MXPA01008489A - Adipose-derived stem cells and lattices - Google Patents

Abstract

The present invention provides adipose-derived stem cells and lattices. In one aspect, the present invention provides a lipo-derived stem cell substantially free of adipocytes and red blood cells and clonal populations of connective tissue stem cells. The cells can be employed, alone or within biologically-compatible compositions, to generate differentiated tissues and structures, both in vivo and in vitro. Additionally, the cells can be expanded and cultured to produce hormones and to provide conditioned culture media for supporting the growth and expansion of other cell populations. In another aspect, the present invention provides a lipo-derived lattice substantially devoid of cells, which includes extracellular matrix material from adipose tissue. The lattice can be used as a substrate to facilitate the growth and differentiation of cells, whether in vivo or in vitro, into anlagen or even mature tissues or structures.

Description

"• ** - STEM CELLS AND TISSUE DERIVED CELLS ADIPOSE BACKGROUND OF THE INVENTION In recent years, the identification of mesenchytic stem cells, obtained mainly from bone marrow, has led to advances in the regeneration and differentiation of tissues. Such cells are pluripotent cells found in bone marrow and periosteum, and these have the ability to differentiate into various mesenchymal tissues or connective tissues. For example, you can induce such stem cells derived from bone marrow to develop as myocytes after they are exposed to agents such as 5-azacytine [Wakitani et al., Muscl e Nerve, 18 (12), 1417-26 (1995)]. It has been suggested that such cells are useful for repair tissues such as cartilage tissue, adipose tissue and bone tissue (see for example, US Patents Nos. 5,908,784, 5,906,934, 5,827,740, 5,827,735), and that these also have applications through genetic modification (see for example document 5,591,625). Although the identification of such cells has led to advances in tissue regeneration and differentiation, the use of such cells is hampered by various barriers. An impediment to using such cells is the fact that they are very rare (representing as few as 1 / 2,000,000 cells) and make any procedure to obtain and isolate them difficult and expensive. Of course, obtaining bone marrow is extremely painful for the donor. Furthermore, such cells are difficult to grow without differentiation being induced, unless specifically selected batches of serum are used, which adds additional costs and work to the use of such stem cells. Therefore, there is a need for a source of pluripotent stem cells which can be disposed in an easier way, in particular cells that can be grown without the need for costly preselection of the culture materials. Other advances in tissue engineering have shown that cells can be grown in specially defined cultures to produce three-dimensional structures. In general, spatial definition is achieved by using various grids or matrices lacking cells to support and direct cell growth and differentiation. Although this technique is still in its early stages, experiments in animal models have shown that it is possible to use various reticular materials lacking cells to regenerate whole tissues [see for example, Probst et al., BJU In t. , 85 (3), 362-7 (2000)]. An appropriate reticular material is the secreted extracellular matrix material that is isolated from tumor cell lines [see for example, Engelbreth-Holm-Swarm tumor secreted matrix - "matrigel" (matrix secreted from tumor "matrigel")]. This material contains type IV collagen and growth factors, and provides an excellent substrate for cell growth [see eg, Vukice ic et al. , Exp. Cel l Res, 2 02 (1), 1-8 (1992)]. However, because this material also facilitates the malignant transformation of some cells [see for example, Fridman, et al., In t. J. Cán cer, 51 (55), 740-44 (1992)], this material is not suitable for clinical application. Although other artificial reticular materials have been developed, these could prove to be toxic either to cells or to patients when they are used in the first place. Accordingly, there remains a need for a reticular material that is suitable for use as a substrate in the cultivation and development of cell populations.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides stem cells and reticles derived from adipose tissue. In one aspect, the present invention provides a stem cell derived from adipose tissue substantially free of adipocytes and red blood cells and clonal populations of connective tissue stem cells. The cells can be used, alone or with biologically compatible compositions, to generate differentiated tissues and structures, both in vi and vi vi. Additionally, the cells can be expanded and cultured to produce hormones and to provide conditioned culture media that support the growth and expansion of other cell populations. In another aspect, the present invention provides a network derived from adipose tissue substantially free of cells, which includes extracellular matrix material from adipose tissue. The reticle can be used as a substrate to facilitate cells to grow and differentiate, either in vi ve or in vi tro, as an anlage or even as mature tissues and structures. Considering the abundance of adipose tissue, the cells and reticles of the present invention represent an affordable source of pluripotent stem cells. In addition, because the cells can be cultured in undifferentiated state under culture conditions that do not require preselected batches of serum, the cells of the invention can be maintained at significantly lower expense than for the other types of stem cells. These and other advantages of the present invention, as well as additional features of the invention, will be apparent from the accompanying drawings and in the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION One aspect of the invention pertains to a stem cell derived from adipose tissue. Preferably, the stem cell is substantially free of other cell types (for example, adipocytes, red cells, other esthetic cells, etc.) and of extracellular matrix material; more preferred, the stem cell is completely free of such other cell types and matrix material. Preferably, the cells of the invention are obtained from the adipose tissue of a primate, and more preferably from a higher primate (for example a baboon or an ape). Typically, the cell of the invention will be obtained from adipose tissue of human, using methods such as those described in the present invention. Although the cells of the invention can be any type of stem cell, for use in tissue engineering it is desired that the cell be of sodérmico origin. Generally such cells, when isolated, retain two or more phenotypes of esodimeric or mesenchymal development (ie, these are pluripotencial). In particular, such cells generally have the capacity to develop as mesodermal tissues, such as mature adipose tissue, bone tissue, various tissues of the heart (pericardium, epicardium, epimyocardium, myocardium, pericardium, valve tissue, etc.). ), connective dermal tissue, hemangic tissues (eg, corpuscles, endocardium, vascular epithelium, etc.) muscle tissues (including skeletal muscle, cardiac muscles, smooth muscles, etc.), urogenital tissues (eg, kidney, pronephros, ethereal, mesonephically rich conduits, metanephric diverticulum, ureters, renal pelvis, collecting tubules, epithelium of female reproductive tract structures, particularly the oviducts, uterus and vagina), tissues of the pleura and peritoneum, viscera, mesodermal glandular tissues ( for example, tissues of the adrenal cortex) and stromal tissues (for example, bone marrow). Of course, as long as the cell can retain the potential to develop as mature cells, it can also achieve its phenotypic developmental potential by differentiating itself as an appropriate precursor cell (eg, a pre-adipocyte, a pre-myocyte, a pre-adipocyte, a - I do not know, etc.). In addition, depending on the culture conditions, the cells may also present developmental phenotypes such as embryonic, fetal, hematopoietic, neurological, or neuralgiagenic development phenotypes. In this sense, the cell of the invention can have two or more development phenotypes such as the phenotypes of adipogenic, chondrogenic, cardiogenic, dermatogenic, hematopoietic, hemangiogenic, myogenic, nephrogenic, neurogenic, neuralgiagenic, urogeni togenic, osteogenic, pericardiogenic development phenotypes. , peri tonogenic, pleurogenic, splancogenic (creator of viscera), and stromal. Although such cells can retain two or more of these developmental phenotypes, preferably, such cells have three or more such developmental phenotypes (eg, four or more phenotypes of esodimeric or mesenchymal development), and some types of stem cells of the invention have the potential to acquire any mesodermal phenotype through the process of differentiation. The stem cell of the invention can be obtained from adipose tissue by any appropriate method. A first step in any such method requires the isolation of adipose tissue from the source of animal origin. The animal may be alive or dead, as long as the adipose stromal cells within the animal are viable. Typically, human adipose stromal cells are obtained from living donors, using well-recognized protocols such as surgical lipectomy or by suction. Indeed, because liposuction procedures are so common, effluents from liposuction are a particularly preferred source from which the cells of the invention can be obtained. Regardless of how it is obtained, the adipose tissue is processed to separate the stem cells from the remnant of the material. In one of the protocols, the adipose tissue is washed with physiologically compatible saline solution [eg, phosphate buffered saline (PBS)] and then vigorously shaken and allowed to settle, a step that eliminates loose matter (eg, damaged tissue, blood, erythrocytes, etc.) from adipose tissue. Therefore, the washing and sedimentation steps are usually repeated until the supernatant is relatively clean of debris. The remaining cells will generally be present in clumps of various sizes and the protocol proceeds using calculated steps to