MXPA97005612A - Induction directed by lineage of the differentiation of human mesenquimatosas cells of ori - Google Patents

Abstract

The present invention relates to methods for induction directed by in vitro or ex vivo lineage of isolated culture-expanded human mesenchymal basal cells comprising the contacting of mesenchymal basal cells with an effective bioactive factor to induce the differentiation of the same in a lineage of choice, as well as compositions that include human mesenchymal basal cells expanded by culture, isolated and effective bioactive factors to induce induction by directed lineage. The method is also presented which also includes the introduction of such lineage-induced mesenchymal basal cells expanded by culture in a host from which they have been originated for the purpose of regeneration or repair of mesenchymal tissue.

Description

INDUCTION DIRECTED BY LINEAGE OF THE DIFFERENTIATION OF HUMAN MESENCHIMATUS CELLS OF ORIGIN The present invention provides methods for directing the original esenchymatase cells cultured in vitro to differentiate into specific cell lineages before their implantation for the therapeutic treatment of pathological conditions. in humans and other species or at the time of said implantation. The mesenchymal cells of origin < MSCs) are blasts-like cells or formative pluripotent embryos that are found in the bone marrow, blood, dermis, and period that can be differentiated into specific types of connective or mesenchymal tumors including adipose, bone, connective tissues, and bone. rt i lagenous, elastic, muscular, and fibrous. The specific pathway of differentiation that these cells take depends on various influences from mechanical influences and / or endogenous bioactive factors, such as growth factors, cytokines, and / or local microenvironmental conditions established by host tissues. Even when these cells are normally present at very low frequencies in the bone marrow, a process to isolate, purify, and mitotically expand the population of these cells in cell culture is reported in Caplan et al., North American Patents No 5,197,985 and 5,226,914.

In prenatal organisms, the differentiation of MSCs into specialized connective tissue cells is well established; for example, mesenchymal cells of chicken, mouse or human origin differentiate into cartilage, bone and other connective tissues < l-5). In addition, it has also been shown that a cloned rat fetal calvarial cell line differs in muscle, fat, cartilage, and bone (6). The existence of MSCs in postnatal organisms has not been studied extensively in order to show the differentiation of post-brionic cells in several mesodermal phenotypes. The few studies carried out involve the formation of bone and cartilage by cells of the bone marrow after their placement in diffusion chambers and in transplants in vivo (7,8). Recently, it has been shown that cells derived from the bone marrow from young rabbits < B00-1000 g) form osteogenic adipocytic cells in vivo (9) and it has been shown that cells are from bone marrow cloned from po- taneous mice forming adipocytes and osteogenic cells (10). In the same way, cells from chicken peri-steppe have been isolated, expanded in culture and, under conditions of high density in vitro, they were shown to differentiate into cartilage and bone (11). It has been shown that mesenchymal cells derived from rat bone marrow have the ability to differentiate into osteoblasts and chondrocytes when implanted in vivo (12.6). It has never been observed that cells from several marrow sources of postnatal organisms have iogenic properties, and the ultinuclear appearance is the most easily recognized culture in culture. In a first aspect, the invention provides a method for effecting lineage-directed induction of isolated, expanded culture-expanded mesenchymal cells of human origin, comprising contacting the mesenchymal cells of origin with a bioactive factor or a combination thereof. effective factors to induce the differentiation of the same in a lineage of choice. More particularly, this method is a method in which the bioactive factor induces the differentiation of such cells into a selected mesenchymal lineage within the group consisting of osteogenic, chondrogenic, tendonogenic, 1togenic, iogenic, stromal, spinal and adipsgenic lineages. dermogenic Preferably, the cells come into contact ex vivo with one or several bioactive factors in this aspect, whereby a method is provided without any risk of associating with the in vivo administration of