit, vimentin and / or CD90. In certain embodiments of the compositions containing stromal stem cells derived from adipose tissue, less than about 15%, about 10%, about 5%, and preferably about 4%, 3%, 2% or 1%. % of the stem cells express the markers CD34, factor VIII, alpha-actin, desmin, S-100 and / or keratin. A population of stromal stem cells derived from adipose tissue expressing the c-Kit, vimentin and CD90 markers and not expressing the CD34, factor VIII, alpha-actin, desmin, S-100 and keratin markers are also provided. The phenotypic characterization of a population of cells by surface markers can be carried out either by individual staining of the cells (flow cytometry) or by preparing histological sections of the population in situ, performed according to normal methods. The determination of the expression profile of the surface markers by antibodies, characterization of immunophenotype, can be direct, using a labeled antibody, or indirectly, using a second labeled antibody against the primary specific antibody of the cellular marker, thus achieving an amplification of the signal. On the other hand, the presence or absence of binding to the antibody can be determined by different methods including, but not limited to, immunofluorescence microscopy and radiography. Similarly, it is possible to monitor antibody binding levels by flow cytometry, a technique that allows the levels of fluorochrome to be correlated with the amount of antigens present on the surface of the cell specifically bound to labeled antibodies. . The differential expression of a series of surface markers in a population of cells provides a method for the identification and isolation of said population. In certain embodiments, compositions containing stromal stem cells derived from adipose tissue are suspensions of stromal stem cells derived from adipose tissue in various solutions or materials, for example for use as pharmaceuticals or biomaterials, as described with greater detail below. In one embodiment, the subject cell composition comprises a suspension of the stromal stem cells derived from adipose tissue in Ringer's solution and HSA. In another embodiment, the cell composition comprises a suspension of the stromal stem cells derived from target adipose tissue in a material, such as a polymer, glue, gel, etc. Such suspensions can be prepared, for example, by stripping the stromal stem cells derived from the adipose tissue object of the culture medium and resuspending them in the desired solution or material. The cells can be separated by sedimentation and / or change of the culture medium, for example, by centrifugation, filtration, ultrafiltration, etc.

The concentration of stromal stem cells derived from target adipose tissue in the subject compositions containing stromal stem cells derived from adipose tissue can be at least about 5 x 10 6 cells / ml, at least about 10 x 10 6 cells / ml, at least about 20 x 106 cells / ml, at least about 30 x 106 cells / ml or at least about 40 x 106 cells / ml.

Accordingly, another aspect of the present invention relates to the progeny of the stromal stem cells derived from adipose tissue object, for example those cells that have been derived from stromal stem cells derived from adipose tissue. Such progeny may include subsequent generations of stromal stem cells derived from adipose tissue, as well as lineage-directed cells generated by inducing differentiation of stromal stem cells derived from adipose tissue after isolation from the explant, for example, induced in vitro. In certain embodiments, the cells of the progeny are obtained after about 2, about 3, approximately 4, about 5, about 6, about 7, about 8, about 9 or about 10 passes from the original population. However, the cells of the progeny can be obtained after any number of passes from the original population. In certain embodiments, the compositions containing stromal stem cells derived from adipose tissue of the invention will be provided as part of a pharmaceutical preparation, eg, a sterile preparation, free from the presence of viruses, bacteria and other unwanted pathogens, as well as pyrogen-free. That is, for administration to human beings, the subject compositions must comply with standards of sterility, pyrogenicity, as well as purity and general safety required by the regulations of the Office of Biological Products of the FDA. In certain embodiments, such compositions containing stromal stem cells derived from adipose tissue can be used for transplantation in animals, preferably mammals, and even more preferably humans. The cells may be preferably autologous, but also allogeneic or xenogenic with respect to the host of the transplant. Due to difficulties in obtaining sufficient autologous stem cells, stromal stem cells derived from adipose tissue from allogeneic donors can be a valuable alternative source of stem cells for therapeutic use. It is known in the art that bone marrow stromal stem cells and stromal cells derived from adipose tissue do not elicit an allogeneic lymphocyte response in vitro and consequently, allogeneic adipose derived stromal stem cells derived from a donor may be used theoretically for any patient, regardless of MHC incompatibility. Methods of administering the compositions containing stromal stem cells derived from adipose tissue to subjects, particularly human subjects, which are described in detail herein, include injection or implantation of the cells at target sites in the subjects, the cells can inserted into a delivery device that facilitates the introduction, by injection or implantation, of the cells in the subjects. Such delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject. In a preferred embodiment, the tubes additionally have a needle, for example, a syringe, by means of which the compositions containing stromal stem cells derived from adipose tissue can be introduced into the subject in a desired location. Compositions containing stromal stem cells derived from adipose tissue can be inserted into a delivery device of this type, for example, a syringe, in different forms. For example, compositions containing stromal stem cells derived from adipose tissue include stromal stem cell compositions derived from adipose tissue that are suspended in a solution or embedded in a support matrix when contained in a delivery device for this. kind.

- - Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid to the extent that syringability is readily available. Preferably, the solution is stable under the conditions of manufacture and storage and is protected against the contaminating action of microorganisms such as bacteria and fungi by the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. Solutions can be prepared which are stromal stem cell compositions derived from the adipose tissue of the invention incorporating stromal stem cells derived from adipose tissue such as described herein in a pharmaceutically acceptable carrier or diluent and, as required, other components listed above, followed by a filter sterilization. Some examples of materials and solutions that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and waxes for suppositories; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) solutions with buffered pH; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. In certain embodiments, compositions containing stromal stem cells derived from adipose tissue further comprise an adhesive. In certain embodiments, the adhesive is a fibrin-based adhesive, such as a fibrin gel or fibrin glue or fibrin-based adhesive or polymer, or other tissue adhesive or surgical glue, such as, for example, cyanoacrylate, collagen, thrombin and polyethylene glycol. Other materials that can be used include, but are not limited to, calcium alginate, agarose, types I, II, IV or other isoforms of collagen, poly (lactic acid) / poly (glycolic acid), hyaluronate derivatives or other materials ( Perka C. et al. (2000) J. Biomed, Mater.Res. 49: 305-311; Sechriest VF., Et al. (2000) J. Biomed, Mater.Res.49: 534-541; Chu CR ef al. (1995) J. Biomed, Mater, Res. 29: 1147-1154, Hendrickson DA, et al (1994) Orthop Res 12: 485-497). In other embodiments, the adhesive is a liquid bandage, wherein the compositions containing stromal stem cells derived from adipose tissue of the method are mixed with the liquid bandage material. A "liquid bandage" is a solution comprising a compound, for example a polymeric material, which is applied to a wound with a spray or brush, followed by removal of the solvent by evaporation to provide a protective layer over the wound. Also provided herein are methods for preparing compositions containing stromal stem cells derived from adipose tissue comprising compounds or materials for use in the repair of fistulas or wounds. In one embodiment, a method of preparing such materials comprises suspending the stromal stem cells derived from adipose tissue of a subject composition of cells with the material. In one embodiment, the stromal stem cells derived from adipose tissue are separated by sedimentation from the culture medium and resuspended in a fibrin gel or glue. Fibrin glues and gels and other fibrin-based adhesives and polymers are well known in the art and are commercially available. For example, a commercially available fibrin glue kit is Tissucol® Duo 2.0, and other commercially available fibrin sealants include Crosseal®, TISSEEL VH Fibrin Sealant®, and the like. Compositions containing stromal stem cells derived from adipose tissue of the invention can also be used to coat a support, for example a medical device. For example, the support may be a suture, thread, meniscus repair device, rivet, tack, staple, screw, bone plate, bone plate system, surgical mesh, patch, for example a repair patch, cardiovascular patch or patch. pericardial, sling, orthopedic nail, adhesion barrier, endoprosthesis, guided tissue repair / regeneration device, articular cartilage repair device, nerve guide, tendon repair device, atrial septal defect repair device, filler or filler, vein valve, bone marrow structure, meniscus regeneration device, ligament and tendon graft, cell implant eyepieces, spinal fusion cage, cutaneous substitute, dural substitute, bone graft substitute, bone peg, wound dressing, glue, polymer or hemostatic clamp. Supports in which the compositions containing stromal stem cells derived from adipose tissue can be incorporated or embedded or on which the compositions containing stromal stem cells derived from adipose tissue can be coated include matrices that are compatible with the receptor and that are They degrade to give products that are not harmful to the recipient. Biodegradable natural and / or synthetic matrices are examples of such matrices.