degrade the coarse structure while at the same time minimizing damage to the cells themselves. One method to achieve this end is to treat the clumps of washed cells with an enzyme that weakens or destroys the junctions between the cells (eg, collagenase, dispase, trypsin, etc.). The amount and duration of such enzymatic treatment will vary, depending on the conditions used, but in general, the use of such enzymes is known in the art. Alternatively or in conjunction with such enzymatic treatment, lumps of cells can be degraded using other treatments, such as mechanical agitation, sonic energy, thermal energy, etc. If degradation is achieved by enzymatic methods, it is desirable to neutralize the enzyme after an appropriate period, to minimize the deleterious effects on the cells. Typically, the degradation step produces a slurry or suspension of aggregated cells (generally liposomes) and a fluid fraction that usually contains free stromal cells (e.g., red blood cells, smooth muscle cells, endothelial cells, fibroblast cells and mother cells) . The next step in the separation procedure is to separate the aggregated cells from the stromal cells. This can be achieved by centrifugation, which forces the stromal cells to become a pellet covered by a supernatant. The supernatant can then be discarded and the pellet suspended in a physiologically compatible fluid. In addition, the suspended cells typically include erythrocytes, and in most protocols it is desirable to use them. Methods for selectively lysing erythrocytes are known in the art, and any suitable protocol (e.g., incubation in a hypertonic or hypotonic medium) can be used. Of course, if the erythrocytes are lysed, then the remaining cells must be separated from the lysed material, for example by filtration or centrifugation. Of course, regardless of whether the erythrocytes are lysed or not, the suspended cells can be washed, re-centrifuged and resuspended, one or more successive times, to obtain a higher purity. Alternatively, the cells can be separated using a cell sorter or based on the size and granularity of the cell, the stem cells being relatively small and non-granular. Telomerase expression can also serve as a specific marker for stem cell. These can also be separated immunohistochemically, for example by visual inspection or using magnetic beads. Any of the steps and methods for isolating the cells of the invention can be performed manually, if desired. Alternatively, the method of isolating such cells can be facilitated by the use of an appropriate device, many of which are known in the art (see, for example, US Patent No. 5, 786, 207). After final isolation and re-suspension, cells can be cultured, and if desired, they can be evaluated for number and viability to evaluate yield. In desired form, the cells can be cultured without differentiation using standard culture media (e.g., DMEM, typically supplemented with 5-15% (e.g., 10%) of serum (e.g., fetal bovine serum, horse serum, etc.) .). Preferably the cells can be passed at least five times in such a medium without differentiation, but while retaining their developmental phenotype, and more preferred, the cells can be passed at least 10 times (per example at least 15 times or even at least 20 times) without losing its development phenotype In this way, it is possible to grow the cells of the present invention without inducing differentiation and without the need for batches of serum especially selected, as is generally the case for mesenchymal stem cells (e.g., those obtained from marrow.) Methods for measuring viability and performance are known in the art (for example mplo, the exclusion method with blue ripán). After isolating them, the stem cells are further separated by identification of phenotypes, to identify those cells that have two or more of the aforementioned development phenotypes. Typically, the stromal cells are plated at a desired density such as between about 100 cells / cm "up to about 100,000 cells / cm" such as for example between about 500 cells / c 2 and 50,000 cells / cm2 or more particularly, between about 1,000 cells / cm2 to about 20,000 cells / cm2). If they are sown at lower densities (for example about 300 cells / cm 2), the cells can be clonally isolated more easily. For example, after a few days, the cells plated at such densities will proliferate as a population. Such cells and populations can be expanded clonally, if desired, using an appropriate method to clone cell populations. For example, a population of cells that have already proliferated can be physically selected and planted on a separate plate (or in the cavity of a multi-cavity plate). Alternatively, the cells can be subcloned into a multi-cavity plate up to a statistical ratio to make it easier to place a single cell in each cavity (e.g., from about 0.1 to about 1 cell / cavity, or even about 0.25 to about 0.5 cells / cavity, such as 0.5 cells / cavity). Of course, the cells can be cloned by plating them at a low density (for example in a Petri dish or other appropriate substrate) and isolating them from the other cells using devices such as rings for cloning. Alternatively, when a radiation source is available, the clones can be obtained by allowing the cells to grow as a monolayer and then cover one of the cells and irradiate the rest of the other cells witthe monolayer. The surviving cell will later grow as a clonal population. Although the production of a clonal population can be expanded in any appropriate culture medium, a preferred culture condition for cloning stem cells (such as the clone cells of the invention or other stem cells) is approximately 2/3 of F12 + medium. % of serum (preferably bovine fetal serum) and about 1/3 of standard medium that has been conditioned with stromal cells (for example, cells from the vascular fraction estomática of the material aspirated by liposuction), determining volumetrically the relative proportions . In any case, whether they are clonal or not, the isolated cells can be cultured to an appropriate point at which their developmental phenotype can be established. As mentioned, the cells of the invention have at least two of the aforementioned development phenotypes. In this way, one or more cells obtained from a given clone can be treated to establish whether or not they possess such development potentials. One type of treatment is to cultivate the cells of the invention in culture media that have been conditioned by exposure to mature cells (precursors thereof) of the respective type to be differentiated (for example, media conditioned by exposure to myocytes can induce myogenic differentiation, media conditioned by exposure to cardiac valve cells can induce differentiation in cardiac valve tissue, etc.). Of course, defined means can also be used to induce differentiation. For example, the phenotype of adipogenic development can be assessed by exposing the cell to a medium that facilitates adipogenesis, for example, that contains a glucocorticoid (such as, for example, 1-butyl-1-methoxant ina, dexamethasone, hydrocortisone, cort sona, et c.), insulin, a compound that raises the intracellular levels of cAMP (for example, dibut i ri 1 -AMPc, 8-CPT-cAMP (8- (4) -clorophophenylthio) -adenosine 3 ', 5' cyclic monophosphate "; 8-bromine-AMPc; dioctioli-AMPc, forskolin, etc.), and / or a compound that inhibits the degradation of cAMP (for example, a phosphodiesterase inhibitor such as meth i 1 -isobutylxin ina, theophylline, caffeine, indomethacin and the like) . Therefore, exposure of the stem cells to an amount of insulin between about 1 μM and about 10 μM in combination with about 10 ~ 9 M to about 10"6 (for example, approximately 1 μM) of dexamethasone can induce adipogenic differentiation. Such a medium can also include, if desired, other agents such as indomethacin (for example, between about 100 μM and about 200 μM), and preferably the medium is serum free. The osteogenic development phenotype can be assessed by exposing the cells to an amount between about 10"7 M and about 10" 9 M dexamethasone (e.g., about 1 μM) in combination with about 10 μM to about 50 μM ascorbate-2. - phosphate and between about 10 nM and about 50 nM of β-glycerophosphate, and the medium can also include serum (for example bovine serum, horse serum, etc.). Myogenic differentiation can be induced by exposing the cells to an amount between about 10 μM and about 100 μM hydrocortisone, preferably in a medium enriched with serum (containing, for example, between about 10% and about 20% serum (either bovine, horse or a mixture thereof)). Chondrogenic differentiation can be induced by exposing the cells to an amount between about 1 μM and about 10 μM 15 insulin and between about 1 μM to about 10 μM transferrin, between about 1 ng / ml and 10 ng / ml factor Transforming growth ßl (TGF-ßl for its acronym in English) and between approximately 10 nM and approximately 20 nM ascorbat or - 2 - fos fat or (50 nM). For chondrogenic differentiation, the cells are preferably cultured with high density (for example at a density of approximately several million cells / ml or using micromass culture techniques), and also in the presence of low amounts of serum (for example between about 1% up to about 5%). Cells can also be induced to acquire a more immature developmental phenotype (for example a fetal or embryonic phenotype). Such induction is achieved by exposing the cells of the invention to conditions that mimic those found in fetuses and embryos. For example, the cells or populations of the invention can be co-cultured with cells isolated from fetuses or embryos, or in the presence of fetal serum. Along with these lines, the cells can be induced to differentiate as any of the aforementioned mesodermal lineages by counting them with mature cells of the respective type, or with precursors thereof. Thus, for example, the myogenic differentiation can be induced by culturing the cells of the invention with myocytes or precursors, and similar results can be achieved with respect to the other types of tissues mentioned in the present invention. Other methods for inducing differentiation are known in the art, and many of these may be employed, as appropriate.