bioacid factors. In another aspect, the method of the present invention further provides the administration to an individual that requires such treatment of cultured, isolated, expanded mesenchymal cells of human origin and an effective bisactive factor to induce the differentiation of such cells into a lineage of choice. Preferably, the mesenchymal cells of origin and the biactive factor are administered together or. they may alternatively be administered separately. Particularly, this aspect of the method comprises administering a bioactive factor ion to an individual to whom it has been administered, a preparation comprising mesenchymal cells of isolated autologous human origin being administered or administered. In another aspect, the present invention provides a method for inducing the in vivo production of human cytokines in an individual that requires them comprising the administration to the individual of mesenchymal cells of human origin expanded by isolated cultures and an effective bioactive factor for induce the differentiation of such cells in a mesenchymal lineage that produces descending cytokines in such an individual. Preferably, the mesenchymal cells of origin and the bioactive factor are administered together or, alternatively, they can be administered separately. In specific preferred examples of these aspects, the bioactive factor is an orfogenic bone protein and the human MSCs are directed in the chondrogenic lineage.; the bioactive factor is interleukin 1 and human MSCs are directed in the stromal cell lineage (preferably interleukin 1 is interleukin lalfa); the bioactive factors are dexamethasone, ascorbic acid-2-phosphate and beta-glycerophosphate and human MSCs are targeted in the osteogenic lineage; or else the bioactive factor is selected from the group consisting of 5-azacytine, 5-azadeoxycytidine and analogs of any of them and the mesenchymal cells of origin -humans are directed in the myogenic lineage. Another aspect of the present invention provides a composition comprising mesenchymal cells of human origin expanded by culture, isolated and a bioactive factor, or a combination, effective to induce the differentiation of such cells in a lineage of choice. Preferably, the composition further comprises a tissue culture medium. Alternatively, the composition may comprise a medium suitable for administration to an animal, especially a human being, which requires such administration. This aspect of the invention also provides specific modalities using the bioactive factors identified above for induction of lineage in the lineages associated therewith as described above. Figure 1 illustrates diagrammatically the process esengénico by means of which the mesenquimatosas cells of origin are different in several routes of lineage. Figure 2 illustrates diagrammatically the osteogenic differentiation pathway. Figure 3 graphically demonstrates the increase in alkaline phosphatase activity as a function of time in cultures, in the initial studies reported in the example 1. Figure 4 shows the results of the subsequent studies reported in example 1. Figure 5 illustrates diagrath between the pathway of chondrogenic differentiation. Figure 6 shows the extent of cytokine expression of mesenchymal cells of human origin, with and without stimulation by interleukin-l, based on the experiments of example 4. Figures 7A and 7B. (A) Phase contrast micrograph of live culture of MSCs showing the uninucleated cells derived after exposure to 5-aza-CR. This micrograph shows a culture 2 weeks after treatment with 10 μM of 5-aza-CR. Many nuclei (arrow) in the cell can be observed, but striae are not visible. (B) Phase contrast micrograph of live culture of normal rat fetal muscle cells prepared from the hind paws of 17 day old rat fetuses. As in the case of myotubes derived from bone marrow MSCs, no apparent stria is observed. The scale bar is 50 μm. Figure 8: Unofluorescence staining for muscle specific myosin in myotubes derived from rat bone marrow MSCs after exposure to 5-aza-CR. Myosin antibodies do not visualize cross striae, but the antibodies clearly illuminate longitudinal fibers. Scale bar: 30 μm. 