Natural biodegradable matrices include plasma clots, for example, derived from a mammal, and collagen matrices. Synthetic biodegradable matrices include synthetic polymers such as polyanhydrides, polyorthoesters and poly (lactic acid). Other examples of synthetic polymers and methods of incorporating or embedding cells in those matrices are known in the art. See, for example, U.S. Patent No. 4,298,002 and U.S. Patent No. 5,308,701. These matrices provide support and protection for fragile cells in vivo. The support can be coated with cells in any manner as known to one skilled in the art, for example, by soaking, spraying, painting, printing, etc. In one embodiment, the support is a suture, staple, absorbable yarn, non-absorbable yarn, natural yarn, synthetic yarn, monofilament yarn or multifilament yarn (also referred to as braids). Preferred methods of preparing sutures and other supports used to close wounds coated with stromal stem cells derived from adipose tissue are disclosed in U.S. Patent Application No. 11 / 056,241"Biomaterial for Suturing", filed on February 14, 2005 , application that is incorporated by reference in its entirety. The compositions containing stromal stem cells derived from adipose tissue disclosed herein represent novel compositions that can be used with the methods disclosed in U.S. Patent Application No. 11 / 056,241. In addition, in any of the compositions containing stromal stem cells derived from adipose tissue, at least one therapeutic agent may be incorporated into the composition. For example, a composition may contain an analgesic, to help treat inflammation or pain at the site of the fistula or wound, or an anti-infective agent to prevent infection of the site treated with the composition.

More specifically, non-limiting examples of useful therapeutic agents include the following therapeutic categories: analgesics, such as non-steroidal anti-inflammatory drugs, opiate agonists and salicylates; anti-infective agents, such as anthelmintics, antianaerobic, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, various ß-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacteria , antimicrobial antituberculosis, antiprotozoal, anti-malarial antiprotozoal, antiviral agents, antiretroviral agents, scabicides, anti-inflammatory agents, corticosteroid anti-inflammatory agents, antipruritics / local anesthetics, topical anti-infectives, topical anti-fungal antifungal, antiviral topical infectious; electrolytic and renal agents, such as acidifying agents, alkalizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte renewals, and uricosuric agents; enzymes, such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal antiinflammatory agents, salicylate gastrointestinal antiinflammatory agents, antiulcer agents antacids, antiulcer agents gastric acid pump inhibitors, antiulcer agents of the gastric mucosa, anti-ulcer agents blockers of H2, colelitolytic agents, digestive , emetics, laxatives and stool softeners, and prokinetic agents; general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, intravenous barbiturate anesthetics, intravenous benzodiazepine anesthetics, and intravenous anesthetics of opiate agonists; hormones and hormone modifiers, such as abortifacients, suprarenal agents, adrenal corticosteroid agents, androgens, antiandrogens, immunobiological agents, such as immunoglobulins, immunosuppressants, toxoids and vaccines; local anesthetics, such as local amide anesthetics and local ester anesthetics; musculoskeletal agents, such as anti-inflammatory anti-inflammatory agents, anti-inflammatory agents of corticosteroids, anti-inflammatory agents of gold compound, anti-inflammatory immunosuppressive agents, non-steroidal anti-inflammatory drugs (NSAIDs), salicylate anti-inflammatory agents, minerals; and vitamins, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin K. Preferred classes of useful therapeutic agents of the above categories include: (1) analgesics in general, such as lidocaine or derivatives thereof , and analgesics of non-steroidal anti-inflammatory drugs (NSAIDs), including diclofenac, ibuprofen, ketoprofen, and naproxen; (2) analgesics of opiate agonists, such as codeine, fentanyl, hydromorphone and morphine; (3) salicylate analgesics, such as aspirin (AAS) (enteric coated AAS); (4) H-i blocking antihistamines, such as clemastine and terfenadine; (5) anti-infective agents, such as mupirocin; (6) anti-anaesthobic anti-infectives, such as chloramphenicol and clindamycin; (7) anti-infective antifungal antibiotics, such as amphotericin b, clotrimazole, fluconazole and ketoconazole; (8) anti-infective macrolide antibiotics, such as azithromycin and erythromycin; (9) anti-infective various ß-lactam antibiotics, such as aztreonam and imipenem; (10) anti-infective penicillin antibiotics, such as nafcillin, oxacillin, penicillin G and penicillin V; (11) antiinfective quinolone antibiotics, such as ciprofloxacin and norfloxacin; (12) anti-infective tetracycline antibiotics, such as doxycycline, minocycline and tetracycline; (13) Antimicrobial anti-tuberculosis drugs such as isoniazid (INH) and rifampin; (14) antiprotozoal antiinfectives, such as atovaquone and dapsone; (15) anti-malarial antiprotozoal anti-infectives, such as chloroquine and pyrimethamine; (16) antiretroviral anti-infectives, such as ritonavir and zidovudine; (17) antiviral antiinfective agents, such as acyclovir, ganciclovir, interferon alfa and rimantadine; (18) topical antifungal anti-infectives, such as amphotericin B, clotrimazole, miconazole and nystatin; (19) topical antiviral anti-infectives, such as acyclovir; (20) electrolyte and renal agents, such as lactulose; (21) loop diuretics, such as furosemide; (22) potassium-sparing diuretics, such as triamterene; (23) thiazide diuretics, such as hydrochlorothiazide (HCTZ); (24) uricosuric agents, such as probenecid; (25) enzymes such as RNase and DNase; (26) antiemetics, such as prochlorperazine; (27) salicylate gastrointestinal anti-inflammatory agents, such as sulfasalazine; (28) anti-ulcer agents inhibiting the gastric acid pump, such as omeprazole; (29) H2-blocking anti-ulcer agents, such as cimetidine, famotidine, nizatidine and ranitidine; (30) digestives, such as pancrelipase; (31) prokinetic agents, such as erythromycin; (32) local ester anesthetics, such as benzocaine and procaine; (33) anti-inflammatory agents of musculoskeletal corticosteroids, such as beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone and prednisone; (34) musculoskeletal anti-inflammatory immunosuppressants, such as azathioprine, cyclophosphamide and methotrexate; (35) musculoskeletal non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketorolac and naproxen; (36) minerals, such as iron, calcium and magnesium; (37) Vitamin B compounds, such as cyanocobalamin (vitamin B12) and niacin (vitamin B3); (38) vitamin C compounds, - - such as ascorbic acid; and (39) vitamin D compounds, such as calcitriol. In certain embodiments, the therapeutic agent may be a growth factor or another molecule that affects cell differentiation and / or proliferation. Growth factors that induce states of final differentiation are well known and can be selected from any of such factors that have been shown to induce a state of final differentiation. Growth factors for use in methods described herein can be, in certain embodiments, variants or fragments of a naturally occurring growth factor. For example, a variant can be generated by making conservative amino acid changes and testing the resulting variant in one of the functional assays described above or another functional assay known in the art. Conservative amino acid substitutions refer to the ability to exchange residues that have similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; a group of amino acids that have aliphatic-hydroxyl side chains is serine and threonine; A group of amino acids having side chains containing amide is asparagine and glutamine; a group of amino acids that have aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids that have basic side chains is lysine, arginine and histidine; and a group of amino acids that have side chains containing sulfur is cysteine and methionine. Conservative amino acid substitution groups are: valine-leucine -soleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. As will be appreciated by those skilled in the art, variants or fragments of polypeptide growth factors can be generated using - - conventional techniques, such as mutagenesis, including creating mutation / point mutations (s) differentiated (s), or by truncation. For example, the mutation can result in variants that substantially retain the same, or simply a set of, the biological activity of a polypeptide growth factor from which they are derived. 3. Methods of preparation of novel compositions containing stromal stem cells derived from adipose tissue Methods of preparation of stromal stem cells derived from adipose tissue constituting the compositions described above containing stromal stem cells derived from adipose tissue are also provided. In one embodiment, a method comprises: (a) collecting adipose tissue from a subject; (b) obtaining a cell suspension by enzymatic digestion; (c) sedimenting the cell suspension and resuspending the cells in a culture medium; (d) culturing the cells for at least about 10 days; and (g) expanding the cells during at least two culture passages. Preferably, the stromal stem cells derived from adipose tissue are isolated from the adipose tissue of the subject into which the final compositions containing stromal stem cells derived from adipose tissue must be introduced. However, stromal stem cells can also be isolated from any organism of the same species or a different one from that of the subject. Any organism with adipose tissue can be a possible candidate. Preferably, the organism is a mammal, most preferably the organism is a human being. In certain embodiments, the cells are cultured for at least about 15, at least about 20 days, at least about 25 days or at least about 30 days. It is preferable that the cells expand in culture for a longer time to improve the homogeneity of the cell phenotype in the cell population. In certain embodiments, the cells are expanded in culture for at least three culture passages or "one pass is made at least three times". In other embodiments, a passage is made to the cells at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times or at least ten times. It is preferable that a passage is made to the cells more than three times to improve the homogeneity of the cell phenotype in the cell population. In fact, the cells can expand in culture indefinitely as long as the homogeneity of the cell phenotype improves and the differential capacity is maintained. The cells can be cultured by any technique known in the art for stem cell culture. A discussion of the various culture techniques, as well as their scaling, can be found in Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 4th edition, Wiley-Liss 2000. In certain embodiments, cells are grown by monolayer culture. In one embodiment, the cells are cultured and passages are performed as described in Example 1 below. Any medium that can support stromal cells in tissue culture can be used. Media formulations that will support the growth of fibroblasts include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha-modified minimal essential medium (.alpha. MEM) and Roswell Park Memorial Institute 1640 medium (medium).