After culturing the cells in the differentiation inducing medium for a period • appropriate (for example several days up to a week or more), the cells can be evaluated to determine if, in fact, they have differentiated or not to acquire physical qualities of a certain type of cell. One measure of differentiation per se is the length of telomeres, undifferentiated stem cells have telomeres • 10 longer than the differentiated cells. Therefore the cells can be evaluated for the level of telomerase activity. Alternatively, RNA or proteins can be extracted from the cells and evaluated (by Northern hybridization, RTPCR, Western blot analysis, etc.), with respect to the presence of markers that • indicate the desired phenotype. Of course, the cells can be tested immunohistochemically or can be stained, using specific stains for tissue. Thus, for example, to evaluate the adipogenic differentiation, the cells can be stained with specific stains for fat (eg, Red O-oil, safarin red, black sweat, etc.), or can probe to assess the presence of factors related to adipose tissue (for example, type IV collagen, PPAR-gamma, adipsin, lipoprotein lipase, etc.). Similarly, osteogenesis can be evaluated by staining the cells with bone-specific stains, for example alkaline phosphates, von Kossa, etc.) or can be probed for the presence of specific markers of bone tissue (eg, osteocalcin). , osteonectin, osteopontin, collagen type I, morphogenic bone proteins, cbfa, etc.). Myogenesis can be assessed by identifying classical morphological changes (eg, polynuclear cells, syncytia formation, etc.), or it can be evaluated biochemically with respect to the presence of muscle-specific factors (eg myo-D, heavy chain). of myosin, NCAM, etc.). Chondrogens can be determined by staining the cells using specific stains for cartilage (eg, blue alcian) or probing the cells with respect to the expression / production of specific cartilage molecules (eg glycosaminoglycans and sulphated proteoglycans (eg, keratin, chondroitin). , etc.) in the middle, type II collagen, etc.). Other methods for evaluating the developmental phenotype are known in the art, and any of these is appropriate. For example, cells can be classified by size and granularity. In addition, the cells can be used to generate monoclonal antibodies, which are then used to evaluate whether or not they bind preferentially to a certain type of cells. The correlation of antigenic character could confirm that the mother cell has differentiated along a certain development trajectory. Although the cell may be solitary and isolated from the others, it is preferably within a population of cells, and the invention provides a defined population that includes the cell of the invention. In some modalities, the population is heterogeneous. Thus, for example, the population may include support cells that provide factors for the cells of the invention. Of course, the stem cells of the invention can themselves serve as support cells for cultivating other types of cells (such as other types of stem cells, for example, such as neuronal stem cells (NSC)). , hematopoietic stem cells (HPC, in particular CD34 + stem cells), embryonic cells, (ESC) and mixtures thereof), and the population could include such cells. In other embodiments, the population is substantially homogeneous, consisting essentially of the stem cells of the invention obtained from the adipose tissue. Because the cells of the invention can be cloned, a substantially homogeneous population containing the cells could be clonal. Indeed, the invention also relates to any defined population consisting essentially of mesodermal stem cells, connective tissue stem cells, or mixtures thereof. In this embodiment, cells can be derived from adipose tissue or derived from other mesodermal tissues or connective cell tissues (e.g., bone marrow, muscle, etc.) using methods known in the art. After isolating them, the cells can be expanded in clonal form as described in the present invention. The cells of the invention (and the cell populations) can be used for a variety of purposes. As already mentioned, cells can support the growth and expansion of other cell types, and the invention pertains to methods to achieve this end. In one aspect, the invention pertains to a method of conditioning the culture medium using the stem cells of the invention and the conditioned medium produced by said method. The medium is conditioned by exposing a desired culture medium to the cells under conditions sufficient for the cells to condition it. Typically, the medium is used to support the growth of the cells of the invention, which secrete hormones, cell matrix material and other factors into the culture medium. After an appropriate period (for example, from one to a few days), the culture medium containing the secreted factors can be separated from the cells and stored for future use. Of course, the cells and populations of the invention can be successfully reused to condition the medium, as desired. In other applications (for example, to co-culture the cells of the invention with other cell types), the cells can remain within the conditioned medium. The invention therefore provides a conditioned medium that is obtained using this method, which may contain the cells of the invention or may be substantially free of the cells of the invention, as desired. The conditioned medium can be used to support the growth and expansion of the desired cell types, and the invention provides a method for growing cells (in particular stem cells) using the conditioned medium. The method involves maintaining a desired cell in the conditioned medium under conditions for the cell to remain viable. The cell can be maintained under any suitable condition for cultivation, such as those known in the art. In desired form, the method allows successive rounds of mitotic division of the cell to form an expanded population. The exact conditions (for example, temperature, C02 levels, agitation, presence of antibiotics, etc.) will depend on the other constituents of the medium and the type of cell. However, the optimization of these parameters is within the ordinary skill in the art. In some embodiments, it is desirable that the medium be substantially free of cells derived from adipose tissue used to condition the medium as described in the present invention. However, in other embodiments, it is desired that cells derived from adipose tissue remain in the conditioned medium and cocultivate with the cells of interest. Indeed, because the cells derived from adipose tissue of the invention can express cell surface mediators of intercellular communication, it is often desired that the cells of the invention and the other desired cells are co-cultured under conditions in which Two types of cells are in contact. This can be achieved, for example, by seeding the cells as a heterogeneous population of cells on a suitable substrate for culture. Alternatively, the cells of the invention derived from adipose tissue can be grown first to confluence, which serve as a substrate for the second desired cells to be cultured under the conditions of the medium. In another embodiment, adipose-derived cells of the invention can be genetically modified, for example, to express exogenous genes or to repress the expression of endogenous genes, and the invention provides a method for genetically modifying such cells and populations . According to this method, the cell is exposed to a vector for gene transfer comprising a nucleic acid that includes a transgene, in such a way that the nucleic acid is introduced into the cell under appropriate conditions for the transgene to be expressed inside the cell. Generally, the transgene is an expression cassette that includes a coding polynucleotide linked in operable form to an appropriate promoter. The encoding polynucleotide may encode a protein, or it may code for biologically active RNA (e.g., antisense RNA or a ribozyme). In this way, for example, the encoding polynucleotide can encode a gene that confers resistance to a toxin, a hormone (such as peptide growth hormones, hormone releasing factors, sex hormones, adrenocorticotropic hormones, cytokines (eg, interferins, interleukins', lymphokines), etc.), a portion for intracellular signaling bound to the cell surface (eg, cell adhesion molecules, hormone receptors, etc.), a factor that promotes a lineage determined of differentiation, etc. Of course, in cases where it is desired to use gene transfer technology to deliver a given transgene, its sequence will be known. Within the expression cassette, the encoding polynucleotide is operably linked to an appropriate promoter. Examples of suitable promoters include prokaryotic promoters and viral promoters (e.g., ret roviral ITRs, LTRs, immediate initial viral promoters (IEp), such as herpes virus IEp (e.g. ICP4-IEp and ICPO- IEp), IEp of cytomegalovirus (CMV) and other viral promoters, such as the Rous Sarcoma Virus (RSV) promoters and the Murmo Leukemia Virus promoters). Other suitable promoters are eukaryotic promoters, such as enhancers (e.g., rabbit ß-globma regulatory elements), constitutively active promoters (e.g., the β-actma promoter, etc.), signal-specific promoters (e.g., inducible promoters such as a promoter that responds to RU486 , etc.), and tissue-specific promoters. It is within the field of the art to select an appropriate promoter to drive the expression of genes in a predefined cellular context. The expression cassette may include more than one encoding polynucleotide, and this may include other elements (eg, polyadenylation sequences, sequences encoding for a membrane insertion signal or a secretion leader, ribosome entry sequences, transcriptional regulatory elements (eg enhancers, silencers, etc.), and the like, as desired.The expression cassette containing the transgene must be incorporated into a suitable genetic vector to deliver the transgene to the cells. desired final application, any of such vectors can also be used in the same way to genetically modify the cells (eg, plasmids, naked DNA, viruses such as adenovirus, adeno-associated virus, herpes virus, lentivirus, papi lomavirus, retrovirus , etc.) Any construction method can be used for the desired expression cassette within s vectors, of which many are known in the art (e.g., direct cloning, homologous recombination, etc.). Of course, the choice of vector will largely determine the method used to introduce the vector into cells (e.g., by protoplast fusion, calcium phosphate precipitation, gene gun, electroporation, infection with viral vectors, etc.) , which are generally known in the art. Genetically altered cells can be used as bioreactors to produce the product of a transgene. In other embodiments, genetically modified cells are used to deliver the transgene and its product to an animal. For example, cells, once genetically modified, can be introduced into the animal under sufficient conditions for the transgene to be expressed in vivo. In addition to serving as useful targets for genetic modification, many of the cells and populations of the present invention secrete hormones (e.g., cytokines, peptide or other growth factors (e.g., monobutyrin), etc.). Some of the cells naturally secrete such hormones after initial isolation, and other cells can be genetically modified to secrete hormones, as discussed in the present invention. The cells of the present invention that secrete hormones can be used in a variety of contexts in vi and in vi tro. For example, such cells can be used as bioreactors to provide an accessible source of a given hormone, and the invention pertains to a method for obtaining hormones from said cells. According to the method, the cells are cultured under appropriate conditions so that they secrete the hormone in the culture medium. The medium is harvested after an appropriate time interval, and preferably periodically, and processed to isolate the hormone from the medium. To purify the hormone from the medium, any standard method (eg, gel or affinity chromatography, dialysis, ligation, etc.) can be used, many of which are known in the art. In other embodiments, the cells (and populations) of the present invention that secrete hormones can be used as therapeutic agents. In general, such methods involve the transfer of the cells to the desired tissue, either in vi tro (for example, as a graft before implanting or grafting) or in vivo, to an animal tissue directly. The cells can be transferred to the desired tissue by any appropriate method, which will generally vary in accordance with the type of tissue. For example, the cells can be transferred to a graft by bathing the graft (or infusing it) with culture medium containing the cells. Alternatively, the cells can be seeded into the desired site within the tissue to establish a population. The cells can be transferred to the sites in vi ve using devices such as catheters, trocars, cannulae, stents (which can be seeded with the cells), etc. For these applications, the cell preferably secretes a cytosine or a growth hormone such as human growth factor, fibroblast growth factor, nerve growth factor, insulin-like growth factors, hematopoietic stem cell growth factors. , members of the fibroblast growth factor family, members of the platelet-derived growth factor family, vascular or endothelial cell growth factors, members of the TGFb family (including bone morphogenic factor), or specific enzymes for congenital disorders (for example dystrophin). In one application, the invention provides a method for promoting the closure of a wound within a patient using such cells. According to the method, the cells of the invention that secrete the hormone are transferred to the vicinity of a wound under conditions sufficient for the cell to produce the hormone. The presence of the hormone in the vicinity of the wound promotes the closure of the wound. The method promotes the closure of wounds both external (for example on the surface) and internal. Wounds for which the method of the present invention is useful for promoting closure include but are not limited to scrapes, avulsions, blow wounds, burn wounds, bruises, gunshot wounds, cut wounds, open wounds, penetrating wounds, perforation wounds, aggression wounds, stab wounds, surgical wounds, subcutaneous wounds, or tangential wounds. The method does not need to achieve complete healing or closure of the wound; it is sufficient that the method promotes any degree of wound closure. In this sense, the method can be used alone or as an auxiliary for other methods for the healing of injured tissue. In the cases in which the cells of the invention secrete an angiogenic hormone (for example, vascular growth factor, vascular and endothelial cell growth factor, etc.), these (as well as the populations that contain them) can be Use to induce angiogenesis within tissues. In this way, the invention provides a method for promoting neovascularization within the tissue using said cells. In accordance with this method, the cell is introduced into the desired state under conditions sufficient for the cell to produce the angiogenic hormone. The presence of the hormone within the tissue promotes neovascularization within the tissue. Because the stem cells of the invention have a developmental