'Figures 9A-9D: Miotubes derived from rat bone marrow MSCs 2 weeks ((A) and (B)) and 5 weeks ((C) and (D) > after exposure to 5-aza-CR. the phase contrast micrograph ((A)) and (O) and the inosfusion staining for myosin ((B) and (D)). (A) and (B), (C) and (D) are The same visual fields Myotubes 2 weeks after exposure to 5-aza-CR are stained with anti-iosin antibody, but 5 weeks after exposure are not 50 μM scale bar Figures 10A-10B: Micrograph of the MSCs treated with 5- aza-CR containing droplets in their cytoplasm, this culture was stained with Sudan Black (A) Adipacits groups were observed (arrows), 200 μM scale bar (B) The droplets were stain from brown to black (arrows), suggesting that these droplets are lipids, 100 μM scale bar Figure 11: Phase contrast micrograph of live culture of myogenic cells derived from bone marrow MSCs rat after exposure to 5-aza-CR. After exposure to 5-aza-CR, these cells were cultured with 4ng / mk of bFGF for 10 days. Large myotubes can be observed; scale bar 300 μm. Figures 12A-12D graphically illustrate the expression of S-CSF, GM-CSF, M-CSF and SCF, respectively, observed in the experiments reported in example 6. Figures 13A-13C graphically illustrate the expression of LIF, IL -6 and IL-11, respectively observed in the experiments reported in example 6. Figure 14 graphically illustrates the dose-dependent induction of IL-lalpha of GM-CSF expression observed in the experiments reported in example 6. This invention has multiple uses and advantages. The first consists of the ability to direct and accelerate the differentiation of MSCs before their implantation back into autologous hosts. For example, MSCs are digested in vitro to become osteogenic cells that synthesize the bone matrix at an implant site more rapidly and uniformly than MSCs that must first be recruited into the lineage and then progress through the essential steps of differentiation. Such ex vivo treatment also provides a uniform and controlled application of biactive factors to purify MSCs, leading to a uniform lineage dedication and differentiation. The in vivo biadisponibi1ity of endogenous bioactive factors can not be ensured or controlled so easily. A pre-processor step such as the one presented here avoids this problem. In addition, by treating MSCs before implantation, potentially harmful side effects associated with systemic or local administration of exogenous bioactivss factors are avoided. Another use of this technique is in the ability to direct tissue regeneration based on the stage of differentiation in which the cells are located at the time of implantation. That is to say, in relation to bone and cartilage, the state of the cells at the time of implantation can control the type of final tissue formed. Hypertrophic chondrocytes mineralize their matrix and eventually prepare the pathway for vascular invasion, which ultimately results in new bone formation. Clearly, MSCs implanted for the purpose of restoring normal hyaline cartilage should not progress throughout the lineage. However, implants designed to repair superficial articular defects and the underlying subcandral bone may benefit from a two-component system where cells in the area of future bone are directed ex vivo to become hypertrophic chondrocytes, while cells in the area of The future articulation surface is only directed to become chondroblasts. In the area of stroma 1 reconstitution, the ex vivo control of differentiation can optimize MSC cell populations for the production of specific cytokines for steps necessary for the needs of the individual. Muscle morphogenesis can similarly be directed to create fibers of fast or slow twitching, depending on the indication. ISOLATION AND PURIFICATION OF ORIGINAL CELLS MESENCHI HUMAN ATOSES The mesenchymal cells of human origin isolated and purified according to what is described here can be derived, for example, from bone marrow, blood, dermis or periosteum. When obtained from the bone marrow, it can be marrow from several different sources including caps of femoral bone cancellous bone pieces obtained from patients with degenerative joint disease during a surgical intervention to replace the hip or knee, or from aspirated marrow obtained from normal donors and oncology patients with harvested marrow for a future bone marrow transplant. The harvested marrow is then prepared for cell culture. The isolation process involves the use of a specially prepared medium containing agents that allow not only the growth of the mesenchymal cells of origin without differentiation but also the direct adherence of only the mesenchymal cells of origin on the plastic or glass surface of the culture vessel. By creating a means that allows the selective fixation of the mesenchymal cells of desired origin that are present in the mesenchymal tissue samples in very small amounts it is possible to separate the mesenchymal cells of origin from