RPMI 1640) and the like. Normally, 0 to 20% fetal bovine serum (FBS) or 1-20% horse serum will be added to the above media in order to support the growth of stromal cells and / or chondrocytes. However, a defined medium can be used if the growth factors, cytokines and hormones in FBS needed for stromal cells and chondrocytes are identified and provided in appropriate concentrations in the growth medium. The media useful in the methods of the invention may contain one or more compounds of interest, including, but not limited to, differentiating or mitogenic antibiotic compounds for stromal cells. The cells will be grown at temperatures between 31 ° C to 37 ° C in a humidified incubator. The carbon dioxide content will remain between 2% to 10% and the oxygen content between 1% and 22%. The cells can remain in this environment for periods of up to 4 weeks. Antibiotics with which the medium can be supplemented include, but are not limited to, penicillin and streptomycin. The concentration of penicillin in the chemically defined culture medium is from about 10 to about 200 units per ml. The concentration of streptomycin in the chemically defined culture medium is from about 10 to about 200 Dg / ml. Stromal stem cells derived from adipose tissue can be transfected or transduced stably or transiently with a nucleic acid of interest using a plasmid, viral or alternative vector strategy. Nucleic acids of interest include, but are not limited to, those that code for gene products that enhance the production of extracellular matrix components found in the type of tissue to be repaired, for example intestinal wall or vaginal wall. Transduction of viral vectors carrying regulatory genes into stromal stem cells can be performed with viral vectors (adenovirus, retrovirus, adeno-associated virus or other vector) purified by banding with cesium chloride or another method at a multiplicity of infection (viral units: cell) of between 10: 1 and 2000: 1. The cells will be exposed to the virus in serum-free medium or containing serum in the absence or presence of a cationic detergent such as polyethylene imine or Lipofectamine ™ for a period of 1 hour to 24 hours (Byk T. et al. (1998) Human Gene Therapy 9: 2493-2502; Sommer B. et al. (1999) Calcif. Tissue Int. 64: 45-49). Other suitable methods for transferring vectors or plasmids into stem cells include lipid / DNA complexes, such as those described in U.S. Patent Nos. 5,578,475; 5,627,175; 5,705,308; 5,744,335; 5,976,567; 6,020,202 and 6,051,429. Suitable reagents include lipofectamine, a 3: 1 (w / w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) (Chemical Abstracts record name: N- [2- (2,5-bis [(3-aminopropyl) amino] -1-oxopentyl]. amino) ethyl] -N, N-dimethyl-2,3 trifluoroacetate -bis (9-octadecenyloxy) -1-propanaminium), and the neutral lipid dioleoyl-phosphatidylethanolamine (DOPE) in membrane-filtered water. By way of example, the formulation Lipofectamine 2000 ™ (available from Gibco / Life Technologies No. 11668019) is provided. Other reagents include: FuGENETM 6 transfection reagent (a combination of lipids in non-liposomal form and other compounds in 80% ethanol, available from Roche Diagnostics Corp. No. 1814443); and transfection reagent LipoTAXITM (a lipid formulation from Invitrogen Corp., No. 204110). The transfection of stem cells can be carried out by electroporation, for example, as described in M.L. Roach and J.D. McNeish (2002) Methods in Mol. Biol. 185: 1. Viral vector systems suitable for producing stem cells with stable genetic alterations can be based on adenovirus and retrovirus, and can be prepared using commercially available virus components. Transfection of plasmid vectors carrying regulatory genes into stromal stem cells can be introduced into cells in monolayer cultures by the use of calcium precipitation by calcium phosphate or cationic detergent methods (Lipofectamine ™., DOTAP) or three-dimensional cultures by incorporation of the plasmid DNA vectors directly into the biocompatible polymer (Bonadio J. et al. (1999) Nat. Med. 5: 753-759). For monitoring and detection of functional proteins encoded by these genes, the plasmid or viral DNA vectors will contain an easily detectable marker gene, such as the green fluorescent protein or beta-galactosidase enzyme, both of which can be tracked by histochemical means. 4. Methods of treating fistulas and wounds Another aspect of the invention relates to a novel method for using stromal stem cells derived from adipose tissue in the treatment of fistulas and wounds. In preferred embodiments, the stromal stem cells derived from adipose tissue are derived from the adipose tissue of the subject to be treated. In other preferred embodiments, the stromal stem cells derived from adipose tissue constitute a composition containing stromal stem cells derived from adipose tissue described herein. However, other preparations of stromal stem cells derived from adipose tissue can be used in the methods described herein, for example such as those described in U.S. Patent Nos. 6,777,231 and 6,555,374 and U.S. Patent Application Number 11 / 065,461"Identification and isolation of multipotent cells from non-osteochondral mesenchymal tissue", filed on February 25, 2005 In one embodiment, a method of treating a fistula in a subject comprises: (a) closing the internal orifice with a suture and (b) administer at least about 10 x 106, at least about 20 x 106, at least about 30 x 106 or at least about 40 x 10 6 stromal stem cells derived from adipose tissue, for example, in a composition that contains stromal stem cells derived from adipose tissue of the invention, to the internal orifice closed with suture. In certain embodiments, for example, in which the first administration of cells is insufficient, the method may further comprise: (c) administering a second dose of at least about 20 x 10 6 cells, at least about 30 x 10 6 or at least about 40 x 106 stromal stem cells derived from adipose tissue, for example, in a composition containing stromal stem cells derived from adipose tissue of the invention, to the internal orifice closed with suture. In another embodiment, the composition containing stromal stem cells derived from adipose tissue used in the method is one in which at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or preferably at least about 96%, 97%, 98% or 99% of the stem cells express the CD9 markers, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59 CD90 and / or CD105. In another embodiment, the composition containing stromal stem cells derived from adipose tissue used in the method is one in which at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or preferably at least about 96%, 97%, 98% or 99% of the stem cells express the markers c- Kit, vimentin and / or CD90. Common methods of administration of the cells of the present invention to subjects, particularly human subjects, some of which are described in detail herein, include injection or implantation of the cells at target sites in the subjects, the cells of the invention they can be inserted into a delivery device that facilitates the introduction, by injection or implantation, of the cells in the subjects. Such delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject. In a preferred embodiment, the tubes additionally have a needle, for example, a syringe, through which the cells of the invention can be introduced into the subject in a desired location. The cells of the invention can be inserted into a delivery device of this type, for example, a syringe, in different ways. For example, the cells can be suspended in a solution or embedded in a support matrix when they are contained in such an administration device. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid as long as there is easy syringability. Preferably, the solution is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi by the use, for example, of parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. The solutions of the invention can be prepared by incorporating progenitor cells as described herein in a pharmaceutically acceptable carrier or diluent and, as required, other components listed above, followed by filter sterilization. In other embodiments, a method of treating a fistula in a subject comprises: (a) closing the internal orifice with a suture comprising stromal stem cells derived from adipose tissue, by example, of an object composition containing stromal stem cells derived from adipose tissue. Such cell-coated sutures in the subject compositions containing stromal stem cells derived from adipose tissue are described in detail in U.S. Patent Application No. 11 / 056,241, filed on February 14, 2005, which is incorporated herein by reference. reference. In some embodiments, the methods may further comprise: (d) scraping at least one channel of the fistula in depth and (e) filling said fistula channel with a material. In certain embodiments, the method may further comprise administering at least about 10 x 10 6 stromal stem cells derived from adipose tissue, eg, from a subject composition of cells, to the material.