phenotype, they can be used in tissue engineering. In this regard, the invention provides a method for producing animal matter which comprises keeping the cells of the invention under sufficient conditions for them to expand and differentiate to form the desired material. The material may include mature tissues, or even whole organs, including tissue types in which the cells of the invention can be differentiated (as indicated above in the present invention). Typically, such material will comprise adipose tissue, cartilage tissue, heart tissue, connective tissue of the dermis, blood tissue, muscle tissue, kidney, bone, pleural, visceral or vascular tissue and the like. More typically, the material will comprise combinations of these types of fabric (e.g., more than one type of fabric). For example, the material may comprise all or a portion of an animal organ (e.g., a heart, a kidney) or a limb (e.g., a leg, a wing, an arm, a hand, a foot, etc.). . Of course, as long as the cells can be divided and differentiated to produce such structures they can also form the anlage of such structures. In the initial stages, such angling could be conserved by cryogenesis to generate in the future said mature structure or desired organ. To produce such structures, the cells and populations of the invention are maintained under appropriate conditions so that they expand and divide to form the desired structures. In some applications, this is accomplished by transferring the cells to an animal (i.e. in vi vo) typically at a site where the new material is desired. Thus, for example, the invention can make it easier to regenerate tissues (e.g., bone, muscle, cartilage, tendon tissue, adipose, etc.) within an animal in which the cells are implanted in such tissues. In other modalities, and in particular to create anlage, the cells can be induced to differentiate and expand into tissues. In such applications, cells are grown on substrates that facilitate formation in three-dimensional structures that lead to tissue development. In this way, for example, the cells can be cultured or seeded on a biocompatible network, such as one that includes extracellular matrix material, synthetic polymers, cytokines, growth factors, etc. Such a grid can be molded into the desired shapes to facilitate the development of • types of tissue. In addition, at least at an early stage during such a culture, the medium and / or the substrate are supplemented with factors (eg, growth factors, cytokines, extracellular matrix material, etc.) that make it easier to develop the types and structures • 10 appropriate tissue. Indeed, in some embodiments, it is desired to co-culture the cells with mature cells of the respective tissue type, or precursors thereof, or to expose the cells to the respective conditioned medium, as discussed in the present invention. To facilitate the use of cells and populations derived from adipose tissue of the invention to produce such animal matter and tissues, the invention provides a composition that includes the cells (and populations) of the invention and the biologically compatible network. In general, the grid is formed from polymeric material, having fibers in the form of a mesh or sponge, with spaces typically of the order of 25 between about 100 μm and about 300 μm. Such a structure provides a sufficient area over which the cells can grow and proliferate. In desired form, the reticle is biodegradable with time, in such a way that it will be absorbed in the animal matter as it develops. Therefore, appropriate polymeric lattices can be formed from monomers such as glycolic acid, lactic acid, propyl fumarate, caprolactone, hyaluron, hyaluronic acid and the like. Other lattices may include proteins, polysaccharides, polydihydroxylic acids, polyaldoses, polydides, polyphosphazenes, or synthetic polymers (in particular biodegradable polymers). Of course, an appropriate polymer to form such a lattice may include more than one monomer (for example, combinations of the monomers indicated). In addition, the grid may also include hormones, such as growth factors, cytokines and morphogens (eg, retinoic acid, arachidomic acid, etc.), molecules of the desired extracellular matrix (eg, fibronectm, lammine, collagen, etc.). ), or other materials (eg, DNA, viruses, other cell types, etc.) as desired. To form the composition, the cells are introduced into the grid in such a way that they, by permeation, occupy the interstitial spaces therein. For example, the matrix can be immersed in a solution or suspension containing the cells, or these can be applied to the matrix by infusion or injection. A particularly preferred composition is a hydrogel formed by interlacing a suspension that includes the polymer and that also has the cells of the invention dispersed therein. This method of formation allows the cells to disperse throughout the lattice, facilitating a more uniform permeation of the lattice with the cells. Of course, the composition may also include mature cells of a desired phenotype or precursors thereof, in particular to enhance the induction of the stem cells of the invention so that they are appropriately differentiated within the lattice (e.g. effect of co-culturing such cells within the reticulum). The composition can be used in any suitable form to facilitate the growth and generation of the desired tissue types, structures, or anlage. For example, the composition can be constructed using three-dimensional or stereotactic modeling techniques. Thus, for example, one layer or domain within the composition may be populated by mast cells for osteogenic differentiation, and another layer or domain within the composition may be populated with mast cells for myogenic and / or chondrogenic development. Juxtaposing such domains with one another facilitates the molding and differentiation of complex structures including multiple tissue types (e.g., bone surrounded by muscle, such as found in a limb). To direct the growth and differentiation of the desired structure, the composition can be grown ex vvo in a bioreactor or incubator, as appropriate. In other embodiments, the structure is implanted directly into the host animal at the site in which it is desired to develop the tissue or structure. Even in another embodiment, the composition can be grafted to a host (typically an animal such as a pig, baboon, etc.) where it will grow and mature until ready to be used. After this, the mature structure (or anlage) is removed from the host and implanted in the host, as appropriate. Appropriate lattices for inclusion in the composition can be obtained from any suitable source (eg, matrigel), and there are some commercial sources from which appropriate lattices can be obtained (for example, appropriate lattices of polyhydric acid can be obtained). icolico from sources such as Ethicon, NJ). Another appropriate network can be obtained from the adipose tissue-free cell portion - i.e. adipose tissue extracellular matrix material substantially free of cells, and the invention provides such a network derived from adipose tissue. Typically, such adipose-derived lattices include proteins such as protoglycans, glycoproteins, hyaluronts, fibrils, collagens (type I, type II, type III, type IV, type V, type VI, etc.), and similar, which serve as excellent substrates for cell growth. Additionally, such adipose tissue derived lattices may include hormones, preferably cytokines and growth factors, to facilitate the growth of cells seeded in the matrix. The matrix derived from adipose tissue can be isolated from adipose tissue in a manner similar to that described above, with the exception that it will be present in the cell-free fraction. For example, the adipose tissue or derivatives thereof (eg, a fraction of the cells after centrifugation, as discussed above), can be subjected to thermal or sonic energy and / or enzymatic processing to recover the matrix material. Furthermore, it is desired that the cellular fraction of adipose tissue be broken, for example by treating it with lipases, detergents, proteases and / or by mechanical or sonic breaking (using for example, a homogenizer or a sonicator): No matter how it is isolated , the material is initially identified as a viscous material, but this may be subsequently treated, as desired, depending on the intended end use. For example, the raw matrix material can be treated (eg, by subjecting it to dialysis or it can be treated with proteases or acids, etc.) to produce an appropriate lattice material. In this way, the lattice can be prepared in a hydrated form or it can be dried or lyophilized in a substantially anhydrous form or powder. After this, you can rehydrate the powder to use it as • a substrate for cell culture, suspending it for example in an appropriate cell culture medium. In this sense, the reticulum derived from adipose tissue can be mixed with other suitable lattice materials, such as those described above. Of course, the The invention pertains to compositions that include the lattice derived from adipose tissue as well as to other cells (in particular other types of stem cells). As discussed earlier, they can be used the cells, populations, reticules and compositions of the invention in the engineering and regeneration of • fabrics. Therefore, the invention pertains to a structure that can be implanted (i.e., an implant) that incorporates any of these characteristics of the invention. The exact nature of the implant will vary according to the use in which it will be placed. The implant may be, or may comprise, as described, mature tissue, or it may include immature tissue and the reticle. Thus, for example, a type of implant can be a bone implant, comprising a population of the cells of the invention that are being subjected to (or primed for) osteogenic differentiation, seeded within a lattice of an appropriate size and dimension, as described above. Such an implant can be injected or grafted into a host to promote the generation or regeneration of mature bone tissue within a patient. Similar implants can be used to promote the growth or regeneration of muscles, fat, cartilage, tendons, etc., within patients. Another type of implants are the anlage (such as those described in the present invention), for example, limb buds, digital buds, developing kidneys, etc., which, once grafted into the patient, will mature as the appropriate structures. Conveniently, the reticulum derived from adipose tissue can be used as part of a cell culture case. Accordingly, the invention provides a kit including the adipose tissue-derived network of the invention and one or more other components, such as moisturizing agents (eg, water, physiologically compatible salt solutions, prepared culture media, serum or derivatives thereof, etc.), substrates for cell culture (eg, boxes, plates, flasks for cell culture, etc.), media for cell cultures (whether in liquid or powder form), antibiotic compounds, hormones and similar. Although the kit may include any such ingredients, it preferably includes all the ingredients necessary to support the culture and growth of the desired cell types after combining them appropriately. Of course, if desired, the kit can also include cells (typically frozen), which can be seeded in the grid as described in the present invention. Although many aspects of the invention relate to tissue growth and differentiation, the invention also has other applications. For example, the reticulum derived from adipose tissue can be used as an experimental reagent, such as in the development of improved grids and substrates for tissue growth and differentiation. The reticulum derived from adipose tissue can also be used in cosmetic applications, for example, to hide wrinkles, scars, subcutaneous depressions, etc., or to increase tissues. For such applications, the grating is preferably stylized and packed into unit dosage forms. If desired, it can be mixed with vehicles (for example, solvents such as glycerin or alcohols), perfumes, antibiotics, colorants, and other ingredients commonly used in cosmetic products. The substrate can also be used in an autologous form or as an allograft, and it can be used as, or be included in, ointments or bandages to facilitate healing wounds. Cells derived from adipose tissue can also be used as experimental reagents. For example, these can be used to help discover agents responsible for the initial events in the differentiation. For example, the cells of the invention can be exposed to a means for inducing a particular line of differentiation and then tested for differential expression of genes (for example by PCR with random primers, or elect oforesis or protein or RNA, etc.) . As for any of the steps for isolating the stem cells or the reticulum derived from adipose tissue of the invention, it provides a kit for isolating such reagents from adipose tissues. The kit can include means for isolating adipose tissue from a patient (e.g., cannula, needle, aspirator, etc.), as well as means for separating the stromal cells (e.g., by the methods described in the present invention). ). For example, the kit can be used as a source of stem cells on one side of the bed which can then be reintroduced by the same individual as appropriate. Therefore, the kit can facilitate the isolation of stem cells derived from adipose tissue to be implanted in a patient in need of regeneration of a desired type of tissue, even in the same procedure. In this regard, the kit can also include a means for differentiating the cells, such as those indicated in the present invention. As appropriate, the cells may be exposed to the medium to prime them to differentiate within the patient as necessary. Of course, the kit can be used as a convenient source of stem cells for in vi tro manipulation (eg, for cloning or differentiation as described in the present invention). In another embodiment, the kit can be used to isolate a grating derived from adipose tissue as described in the present invention EXAMPLES Although one skilled in the art is fully capable of practicing the present invention after reading the above detailed description, the following examples will help elucidate some of its features. In particular, these demonstrate the isolation of a stem cell derived from human adipose tissue substantially free of mature adipocytes, the isolation of a clonal population of such cells, the ability of such cells to differentiate themselves in vi v ein vi tro, and the ability to said cells to support the growth of other types of stem cells. The examples also demonstrate the isolation of a lattice derived from adipose tissue substantially free of cells which can serve as a suitable substrate for cell culture. Of course, because these examples are presented for illustrative purposes only, they should not be used to limit the scope of the invention in any way, but rather should be seen as expanding that field after the previous description of the invention. invention as a whole. The methods used in these examples, such as surgery, cell culture, enzymatic digestion, histology, and molecular analysis of proteins and polyucleotides, are familiar procedures for those skilled in the art. As such, and in the interest of brevity, the experimental details are not described in detail.