the other cells (i.e. red and white cells of the blood, other differentiated mesenchymal cells, etc.) that are present in the mesenchymatous tissue of origin. The bone marrow is the soft tissue that occupies the medullary cavities of the long bones, some of the Haversian channels, and the spaces between trabeculae of spongy or cancellous bone. The bone marrow is of two types, red, which is found in all bones at the beginning of life and in locations limited to adulthood (that is, in spongy bones) and which is related to the production of blood cells (it is say hematopoie is) and hemoglobin (therefore the color red); and yellow, which consists largely of fat cells (hence the yellow color) and connective tissue. As a whole, the bone marrow is a complex tissue that comprises heme topoyetic cells, including cells of origin or heme topoyéticas, and red and white blood cells and their precursors; a group of cells that include mesenchymal cells of origin, fibroblasts, reticulocytes, adipocytes, and endothelial cells that contribute to the network of connective tissue called "stroma". Stromal cells regulate the differentiation of hematopoietic cells through direct interaction through cellular surface proteins and the secretion of growth factors and are involved in the establishment and support of bone structure. Studies using animal models have suggested that the bone marrow contains "pre-stromal" cells that have the ability to differentiate into cartilage, bone and another connective tissue cell. (Beresfard, J.N .: Osteogenic Stem Cells and the Sstro a] System of Bone and Marrow, Clin.Orthop., 240: 270, 1989). Recent evidence indicates that these cells, called cells of stromal origin pluppotentes or esenquimatosas cells of origin, have the capacity to generate vanos different types of cell lines (ie, osteocytes, chondrocytes, adipocytes, etc.) When activated, according to the influence of several bioactive factors. In addition, the mesenchymal cells of origin are present in the tissue in very small amounts with a wide variety of other cells (ie erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, etc.).

As a result, a process has been developed to isolate and purify mesenchymal cells of human origin from tissue prior to differentiation and then grow by expansion of the mesenchymal cells of origin to produce a valuable tool for muscle-skeletal therapy. The object of such manipulation is to greatly increase the number of mesenchymal cells of origin and use said cells to redirect and / or reinforce the normal repair capacity of the body. The mesenchymal cells of origin are expanded in large numbers and applied to areas of connective tissue damage to increase or stimulate in vivo growth to regenerate and / or repair, to improve implant adhesion in various prosthetic devices through activation Subsequent and differentiation or increase the production of hemopoietic cells, etc. Along these lines are contemplated various procedures for transferring, and mobilizing, and activating mesenchymal cells of purified origin, expanded by culture at the repair site, implant, etc., including injection of cells at the site of a skeletal defect. , the incubation of the cells with a prosthesis and the implant of the prosthesis, etc. Therefore, by isolation, purification and expansion of the number of cells before differentiation and then active control of differentiation process by its position at the site of tissue damage or by in vitro pretreatment before transplantation, the mesenchymal cells of origin, expanded by culture can be used for several therapeutic purposes such as for alleviating cellular, molecular and genetic disorders in a large number of metabolic bone diseases, skeletal dysplasias, cartilage defects, damage to ligaments and tendons and other disorders uesculaesquela ales and connective tissue. Several media have been prepared which are especially well suited for the desired selective fixation and which are referred to herein as "complete media" when supplemented with serum as described above. One such medium is an enhanced version of Eagle's Medium-Low Glucose Modified by Dulbecco (DMEM-LG), well known and readily available commercially. The commercial formulation is supplemented with 3700 mg / l of sodium bicarbonate and 10 ml / l of lOOx antibiotic-antifungal containing 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) 25 μg of amphotericin B / ml using penicillin G (sodium salt), streptomycin sulfate, and amphotericin B as FUN6IZ0NE (R) in a 0.85% saline solution.