Preferably, the material is a fibrin-based adhesive or polymer, such as a gel or fibrin glue. In certain embodiments, the dose of at least about 10 x 10 6 stromal stem cells derived from adipose tissue is already comprised within the material, for example, such that the material comprises the composition containing stem cells derived from adipose tissue. In a further embodiment, a method of treating a fistula in a subject comprises: (i) deep scraping at least one channel of the fistula (ii) closing the internal orifice of the scraped channel with a suture administering at least about 10 x 106 , at least about 20 x 106, at least about 30 x 106 or at least about 40 x 10 6 stromal stem cells derived from adipose tissue to the internal orifice closed with suture, for example, in a composition containing stromal stem cells derived from adipose tissue of the invention.

- - In certain embodiments, for example, in which the first administration of cells is insufficient, the method may further comprise: (iv) administering a second dose of at least about 20 x 10 6 cells, at least about 30 x 10 6 or at least about 40 x 106 stromal stem cells derived from adipose tissue to the internal orifice closed with suture, for example, in a composition containing stromal stem cells derived from adipose tissue of the invention. Step (i) is preferably carried out by deep scraping all the channels of the fistula to be treated, for example, a curettage needle is inserted in the path of the fistula, and a rasped induced hemorrhage occurs. the walls of the fistula in order to obtain natural fibrin that will fill the path of the fistula.

Recent clinical studies by the inventors suggest that the natural fibrin produced by this scraping method is a preferred option compared to the use of artificial fibrin sealants, therefore, in a preferred embodiment of the method of the invention, the fistula paths that go to be treated are not filled with such material. Step (iv) is preferably carried out by local administration of the cells, for example a composition containing stromal stem cells derived from adipose tissue, by injection into the walls of the fistula along the path of the fistula . For example, two injections of 10 million cells along a 3-cm pathway of the fistula. The methods of the invention can be used to treat any fistula, including, but not limited to, anorectal fistula or fistula of the anus or fecal fistula, arteriovenous fistula or AV fistula, biliary fistula, fistula of the cervix, craniosinus fistula, enteroenteric fistula, fistula - - enterocutaneous, enterovaginal fistula, gastric fistula, metroperitoneal fistula, peri-lymphatic fistula, pulmonary arteriovenous fistula, rectovaginal fistula, umbilical fistula, tracheoesophageal fistula and vesicovaginal fistula. Preferably, the methods can be used to treat intestinal fistulas, for example those that connect the intestine with itself or with another organ, such as rectovaginal fistula, enteroenteric fistula, enterocutaneous fistula and enterovaginal fistula. In another preferred embodiment, the methods can be used to treat vaginal or uterine fistulas, for example those that connect the vagina or uterus to themselves or to another organ, such as fistula of the cervix, rectovaginal fistula, enterovaginal fistula and vesicovaginal fistula. The fistula can be accessed for surgical repair by any method known in the art, for example, by incision, catheter, etc. In another embodiment, a method of treating a wound in a subject comprises: (a) closing the wound with a suture and (b) administering at least about 10 x 106, at least about 20 x 106, at least about 30 x 106 or at least about 40 x 10 6 stromal stem cells derived from adipose tissue, for example, in a composition containing stromal stem cells derived from the adipose tissue, to the wound closed with suture. In certain embodiments, for example, in which the first administration of cells is insufficient, the method may further comprise: (c) administering a second dose of at least about 20 x 10 6 cells, at least about 30 x 10 6 or at least about 40 x 106 stromal stem cells derived from adipose tissue, for example, in a composition containing stromal stem cells derived from the adipose tissue, to the wound closed with suture. In other embodiments, the wound may be filled with a composition containing stromal stem cells derived from the adipose tissue of the invention, for example a dose of at least about 10 x 10 6 stromal stem cells derived from adipose tissue engulfed within a material, for example, such that the material comprises the cellular composition, in which the material is, for example, an adhesive or glue. In other embodiments, a method of treating a wound in a subject comprises: (a) closing the wound with a suture comprising stromal stem cells derived from adipose tissue, for example, from an object composition containing stromal stem cells derived of adipose tissue. Such cell-coated sutures of the subject compositions containing stromal stem cells derived from adipose tissue are described in detail above and in U.S. Patent Application No. 11 / 056,241, filed on February 14, 2005, which is incorporated herein by reference. as reference. The methods described above may further comprise administering a therapeutic agent to the subject being treated, for example systemically or locally at the suture site. In certain embodiments, stromal stem cells derived from adipose tissue are formulated into a composition containing stromal stem cells derived from adipose tissue containing a therapeutic agent, as described above. In other embodiments, the therapeutic agent is administered separately, for example simultaneously with the methods, before the method is performed, or after the method is performed. In some embodiments, the therapeutic agent is administered to the subject before, during and after the methods are performed on the subject. Therapeutic agents by way of example are described above. In preferred embodiments, therapeutic agents for the treatment of Crohn's disease are administered to the subject. Therapeutic agents for Crohn's disease by way of example are anti-inflammatory agents such as agents comprising mesalamine, immunosuppressive agents such as 6-mercaptopurine and - - azathioprine; biological agents such as infliximab (Remicade®), antibiotics and antidiarrheal agents such as diphenoxylate, loperamide and codeine. In embodiments where allogeneic stem cells are used, a supportive treatment may be required. For example, immunosuppressants can be administered before, during and / or after treatment to prevent GVHD, according to methods known in the art. Prior to administration, the cells can also be modified to suppress an immune reaction of the subject against the cells or vice versa, according to methods known in the art. The dosage of any therapeutic agent will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the agent. Any of the subject formulations can be administered in a single dose or in divided doses. Dosages for the therapeutic agents can be readily determined by techniques known to those skilled in the art or as taught herein. In addition, mixtures of more than one therapeutic agent can be administered, or multiple therapeutic agents administered in separate compositions. The therapeutic agents can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by an implanted reservoir. The term parenteral, as used herein, includes infusion or subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection techniques. The precise time of administration and the amount of any particular agent that will produce the most effective treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of a particular compound, the physiological state of the patient (including age, sex, phase and type). of disease, general physical state, receptivity to a given dosage and type of medication), the route of administration and the like. The guidelines presented in this document can be used to optimize the treatment, for example, to determine the optimal time and / or amount of administration that will not require more than routine experimentation consisting of monitoring the subject and adjusting the dosage and / or dosage. moment. While the subject is being treated, the patient's health can be controlled by measuring one or more of the relevant indices at predetermined times during a 24-hour period. The treatment, including supplement, amounts, time of administration and formulation, can be optimized according to the results of such monitoring. The patient can be re-evaluated periodically to determine the extent of the improvement by measuring the same parameters, with the first such reassessments usually occurring at the end of four weeks from the start of treatment, with subsequent re-evaluations occurring every four to eight weeks during treatment and then every three months thereafter. The treatment can continue for several months or even years, with a typical duration of treatment of a minimum of one month for humans. The adjustments to the amount (s) of the agent administered (s) and possibly the time of administration can be made based on these reassessments. The treatment can be started with smaller dosages that are less than the optimum dose of the compound. Subsequently, the dosage can be increased in small increments, until the optimal therapeutic effect is achieved. The combined use of several therapeutic agents can reduce the dosage required for any individual component because the appearance and duration of the effect of the different components can be complementary. In such combination treatment, the different active principles can be administered together or separately, and simultaneously or at different times throughout the day. The toxicity and therapeutic efficacy of the subject compounds can be determined by conventional pharmaceutical methods in cell cultures or experimental animals, for example, to determine LD50 and ED50. Compositions that show high therapeutic indices are preferred. Although compounds showing toxic side effects can be used, caution should be exercised in designing a delivery system that directs the agents to the desired site in order to reduce side effects. The data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of any therapeutic agent or alternatively of any component therein is preferably in the range of circulating concentrations that include the ED5o with little or no toxicity. The dosage may vary within this range depending on the pharmaceutical form used and the route of administration used. For the agents of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration that includes Cl50 (ie, the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