EXAMPLE I This example demonstrates the isolation of a stem cell derived from human adipose tissue substantially free of mature adipocytes. Raw aspirated liposuction material was obtained from patients undergoing optional surgery. Before initiating liposuction procedures, patients were given epinephrine to minimize contamination with blood from the aspirated material. The aspirated material is cast to separate the associated adipose tissue pieces from the associated liquid waste. The isolated tissue was thoroughly washed with neutral phosphate buffered saline and then enzymatically dissociated with 0.075% p / v collagenase at 37 ° C for approximately 20 minutes under intermittent agitation. After digestion, the collagenase was neutralized, and the suspension was centrifuged at approximately 260g for about 10 minutes, which produced a supernatant with multiple layers and a cell tablet. The supernatant was removed and stored for later use, and the tablet was resuspended in a solution to lyse the erythrocytes and incubated without agitation at approximately 25 ° C for about 10 minutes. After incubation, the medium was neutralized and the cells were again centrifuged at approximately 250g for approximately 10 minutes. After the second centrifugation, the cells were suspended and evaluated for viability (using trypan blue exclusion) and cell number. After this, the cells were plated at a density of up to about 1 x 10 6 cells / 100 mm disc. These were cultured at 37 ° C in DMEM + bovine fetal serum (approximately 10% in approximately 5% C02.) Most of the cells were small, mononuclear, relatively agranular, fibroblast-like, adherent cells that did not contain visible droplets. Lipids Most cells showed negative staining with O-red oil and von Kossa staining Cells were also evaluated for telomerase expression (using a commercially available TRAP test kit), using HeLa cells and cells HN-12 as positive controls Human foreskin fibroblasts and HN-12 cell extracts subjected to heat were used as negative controls Telomeric products were resolved in a 12.5% polyacrylamide cell and the signals were determined by formation of phosphorus images Telomeric ladders representing telomerase activity in stem cells derived were observed s of adipose tissue as well as positive controls. No stairs were observed in the negative controls. Thus, these cells could not be identified as myocytes, adipocytes, chondrocytes, osteocytes, or erythrocytes. These results demonstrate that cells derived from adipose tissue express telomerase activity similar to that previously reported for human stem cells. Subsequently, subpopulations of these cells were exposed to the following means to determine their development phenotype: A high density population was cultured in the chondrogenic medium for several weeks. Histological analysis of the tea culture and sections in paraffin was performed with H &E, alciano blue, toludeno blue, and Goldner's trichrome staining at 2, 7, and 14 days. Immunohistochemistry tests were performed using antibodies against chondroitin 4-sulfate, and keratin sulfate and type II collagen. A quantitative estimate of matrix staining was also performed. The results indicate that cartilaginous spheroidal nodules with a distinct edge of perichondrial cells formed as early as 48 hours after the initial treatment. The control cells without treatment showed no evidence of chondrogenic differentiation. These results confirm that the stem cells have a chondrogenic development phenotype. A population was grown to near confluence and then exposed to the adipogenic medium for several weeks. The population was examined at two and four weeks after plating by colorimetric evaluation of the relative opacity after staining with oil-red 0. It was determined that the adipogenesis was underway at two weeks and that it was fully advanced at four weeks (relative opacity of 1 and 5.3 respectively). Stem cells obtained from bone marrow were used as a positive control, • and these cells presented slightly lower adipogenic potential (relative density of 0.7 and 2.8 respectively). A population was grown to near confluence and then exposed to the osteogenic medium for several weeks. The population examined at two and four weeks after plating them by colorimetric evaluation of the relative opacity after von Kossa staining. It was determined that osteogenesis was underway at two weeks and which was fully advanced at four weeks (relative opacity of 1.1 and 7.3 respectively). Stem cells obtained from bone marrow were used as a positive control, and these cells presented osteogenic potential slightly lower (relative density of 0.2 and 6.6 respectively). A population was grown to near confluence and then exposed to the myogenic medium for several weeks. The population was examined at the first, third and sixth weeks after plating them by evaluation of the muted t cells and the expression of muscle-specific proteins (MioD and myosin heavy chain). Foreskin fibroblasts and skeletal human myoblasts were used as controls. Cells expressing MioD and myosin were found at all times after exposure to the myogenic medium in the stem cell population, and the proportion of such cells increased at 3 and at 6 weeks. Inunucleated mute t cells were observed at 6 weeks. In contrast, fibroblasts did not exhibit any of these characteristics at any time. These results demonstrate the isolation of a pluripotent stem cell derived from human adipose tissue substantially free of mature adipocytes.