. Kits In other embodiments, the invention contemplates kits that include compositions containing stromal stem cells derived from adipose tissue and optionally instructions for their use. The kits comprising the pharmaceutical compositions and biomaterials of the present invention are also within the scope of the invention. The components of the kit can be packaged for the implementation either manually or partially or fully automated of the above methods. Such kits can have a variety of uses, for example, treatment, repair, preparation of biomaterials and other applications.

Examples The invention now generally described will be more readily understood by reference to the following examples, which are included only for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

Example 1: Preparation of stem cells from lipoaspirados with improved homogeneity Adipose tissue was obtained by liposuction, with local anesthesia and general sedation. A hollow blunt-tipped cannula was introduced into the subcutaneous space through a small incision (less than 0.5 cm in diameter). With gentle suction the cannula was moved through the adipose tissue abdominal wall compartment to obtain the mechanical breakage of the fatty tissue. Saline and vasoconstrictor epinephrine were injected into the adipose tissue compartment to minimize blood loss. In this way, 80 to 100 ml of untreated lipoaspirate were obtained from each patient to be treated. He was extensively washed with phosphate-buffered saline (PBS, Gibco BRL, Paisley, Scotland, UK) untreated lipoaspirate to remove blood cells, saline and local anesthetic. The extracellular matrix was digested with a solution of type II collagenase (0.075%, Gibco BRL) in balanced saline solution (5 mg / ml, Sigma, St. Louis, USA) for 30 min. at 37 ° C to release the cellular fraction. The collagenase was then inactivated by adding an equal volume of Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) containing 10% fetal bovine serum (FBS, Gibco BRL). The cell suspension was centrifuged at 250 x g for 10 min. The cells were resuspended in 0.16 M NH4CI and allowed to stand for 10 min. at room temperature (RT) for the lysis of erythrocytes. The mixture was centrifuged at 250 xg, and the cells were resuspended in DMEM plus 10% FBS and 1% ampicillin / streptomycin mixture (Gibco, BRL) and then seeded in 100 mm tissue culture plates at a concentration of 10-30 x 103 cells / cm2. The cells were cultured for 24 h at 37 ° C in an atmosphere of % CO2 in air. The plates were then washed with PBS to remove cell fragments and cells that had not adhered. The cells were maintained in culture in the same medium and in the same conditions until they reached approximately 80% confluence, with renewal of the culture medium every 3 to 4 days. Then a passage of the cells was performed with trypsin-EDTA (Gibco BRL) at a dilution of 1: 3 corresponding to a cell density of about 5-6 x 10 3 cells / cm 2. For transplantation cells were used between passages 1 and 3, with cells being preferred for which more than two passes had been made in order to isolate a population of cells with high homogeneity. Cell characterization was performed using cells in passages 1 to 9.

Example 2.- Characterization of stem cells from lipoaspirates with improved homogeneity To characterize the cells by immunofluorescence staining, the cells were plated at low density in DMEM plus 10% FBS on glass slides in 24-well plates. For the immunohistochemical studies, the cells were washed with PBS and fixed in acetone for 10 min. at -20 ° C. For a-actin staining, the cells were fixed in 4% paraformaldehyde for 10 min. to TA. After blocking with PBS containing 4% goat serum and 0.1% Triton X-100, the cells were incubated at 4 ° C overnight with primary antibodies against the following cell markers at the indicated dilutions [( i) alpha-actin; Dako, Glostrup, Denmark; 1/50; (ii) vimentin; Sigma, St. Louis, USA; 1/200; (iii) CD90; CYMBUS, Biotechnology LTD, Chandlers Ford, Hants, UK; 1/50; (iv) factor VIII; Dako; 1/100; (v) CD34; Chemicon, CA, USA; 1/100; (vi) c-Kit; Chemicon; 1/100; (vii) desmin; Dako; 1/100; (viii) cytokeratin; Dako; 1/100 and (ix) S-100; Dako; 1/50]. The cells were then incubated with appropriate secondary antibodies conjugated to fluorescein isothiocyanate (FITC) or tetramethyl thiocyanate isothiocyanate (TRITC) conjugates (Sigma, 1/50) for 45 min. to TA. For the negative controls the primary antibodies were omitted. The nuclei were counterstained with 4 ', 6-diamidino-2-phenylindole (DAPI). The cells were then mounted in Mobiglow (MoBiTec, Gottingen, Germany) and observed with an Eclipse TE300 epifluorescence microscope (Nikon, Tokyo, Japan). In each case, the number of immunopositive cells in different fields was determined and compared with the numbers of stained nuclei. The randomly selected fields were exported to a computer (Macintosh G3, Apple Computer Ink., Cupertino, CA, USA) through a Spotl camera (Diagnostic Instruments Inc., Tampa, FL, USA). Positive controls for immunostaining with the various antibodies were human aortic smooth muscle cells, human umbilical vein endothelial cells (HUVEC) and human synovial fibroblasts. In passage 1, a high percentage (90-95%) of stromal stem cells derived from adipose tissue expressed vimentin, a marker of the mesenchymal cells of the cytoskeleton (Figure 1). The expression of vicentin was maintained at the same level up to and including passage 9. However, the levels of other markers fell over time. For example, α-actin, which was found in 17% of the cells derived from LPA in passage 1, was no longer detectable in passage 7. The marker of endothelial cells, von Willebrandt factor (factor VIII), and CD34, which is also found on the surface of endothelial cells, were only detected in passages 1 to 3 (7% and 12% of immunopositive cells, respectively). On the contrary, the expression of c-Kit (CD117), a marker of cell proliferation, increased with time, with 99% of immunopositive cells from passage 4 onwards (figure 2). The CD90 fibroblast marker, initially expressed in approximately 80% of the cells derived from APL, was found in 99% of the cells from passage 6 (FIG. 3). The expression of the neuroectodermal marker S100 or the ectodermal keratin marker was not observed in any of the cells derived from APL at any time. The change of the observed markers as the number of passes increases indicates an increase in the homogeneity of the cell preparation obtained. To quantify cell growth, cells were seeded in 24-well plates at a concentration of 5 x 10 3 cells / cm 2.

After the cells had bound to the substrate (3 h), the culture medium was replaced by DMEM supplemented with 1% antibiotics plus 0.5%, 2%, 5% or 10% FBS. Human synovial fibroblasts were also cultured as positive controls to test each batch of serum and their growth rates were determined. The medium was replaced every two days. At 24 h intervals, the cells were fixed with 1% glutaraldehyde and the number of cells per well was determined, after nuclear staining with crystal violet, monitoring the absorbance at 595 nm. A standard curve was constructed to establish the relationship between the number of cells per well and the absorbance at 595 nm (1 ^ = 0.99). Successful adipose tissue-derived stromal cells were isolated and cultured from the seven lipoaspirados (LPA). These cells were grown in culture and passes were made at intervals of 7 to 10 days. In some cases the cells were cryopreserved and thawed before implantation. The growth rate of stromal stem cells derived from adipose tissue (ADCM) was dependent on the serum concentration, with maximum proliferation with FBS at between 5% and 10% (Figure 4). The mean time of doubling the population at these serum concentrations was 37.6 ± 0.6 h, which did not differ significantly from the time of doubling the population of human synovial fibroblasts cultured under the same conditions (35.6 ± 1.4 h; p > 0.05; test of the t; results of three independent experiments). In order to analyze the cells in a less subjective and more normalized manner, the cells were also subjected to a separation analysis of fluorescence activated cells (FACS). In general, flow cytometry analysis allows the detection of surface antigens by antibodies, which bind directly (covalently) or indirectly (secondary antibody labeled with fluorescence) to a fluorescent label. On the other hand, the immunohistochemical analysis described above requires the permeabilization of the cells and the subsequent staining with antibodies. Therefore, the latter requires an optimized protocol individually depending on the antibody and the target protein. In addition, due to the permeabilization of the cell membrane it is not possible to distinguish between proteins - - extracellular and internal markers (not attached to the membrane). That is, with an immunohistochemical analysis it is possible to know if a protein marker is being expressed, but it is not possible to distinguish whether it is being expressed on the cell surface or intracellularly. The protocol used in immunocytometry for the detection of surface antigens is normalized and only requires appropriate negative controls. In addition, the FACS analysis allows an evaluation of the percentage of positive cells (cells expressing the surface antigen) and the level of expression (few or many surface antigens on a cell). These evaluations are only subjective in nature and use immunohistochemistry and can vary from experiment to experiment, which is not the case with FACS analysis. Such immunophenotyping of the cells can be carried out in newly isolated cells and after periods of culture, for example on day 7, after 4 weeks and after 3 months of culture. The analysis of surface markers at different times allows the evaluation of the phenotype homogeneity during cultivation. Examples of this analysis and data demonstrating the phenotype obtained from samples obtained from 3 healthy donors from zero to three months of culture are described in detail in U.S. Patent Application No. 11 / 065,461, filed on February 25, 2005, which is incorporated herein by reference. After isolation by the method described above, the stromal stem cells derived from the adipose tissue of one of the patients were characterized according to the presence / absence of a series of surface markers. To do this, the expression of the following surface markers was monitored by flow cytometry: Integrin: CD11b, CD18, CD29, CD49a, CD49b, CD49e, CD49e, CD49f, CD51, CD61, CD104.

- - Hematopoietic markers: CD3, CD9, CD10, CD13, CD16, CD14, CD19, CD28, CD34, CD38, CD45, CD90, CD133, glycophorin. Receptors of the growth factor: CD105, NGFR. Receptors of the extracellular matrix: CD15, CD31, CD44, CD50, CD54, CD62E, CD62L, CD62P, CD102, CD106, CD146, CD166. Others: CD36, CD55, CD56, CD58, CD59, CD95, HLA-I, HLA-II, β2-microglobulin. The cells to be characterized were harvested by gentle digestion with trypsin, washed with PBS and incubated for 30 minutes at 4 ° C with fluorescein-labeled antibody markers (FITC) or phycoerythrin (PE) against each of the markers. of surface that were going to be analyzed. Cell markers were washed and analyzed immediately using Epics-XL cytometer (Coulter). As controls were used cells stained with nonspecific antibodies of the corresponding isotopes labeled with FITC or PE. From the analysis of the expression profile of surface markers (Figure 7A / 7B), the criteria used to determine which markers define the cell population and allow it to be identified and differentiated with respect to other types of cells are the following: 1 Discard those markers that vary from one sample to another or over time during culture in experiments performed with stromal stem cells derived from adipose tissue from healthy donors in U.S. Patent Application No. 11 / 065,461, filed on February 2005, which is incorporated herein by reference. 2. Select markers as a function of their biological relevance, discarding markers characteristic of specific cell types (for example, CD3 is an exclusive marker for lymphocytes). Applying these criteria the population of multipotent stem cells is characterized as being positive for CD9 +, CD10 +, CD13 +, CD29 +, CD44 +, CD49A +, CD51 +, CD54 +, CD55 +, CD58 +, CD59 +, CD90 + and CD105 +; and lack expression of CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and CD133.

Example 3: Stem cell preparations comprising fibrin glue for use in the treatment of fistula For clinical use cells can be used as prepared above after three or fewer passes (figure 3), but are preferably used after two or more passes as described above to give a cell preparation with superior homogeneity. They were treated with trypsin for 3 min. at 37 ° C cell cultures for clinical use. The trypsin treatment was stopped by the addition of DMEM plus FBS, and the suspension was centrifuged at 110 x g for 5 min. The cells were washed in PBS and the suspension was centrifuged again at 150 x g for 5 min. The cells were resuspended at 3 to 30 x 106 cells / ml in 1 to 2 ml of Ringer's lactate solution and placed in a suitable syringe. Optional human serum albumin (HSA) can optionally be added to the Ringer lactate solution. In some cases half of the cells were resuspended in the thrombin component of a fibrin glue kit (Tissucol® Duo 2.0; Baxter, Madrid, Spain) before the combination of the two components of the kit, in an attempt to improve the obturation of the fistula paths. The use of fibrin glue to fill a fistula opening is known in the art; however, it is not effective as an independent treatment for fistula. The addition of fibrin glue to the - - Adipose-derived stromal stem cell-containing compositions described herein serve to retain the cells locally, and it has been observed that the cells grow well within gels and fibrin tails.

Example 4: Improved guirurgical procedure to repair fistulas using stem cell preparations from lipoaspirates A phase I clinical trial designed to test the viability and safety of autologous stem cell transplants using the stromal stem cell compositions was performed. adipose tissue described above for the treatment of fistulas due to Crohn's disease. The protocol was approved by the Ethics and Clinical Trials Committee of La Paz Hospital on April 12, 2002, and a detailed informed consent form was generated for patients to sign it. The ethical committee was kept informed about the evolution of the study throughout the clinical trial.

Methods Patients were selected according to the following inclusion criteria: over 18 years of age; diagnosis of Crohn's disease at least five years before the trial; presence of one or more complex fistulas due to Crohn's disease (enterocutaneous fistula, suprasphincal fistula and / or rectovaginal fistula) that have not responded to medical treatment and have been treated unsuccessfully by classical surgery at least twice; and agreement to participate by signing the informed consent form. The exclusion criteria were the following: failure to meet the inclusion criteria; mental deficiency; extreme thinness, allergy to local anesthetics; previous diagnosis of cancer; and AIDS. Five patients were included in the study (numbers 001-005). There were three men and two women and the average age was 35.1 ± 2.4 years (range: from 31, 2 to 37.5 years). Nine cellular implants were performed: three in rectovaginal fistulas; five in enterocutaneous fistulas (four different fistulas in one patient, and one in a perianal suprasphincter fistula). All the enterocutaneous fistulas had low flow (less than 50 cc per day) and were located in the abdominal wall (table 1). No patient with total parenteral nutrition, Remicaid, or Octreotride was treated simultaneously with this procedure. Patients 001 and 002 required two liposuction procedures because, after the first liposuction, no stem cell survived cryopreservation. One patient was excluded due to bacterial contamination of cultured cells. Nine fistulas were inoculated in four patients with autologous adipose tissue-derived stromal stem cells (CMDA) in step three or earlier. A total of eight inoculated fistulas were followed weekly for at least eight weeks. In six fistulas, the external opening was covered with epithelium at the end of week 8 and, thus, these fistulas were considered cured (75%). In the other two fistulas, there was only incomplete closure of the external opening with a decrease in outflow (not cured); 25%). No adverse effects were observed in any patient at the end of the follow-up period (at least six months and no more than two years). In the case of enterocutaneous fistulas, all channels were scraped in depth. In the case of rectovaginal fistulas, a vaginal access was used, with the detachment of the posterior vaginal wall. The gap was completely removed and the rectal opening closed with 3/0 absorbable stitches. The rectal mucosa had been damaged by Crohn's disease and was extremely fragile. In the case of perianal fistulas, the main canal was recessed and the rectal orifice was closed with 3/0 absorbable stitches along the sclerotic mucosa.