EXAMPLE 2 This example demonstrates that stem cells derived from adipose tissue do not differentiate in response to 5-azacytine. Stem cells derived from adipose tissue obtained according to example 1 were cultured in the presence of 5-azacytine. In contrast to stem cells derived from bone marrow, exposure to this agent does not induce myogenic differentiation (see Akitani et al., S upra).

EXAMPLE 3 This example demonstrates how to generate a clonal population of stem cells derived from adipose tissue. Isolated cells were plated in accordance with the procedure indicated in Example 1 at approximately 5,000 cells / 100 mm box and cultured for a few days as indicated in Example 1. After a few rounds of cell division, some of the clones were chosen with a ring for cloning and transferred to cavities in a 48 cavity plate. These cells were cultured for several weeks, changing the medium twice a week, until they were approximately between 80% and 90% confluence (at 37 ° C in about 5% C02 in 2/3 of F12 medium + 20% of fetal bovine serum and 1/3 of standard medium that was first conditioned by the cells isolated in Example 1, "medium for cloning"). After this, each culture was transferred to a 35 mm box and allowed to grow, and then transferred back to a 100 mm box and allowed to grow to near confluence. After this, the population of one cell was frozen, and the rest of the populations were plated in plates of 12 cavities, at a density of 1,000 cells / well. The cells were cultured for more than 15 passages in medium for cloning and monitored for differentiation as indicated in example 1. The undifferentiated state of each clone remained constant after successive rounds of differentiation. The populations of the clones were then established and exposed to an adipogenic, chondrogenic, myogenic and osteogenic medium as discussed in example 1. It was observed that at least one of the clones could be differentiated as bone, fatty tissue, cartilage and muscle. when exposed to the respective media, and that most of the clones could be differentiated into at least three types of tissues. The ability of cells to develop as muscle or cartilage also demonstrates the pluripotential character of these stem cells derived from adipose tissue. These results demonstrate that the stem cells derived from adipose tissue can be maintained in an undifferentiated state during many passages without the requirement of pre-selected serum batches in a special form. The results also show that the cells retain their pluripotential character after said intensive passage action, proving that the cells are in fact stem cells and not only obligate precursor cells.