With the use of a needle, in cases of enterocutaneous fistula, cells were injected into the channel wall. In the cases of rectovaginal and perianal fistulas, cells were injected into the rectal mucosa, close to the internal opening closed with suture. In all cases, a blister filled with fluid was observed over the area of the injection after the injection (Figure 5). The number of cells injected ranged from 3 to 30 x 106, depending on the growth of the cultured cells (Figure 3). The time from the beginning of the preparation of the inoculum to the end of the injection was less than 90 minutes in all cases. In the case of enterocutaneous fistulas, the channels were filled with fibrin glue and then the skin was sutured. In the case of rectovaginal fistulas, an advance vaginal flap was constructed. When auxiliary channels were detected they were also filled with fibrin glue. No bandages were applied postoperatively. Fluid ingestion was started twelve hours after the procedure and after six hours solid food. The patient was discharged from one to three days after surgery and follow-up visits were planned in the outpatient clinic. Two histopathological samples were obtained. A sample (patient number 002) was obtained from the area of an enterocutaneous fistula (7 months after implant n ° 2 and 10 days after implant n ° 3). The other sample (patient number 001) of the rectovaginal wall was obtained one year after the first implant (implant No. 1), during the surgical procedure associated with implant No. 6. The samples were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and evaluated. A weekly follow-up was scheduled for eight weeks after the operation. The patients were considered cured when a total epithelialization of the external opening was evident after - - eight weeks, independently of the previous observations. After eight weeks there was a monthly follow-up for at least six months and no more than two years.

Results Five patients were included in the study and seven liposuctions were performed (figure 3). Patient number 003 was removed from the assay during the implant procedure as a result of the discovery of contamination by Gram-positive bacteria from the cultured lipoaspirated cells. Bacteria were identified as Oerkovia xanthineolytica. An enterocutaneous fistula was removed from the study in patient 002 due to the emergency abdominal operation of a new enterovesicular fistula that had resulted in acute septicemia. Laparotomy required resection of the implant site. Therefore, in this case the monitoring schedule of at least eight weeks could not be fulfilled. Nine fistulas were inoculated from four patients with CMDA after three or fewer passes (figure 3). Eight fistulas were considered adequate for retention in the study and for follow-up for at least eight weeks (Figure 3). In six fistulas, the external opening had epithelialized in week 8 and these fistulas were considered cured (75%) (figure 6). The other eight had only incomplete closure of the external opening with a decrease in the outflow, as reported by the patients (25%, not cured, figure 3). There was no direct relationship between the number of injected cells or culture time and success of the procedure. There was also no direct relationship between the patient's age or sex and healing. Subsequent studies have indicated that an initial dose of 20 x 106 cells is adequate. It has been determined that a second dose of 40 x 106 cells can be used in case the first dose fails. Doses of higher cells are preferred since it has been observed that higher cell numbers have a better therapeutic effect in tissue repair.

Surgical and implantation procedures were performed without additional technical difficulty in the nine treated fistulas. No immediate adverse reactions (for example, anaphylaxis, allergic reactions) were observed in any of the cases studied. Two histopathological samples were obtained seven months (enterocutaneous fistula) and one year (rectovaginal fistula) after surgery. No cytological transformation was detected in a complete series of histopathological sections.

Discussion In a previous report we described the successful cell-based treatment of a young woman with a recurrent rectovaginal fistula who had not responded to medical treatment. Therefore, the present phase I clinical trial was designed to evaluate the feasibility and safety of such transplantation of autologous adipose tissue stromal stem cells (with improvements to the original protocol) for the treatment of fistulas due to Crohn's disease that do not respond to treatment, as well as to test the use of adipose tissue stromal stem cells in conjunction with a fibrin glue. The adipose tissue is chosen as a source of stem cells due to its capacity for myogenic differentiation and the fact that fistulas respond well to muscle transplants. In addition, liposuction fat is available in large quantities and can be collected with minimal adverse effects on the patient. Other groups have used stem cells derived from the bone marrow but, in such cases, a cell mobilization procedure is required which can be dangerous for some patients, such as those with a myocardial infarction. In this study, all liposuction procedures gave a number of clinically useful cells with stem cell characteristics. The patients were followed according to the planned program and a complete cure was observed in 6 of 8 procedures. It is important to note that Crohn's disease provides the worst conditions for a surgical access to fistulas due to the fragility of the tissue and the enormous problems associated with healing in these patients. The patients were chosen because they had not responded to medical treatment or at least two previous surgical procedures, but this treatment seemed to be quite effective. However, new outbreaks of Crohn's disease can still produce new fistulas in any given patient, who will need to be treated again using the patient's cryopreserved antigenic cells. The biological mechanism underlying the therapeutic success of the CMDA transplant is unknown. Stem cells can differentiate into connective, muscular or scar tissue. Alternatively, the secretion of growth factors by stem cells may facilitate the healing of wounds. Typical scar tissue was observed in the histopathologically examined fistulas, but there is no way to distinguish the transplanted cells from the local connective tissue. Complete healing was observed in 75% of the cases using this treatment.

Bibliography The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd ed., Ed. - - by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Patent No. 4,683,195 to Mullis et al .; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds, 1984); Transcription And Translation (B. D.

Hames & S. J. Higgins eds. 1984); Culture of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, volumes 154 and 155 (Wu et al., Eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). All publications and patents mentioned herein, including the items listed below, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definition in this document, will dominate. 1. American Gastroenterological Association Medical Position Statement: Perianal Crohn's Disease. Gastroenterology (2003) 125: 1503-1507. 2. Levy C, Tremaine WJ. Inflamm Bowel Dis (2002) 8 (2): 106-11. - - 3. Pennincke F, D'Hoore A, Filez L. Acta Gastroenterol Belg (2001) 64 (2): 223-226. 4. Rius J, Nessim A, Nogueras JJ, Wexner SD. Eur J Surg (2000) 166 (3): 218-222. 5. Mizuno H, Zuk PA, Zhu M, Lorenz HP, Benhaim P, Hedrick MH.

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Equivalents The present invention provides, among other things, methods and compositions for treating and preventing fistulas. Although specific embodiments of the subject invention have been addressed, the above description is illustrative and not restrictive. Many variations of the invention will be apparent to those skilled in the art in reviewing this - - descriptive memory. The appended claims are not intended to claim all of such embodiments and variations, and the full scope of the invention should be determined by reference to the claims, with their full scope of equivalents, and the specification with such variations.