EXAMPLE 4 This example demonstrates that stem cells derived from adipose tissue can support the culture of other types of stem cells. Stem cells derived from human adipose tissue were transferred to 96-well plates at a density of about 30,000 cells / well, cultured for a week and then irradiated. Human CD34 + hematopoietic stem cells isolated from umbilical cord blood were then seeded into the cavities. Co-cultures were maintained on MyeloCult H5100 media, and viability and cell proliferation were monitored subjectively by microscopic observation. After two weeks of co-culture, hematopoietic stem cells were evaluated for CD34 expression by flow cytometry. • 10 After a period of co-culture with static cells, the hematopoietic stem cells formed large colonies of round cells. Flow analysis revealed that 62% of the cells remained CD34 +. Taking based on microscopic observations, stromal cells derived from adipose tissue • human survival and supported the growth of hematopoietic stem cells derived from umbilical cord blood. 20 These results demonstrate that stromal cells from human subcutaneous adipose tissue can support the maintenance, growth and differentiation ex vi ve of other stem cells. 25 EXAMPLE 5 This example demonstrates that stem cells derived from adipose tissue can be differentiated in vi vo. Four groups (AD) of 12 thymus-free mice were implanted subcutaneously with hydroxyapatite / tricalcium phosphate buckets containing the following: Group A contained adipose-derived stem cells that had previously been treated with osteogenic medium as indicated in Example 1. Group B contained stem cells derived from untreated adipose tissue. Group C contained osteogenic medium but not cells. Within each group 6 mice were sacrificed at three weeks and the remaining mice were sacrificed at eight weeks after implantation. The cubes were removed, fixed, decalcified and sectioned. Each section was analyzed for osteocalcin by staining with H & E, Mallory's bone stain and immunot inc. Different regions of osteoid tissue were observed, which show staining for osteocalcin and Mallory bone staining in sections from groups A and B. Substantially more osteoid tissue was observed in groups A and B than in the other groups (ANOVA de p <0.05), but no significant difference in osteogenesis was observed between groups A and B. In addition, a qualitative increase in tissue growth was noted in both groups A and B between 3 and 8 weeks. These results show that stem cells derived from adipose tissue can be differentiated i n vi EXAMPLE 6 This example demonstrates the isolation of a reticulum derived from adipose tissue substantially free of cells. In one protocol, the supernatant retained from Example 1 is subjected to enzymatic digestion for three days in trypsin-EDTA 0.05% / 100 U / ml deoxyribonuclease to destroy the cells. The residue is rinsed daily in saline and a new enzyme is added. After this, the material is rinsed in saline and resuspended in 0.05% collagenase and approximately 0.1% lipase to partially digest the proteins and fats present. This incubation is continued for two days. In another protocol, the retained supernatant from Example 1 was incubated in EDTA to remove any of the epithelial cells. The remaining cells were lysed using a buffer solution containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 0.4 M NaCl, 50 mM Tris-HCl (pH 8) and protease inhibitors and 10 μg / ml of each leupeptin, chemostatin, antipain and pepstatin A. Finally, the tissue was washed exhaustively in PBS without divalent cations. After both preparation protocols, the remaining substance was washed and identified as a gelatinous mass. The microscopic analysis of this material revealed that it did not contain cells, and that it was composed of high amounts of collagen (probably type IV) and a wide variety of growth factors. Preparations of this material have supported cell growth, demonstrating that it is an excellent substrate for tissue culture.

Claims (10)

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the content of the following is claimed as property: CLAIMS

1. - A stem cell derived from mammalian adipose tissue, substantially free of mature adipocytes.

2. The cell according to claim 1, further characterized in that it can be cultured in DMEM + about 10% fetal bovine serum during at least 15 passages without differentiation.

3. - The cell according to claim 2, further characterized by having two or more development phenotypes that are selected from the group of phenotypes of development consisting of phenotypes of adipogenic, chondrogenic, cardiogenic, dermatogenic, hematopoietic, hemangiogenic, myogenic, nephrogenic, neurogenic, neuralgiagenic, urogeni togenic, osteogenic, pericardiogenic, peri tonic, pleurogenic, splancogenic, and stromal.

4. The cell according to any of claims 1-3, further characterized because it is human.

5. - The cell according to any of claims 1-4, further characterized in that it is genetically modified

6. The cell according to any of claims 1-5, further characterized in that it has an intracellular signaling portion attached to the cell. cell surface.

7. The cell according to any of claims 1-5, further characterized in that it secretes a hormone.

8. The cell according to claim 7, further characterized in that the hormone is selected from the group of hormones consisting of cytokines and growth factors.

9. A defined cell population comprising a cell according to any of claims 1-8.

10. The cell population defined according to claim 9, further characterized because it is heterogeneous.

11. The cell population defined according to claim 9 or claim 10, further characterized in that it comprises a stem cell that is selected from the group consisting of neuronal stem cells (NSC), hematopoietic stem cells (HPC), cells embryonic stem, (ESC) and mixtures thereof.

• 12. The cell population defined according to claim 9, further characterized in that it consists essentially of cells according to any of the claims

13. The cell population defined in accordance with claim 9 or with the

• claim 12, also characterized because it is substantially homogeneous. 14.- The defined cell population of

20 according to claim 13, further characterized in that it is clonal. 15. A defined cell population consisting essentially of mesodermal stem cells (MHC), tissue stem cells

25 connective (CTSC), or mixtures thereof, also characterized because the population is clonal. 16. The population according to claim 15, further characterized in that the

• Stem cells have two or more developmental phenotypes that are selected from the group of developmental phenotypes consisting of adipogenic, chondrogenic, cardiogenic, dermatogenic, hematopoietic, hemangiogenic, myogenic, nephrogenic, developmental phenotypes,

10 neurogenic, neuralgiagenic, urogeni togenic, osteogenic, pericardiogenic, peri tonic, pleurogenic, splancogenic, and stromal 17.- A reticulum derived from adipose tissue that comprises matrix matter

15 extracellular adipose tissue substantially free of cells. 18. The reticule derived from adipose tissue according to claim 17, further characterized in that it comprises a protein

20 of human, proteoglycan, glycoprotein, hyaluronin or a fibronectin molecule. 19. The reticule derived from adipose tissue according to claim 17 or claim 18, further characterized in that

25 comprises a collagen selected from the group of collagens consisting of collagens of type I, type II, type III, type IV, type V, type VI. 20. The reticulum derived from adipose tissue according to any of claims 17-19, further characterized in that it comprises a hormone. 21. The reticulum derived from adipose tissue according to claim 20, further characterized in that the hormone is selected from the group of hormones consisting of cytokines and growth factors. 22. The reticule derived from adipose tissue according to any of claims 17-21, characterized in addition because it is substantially anhydrous. 23. The reticulum derived from adipose tissue according to any of claims 17-22, further characterized because it is lyophilized. 24. The reticulum derived from adipose tissue according to any of claims 17-21, further characterized in that it is hydrated 25.- A kit comprising the adipose-derived network according to any of claims 17-24 and one or more components that are selected from the group of components consisting of moisturizing agents,

• substrates for cell culture, media for cell culture, antibiotic compounds and hormones. 26. A composition comprising a cell and the reticulum derived from adipose tissue according to any of claims 10-17-24. 27. A composition comprising the cell according to any of claims 1-8 and a biologically compatible network. 15 28.- A composition comprising the population in accordance with any of the

• claims 9-16 and a biologically compatible network. 29. The composition according to claim 27 or claim 28, further characterized in that the grid comprises polymeric material. 30. The composition according to claim 29, further characterized in that the

The polymeric material is formed from polymeric fibers such as a mesh or sponge. 31. The composition according to claim 29 or claim 30, further characterized in that the polymeric material comprises monomers that are selected from the group of monomers consisting of glycolic acid, lactic acid, propyl fumarate, caprolactone, hyaluron, hyaluronic acid and combinations of the same. 32. The composition according to any of claims 29-31, further characterized in that the polymeric material comprises proteins, polysaccharides, polydihydroxy acids, polyorthoesters, polyanhydrides, polyphosphazenes, synthetic polymers or combinations thereof. . 33. The composition according to any of claims • 29-32, further characterized in that the polymeric material is a hydrogel formed by interlacing a polymer suspension having the cells dispersed therein. 34. The composition according to any of claims 29-33, further characterized in that the grid also comprises a hormone that is selected from the group of hormones consisting of cytokines and factors of

F believes imient o. 35. The composition according to any of claims 29-34, further characterized in that the network is the network derived from adipose tissue according to any of claims 17-24. 36.- A method to obtain a cell

10 genetically modified which comprises exposing the cell according to any of claims 1-8 to a vector for gene transfer comprising a nucleic acid that includes a transgene, with which the acid is introduced

Nucleic acid in the cell under conditions with which the transgene is expressed within the cell. f 37.- The method according to claim 36, further characterized in that the transgene codes for a protein that confers

20 resistance to a toxin. 38.- A method for delivering a transgene to an animal that comprises (a) obtaining a genetically modified cell according to claim 36 or claim 37 and (b)

25 introduce the cell in the animal, in such a way that the gene is expressed in vi vo. 39.- A method to differentiate the cell in accordance with any of the

• claims 1-8 comprising culturing the cell in a morphogenic medium under conditions sufficient for the cell to differentiate. 40.- The method according to claim 39, further characterized in that the medium is an adipogenic, chondrogenic medium,

• .. cardiogenic, dermatogenic, embryogenic, fetal, hematopoietic, hemangiogenic, myogenic, nephrogenic, neurogenic, neuralgiagenic, urogeni togenic, osteogenic, pericardiogenic, peritoneogenic, pleurogenic, splancogenic, or

15 is romogenic. 41.- The method according to claim 39 claim 40

• characterized further because the morphogenic medium is an adipogenic medium and the cell is monitored for

20 to identify the adipogenic differentiation. 42. The method according to claim 39 claim 40, further characterized in that the morphogenic medium is a chondrogenic medium and the cell is monitored to identify the chondrogenic differentiation.

The method according to claim 39 or claim 40, further characterized in that the morphogenic medium is an embryonic or fetal medium and the cell is momtorea to identify the embryonic or fetal phenotype. The method according to claim 39 claim 40, further characterized in that the morphogenic medium is a myogenic medium and the cell is momtorea to identify the myogenic differentiation. 45. The method according to claim 39 claim 40, further characterized in that the morphogenic medium is an osteogenic medium and the cell is momtorea to identify osteogenic differentiation. 46. The method according to claim 39 or claim 40, further characterized in that the morphogenic medium is a stromal medium and the cell is identified to identify the stromal or hematopoietic differentiation. 47. The method according to any of claims 39-46, further characterized in that the cell is differentiated in vi 48. The method according to any of claims 39-46, further characterized in that the cell differentiates in vi vo. 49. A method for producing hormones, comprising (a) culturing the cell according to claim 7 or claim 8 within a medium under conditions sufficient for the cell to secrete the hormone into the medium and (b) isolating the hormone from the middle. 50.- A method for promoting the closure of a wound within a patient that comprises introducing the cell according to claim 7 or the claim in the vicinity of a wound under conditions sufficient for the cell to produce the hormone, thereby The presence of the hormone promotes the closure of the wound. 51. A method for promoting neovascularization within a tissue, comprising introducing the cell according to claim 7 or claim 8 into the tissue under conditions sufficient for the cell to produce the hormone, whereby the presence of the Hormone promotes neovascularization within the tissue. 52.- The method of compliance with the

• claim 51, further characterized in that the tissue is within an animal. 53. The method according to claim 51 or claim 52, further characterized in that the tissue is a graft 10 54. The method according to any of claims 49-53, further characterized in that the hormone is a growth factor that is selected from the group of growth factors that consists of a factor of

15 human growth, nerve growth factor, vascular cell growth factor and endothelial cell, and members of the TGFβ superfamily. 55.- A method for conditioning culture medium that includes exposing a culture medium

Cellular to the cell according to any of claims 1-7 under conditions sufficient for the cell to condition the medium. 56.- The method of compliance with the

25 claim 55, further characterized in that the medium is separated from the cell after it has been conditioned. 57.- The method of compliance with

• any of claims 36-56, further characterized in that the cell is within a population according to any of claims 9-16. 58.- a conditioned culture medium produced in accordance with the method of

• Claim 55 or claim 56. 59. - The conditioned culture medium according to claim 58, further characterized in that it is substantially free of a cell in accordance with any of the

15 claims 1-7. 60.- A method for cultivating a stem cell comprising maintaining a stem cell in the conditioned medium according to claim 58 or claim 59 under

20 conditions for the stem cell to remain viable. 61.- The method according to claim 60, further characterized in that it also comprises allowing successive rounds of

25 mitotic division of the stem cell to form an expanded population of stem cells. 62.- The method according to claim 60, claim 61

• further characterized in that the medium is substantially free of cells derived from adipose tissue according to any of claims 1-7. 63. The method according to any of claims 60-62,

• 10 further characterized in that the medium contains cells derived from adipose tissue according to any of claims 1-7. 64.- The method according to claim 63, further characterized in that

15 are in contact with a stem cell and a cell derived from adipose tissue. 65.- The method according to any of claims 60-64, further characterized in that a stem cell is a

20 hematopoietic stem cell. 66.- A method for producing animal matter that comprises maintaining the composition according to any of claims 18-26 under sufficient conditions so that the

25 cells within the composition expand and differentiate to form matter. 67.- The method according to the rei indication 66, further characterized in that the material comprises a type of tissue that is selected from the group of tissues consisting of adipose, cartilaginous, heart, dermal connective tissue, blood tissue, tissue of muscle, kidney, bone, pleura, and tissues of the viscera and combinations thereof. 68.- The method according to claim 66, claim 67, further characterized in that the material comprises more than one type of fabric. 69.- The method according to any of claims 66-68, further characterized in that the material comprises at least a portion of an animal organ. 70. The method according to claim 66 or claim 68, further characterized in that the material comprises at least a portion of an animal limb. 71. The method according to any of claims 66-70, further characterized in that the composition is maintained in vi tro.

72. - The method according to any of claims 66-70, further characterized in that the composition is introduced into an animal and maintained in vi. 73.- An implant comprising the cell according to any of claims 1-7. 74.- An implant that includes the population in accordance with any of the

10 claims 8-13. 75.- An implant comprising the reticulum derived from adipose tissue in accordance with any of the rei indications 14-16. 76. An implant comprising the composition according to any of claims 17-26. f 1 1. - A kit for isolating stem cells from adipose tissue comprising means for isolating adipose tissue from a patient and means for separating the stem cells from the remnant of adipose tissue. 78.- The kit according to claim 77, further characterized in that it also comprises means for differentiating the stem cells.

The kit according to claim 78, further characterized in that the medium is selected from the group of media consisting of adipogenic, chondrogenic, cardiogenic, dermatogenic, embryonic, fetal, hematopoietic, hemangiogenic, myogenic, nephrogenic, neurogenic, neuralgiagenic, media. Togenetic, osteogenic, pericardiogenic, peritoneogenic, pleurogenic, splancogenic, and stromagenic urogeni.