Claims (55)

1. CLAIMS 1. Composition containing stromal stem cells derived from adipose tissue, wherein at least about 50% of the stromal stem cells derived from adipose tissue comprising the composition express the markers CD9, CD10, CD13, CD29, CD44 , CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105. A composition containing stromal stem cells derived from adipose tissue according to claim 1, wherein at least about 85% of the stromal stem cells derived from adipose tissue comprising the composition express the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105. Composition containing stromal stem cells derived from adipose tissue according to claim 2, wherein at least about 95% of the stromal stem cells derived from adipose tissue comprising the composition express the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105. A composition containing stromal stem cells derived from adipose tissue according to claim 3, wherein at least about 99% of the stromal stem cells derived from adipose tissue comprising the composition express the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105. A composition containing stromal stem cells derived from adipose tissue according to claim 4, wherein less than about 5% of the stromal stem cells derived from adipose tissue comprising the composition express the CD34 markers CD11b, CD14, CD15 , CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and / or CD133. - - 6. Composition containing stromal stem cells derived from adipose tissue according to claim 1, further comprising Ringer's solution and HSA. A composition containing stromal stem cells derived from adipose tissue according to claim 6, wherein the concentration of stromal stem cells derived from adipose tissue comprising the composition is at least about 10 x 10 6 cells / ml. A composition containing stromal stem cells derived from adipose tissue according to claim 7, wherein the concentration of stromal stem cells derived from adipose tissue constituting the composition is at least about 20 x 106 cells / ml. 9. A composition containing stromal stem cells derived from adipose tissue according to claim 1, further comprising an adhesive. 10. A composition containing stromal stem cells derived from adipose tissue according to claim 9, wherein said adhesive is a gel or fibrin glue. 11. Support comprising a composition containing stromal stem cells derived from adipose tissue according to claim 1. 12. Support according to claim 11, wherein said support is a suture. Support according to claim 11, wherein said support is a patch. 14. Method of preparing a composition containing stromal stem cells derived from adipose tissue according to claim 1, comprising: (a) collecting adipose tissue from a subject; (b) obtaining a cell suspension by enzymatic digestion; (c) pellet the cell suspension and resuspend the stromal stem cells derived from adipose tissue in a culture medium; (d) culturing the stromal stem cells derived from adipose tissue for at least about 10 days; and (g) passing the stromal stem cells derived from adipose tissue at least twice. The method of claim 14, wherein the stromal stem cells derived from adipose tissue are cultured for at least 20 days. 16. The method of claim 14, which comprises passing stromal stem cells derived from adipose tissue at least three times. 17. Method of preparing an adhesive for fistula repair, which comprises suspending a composition containing stromal stem cells derived from adipose tissue according to claim 1 with a fibrin-based polymer. 18. Method of treating a fistula in a subject, comprising: (a) closing the internal orifice with a suture and (b) administering at least about 10 x 10 6 stromal stem cells derived from adipose tissue to the inner bore closed with suture . The method of claim 18, wherein the stromal stem cells derived from adipose tissue comprise a composition containing stromal stem cells derived from adipose tissue according to claim 1. Method according to claim 18, further comprising: (d) scrape in depth at least one channel of the fistula; (e) filling said fistula channel with an adhesive. The method of claim 20, further comprising: (f) administering at least about 10 x 10 6 stromal stem cells derived from adipose tissue to the adhesive. 22. The method of claim 20, wherein said adhesive is a gel or fibrin glue. 23. The method according to claim 21, wherein said adhesive further comprises a composition containing stromal stem cells derived from adipose tissue according to claim 1. 24. The method according to claim 18, wherein said method further comprises: (c) administering a second dose of at least about 20 x 10 6 stromal stem cells derived from adipose tissue. 25. The method of claim 18, wherein said method comprises administering at least about 20 x 10 6 stromal stem cells derived from adipose tissue. 26. The method of claim 25, wherein said method further comprises: (c) administering a second dose of at least about 40 x 10 6 stromal stem cells derived from adipose tissue. 27. The method of claim 18, wherein the stromal stem cells derived from adipose tissue are derived from the adipose tissue of the subject. 28. The method of claim 18, wherein the fistula is an anorectal, enteroenteric, enterocutaneous, rectovaginal, or vesicovaginal fistula. 29. The method of claim 18, further comprising administering a therapeutic agent to said subject. 30. The method of claim 29, wherein said therapeutic agent is administered locally to the closed internal orifice with suture. 31. The method of claim 30, wherein said therapeutic agent is administered systhemically to said subject. 32. Method of treating a wound in a subject, comprising: (a) closing the wound with a suture and (b) administering at least about 10 x 10 6 stromal stem cells derived from adipose tissue to the wound closed with suture. 33. The method according to claim 23, wherein the stromal stem cells derived from adipose tissue comprise a cell composition according to claim 1. 34. Stromal stem cell derived from adipose tissue expressing the markers c-Kit, vimentin and CD90 and does not express the markers CD34, factor VIII, alpha-actin, desmin, S-100 and keratin. 35. Stromal stem cell derived from adipose tissue that expresses the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD90 CD90 and CD105 and does not express the markers CD34, CD11b, CD14, CD15 , CD16, CD31, CD34, CD45, CD49f, CD102, CD104, CD106 and / or CD133. 36. Use of stromal stem cells derived from adipose tissue in the manufacture of a pharmaceutical composition for treating a fistula in a subject. 37. Use according to claim 36, wherein said stromal stem cells derived from adipose tissue are derived from the adipose tissue of the subject to be treated. 38. Use according to claim 36, wherein said stromal stem cells derived from adipose tissue comprise a composition containing stromal stem cells derived from adipose tissue according to any one of claims 1 to 10, 34 or 35. 39. Use according to claim 36, wherein said pharmaceutical composition comprises at least about 10 x 10 6 stromal stem cells derived from adipose tissue. 40. Use according to claim 36, wherein said pharmaceutical composition is administered as a first dose to the closed internal orifice of the fistula tract after closing the internal orifice with a suture. 41. Use according to claim 36, wherein said pharmaceutical composition is administered as a first dose to one or more sites in - - the walls of the fistula path after closing the internal hole of the fistula tract with a suture. 42. Use according to claim 40 or 41, wherein said pharmaceutical composition comprises at least about 10 x 10 6 stromal stem cells derived from adipose tissue. 43. Use according to claim 40 or 41, wherein a second dose of said pharmaceutical composition is administered to the closed internal orifice with suture or one or more sites in the walls of the fistula path after said first dose of said pharmaceutical composition. 44. Use according to claim 43, wherein the second dose of said pharmaceutical composition comprises at least about 20 x 10 6 stromal stem cells derived from adipose tissue. 45. Use according to claim 36, wherein said stromal stem cells derived from adipose tissue are comprised in a suture. 46. The use according to claim 36, wherein said stromal stem cells derived from adipose tissue are administered to a material to fill the channel of the fistula. 47. Use according to claim 46, wherein said material is a fibrin-based polymer or an adhesive, such as a gel or fibrin glue. 48. Use according to claim 36, wherein the fistula is an anorectal fistula, fistula of the anus, fecal fistula, arteriovenous fistula, biliary fistula, fistula of the cervix, craniosinus fistula, enteroenteric fistula, enterocutaneous fistula, enterovaginal fistula, gastric fistula, fistula metroperitoneal, perinflaptic fistula, pulmonary arteriovenous fistula, rectovaginal fistula, umbilical fistula, tracheoesophageal fistula or vesicovaginal fistula. 49. Use according to claim 48, wherein the fistula is an anorectal, enterorectal, enterocutaneous, rectovaginal or vesicovaginal fistula. 50. Use according to claim 36, wherein said pharmaceutical composition is administered in combination with a therapeutic agent to the subject to which it is being treated. 51. Use according to claim 50, wherein said therapeutic agent is administered systemically or locally to the site of the suture. 52. Use according to claim 51, wherein said pharmaceutical composition comprises said therapeutic agent. 53. The use of claim 50, wherein said therapeutic agent is administered separately from said pharmaceutical composition comprising said stromal stem cells derived from adipose tissue. 54. The use of claim 50, wherein said therapeutic agent is an anti-inflammatory agent, an immunosuppressive agent, a biological agent, an antibiotic or an antidiarrheal agent. 55. Method of treating a fistula in a subject comprising the steps of: (i) scraping in depth at least one channel of the fistula (ii) closing the internal orifice of the scraped channel with a suture (iii) administering at least about 10 x 10 6 stromal stem cells derived from adipose tissue to the internal orifice closed with suture.