The medium described above is prepared and stored in 90 ml in 100 ml bottles or 450 ml in 500 ml bottles at a temperature of 4 β C until ready for use. For your. Use, add 10 ml or 50 ml of bo or fet fet serum (from selected batches) to the media bottles to provide a final volume of 10% serum. The medium is heated to 30 ° C before use. Regarding this aspect, it was also found that the BGJb medium (Gibco, Grand Island, NY) with lots tested and selected from 10% fetal bovine serum (JR Scientific, Woodland, CA, or other suppliers) was well suited for use in the invention. This medium, which was also a "complete medium", contained factors that also stimulated the growth of mesenchymal cells of indifferentiation origin and allowed selective binding through specific protein binding sites, etc. of only the mesenchymal origin cells on the plastic surfaces of the Petri dishes. In addition, it was also found that the F-12 Nutrient Mixture (Ha) (Gibco, Grand Island, NY) had the desired properties for the selective separation of mesenchymal cells of origin. As indicated above, the complete medium can be used in numerous different isolation processes according to the specific type of initial harvesting processes used to prepare the harvested bone marrow for cell culture separation. Regarding this aspectWhen layers of cancellous bone marrow were used, the marrow was added to complete medium and vortexed to form a dispersion which was then centrifuged to separate the marrow cells from the bone pieces, etc. The marrow cells (which consisted predominantly of red and white blood cells, and a very small amount of mesenchymal cough cells of origin, etc.) were then dissociated into single cells by the sequential passage of complete medium containing the marrow cells to through syringes equipped with a series of size 16, 18, and 20 needles. It was noted that the advantage produced through the use of the mechanical separation process, as opposed to any enzymatic separation process, was that the mechanical process produced few cellular changes while the enzymatic process can cause cellular damage particularly to the protein binding sites necessary for culture adhesion and selective separation, and / or to the protein sites necessary for the production of monoclopals antibodies specific for said cells mesenchymal of origin. The single cell suspension (consisting of approximately 50-100 x 1,000,000 nucleated cells) was then placed in 100 ml dishes for the purpose of selectively separating and / or isolating the mesenchymal cells of origin from the remaining cells found in the suspension. When the aspirated marrow was used as a source of the mast cells of human origin, the original cells of the marrow (which contained few pieces of bone or no piece of bone but a large amount of blood) were added to the complete medium. and fractionated with Percsll gradient (Sigma, St. Louis, MO) described more precisely below in Example 1. The Percoll gradients separated a large percentage of the red blood cells in the morenon-nuclear ectopoietic cells from the fraction of Low density platelets containing the mesenchymal cells of origin derived from the marrow. Regarding this aspect, the fraction of platelets, which contained approximately 30-50 x 1,000,000 cells consisted of an undetermined number of platelets, 30-50 x 1,000,000 cells with nuclei, and only approximately 50-500 mesenchymal cells of origin according to the age of the bone marrow donor. The low density platelet fraction was then placed in the dish in the Petri dish for selective separation based on cell adhesion. Regarding this aspect, bone marrow cells obtained either from cancellous bone or from iliac aspirate (i.e. primary cultures) were cultured in complete medium and allowed to adhere on the surface of the Petri dishes during one a seven days according to the conditions set forth in example 1 below. Since minimal cell fixation was observed after the third day, three days were chosen as the standard period in which the non-adherent cells of the cultures were removed by replacing the original complete medium with fresh complete medium. Subsequent changes of medium were made every four days until the culture dishes became confluent, which normally requires 14 to 21 days. This represented an increase of 1000 to 10,000 times in the number of mesenchymal cells of undifferentiated human origin. The cells were then detached from the culture dishes using a release agent such as for example trypsin with EDTA (acid et i lendia intetraacetic) (0.25% trypsin, 1 M EDTA (IX), Gibco, Grand Island, NY) . The release agent was then deactivated and the mesenchymal cells of undifferentiated cultures grown detached with complete medium were washed for subsequent use. The ability of these undifferentiated cells to enter into discrete lineage pathways is known as the mesengenic process and is represented diagrammatically in Fig. 1. In the mesenhanic process, MSCs are recruited to enter specific multipath lineage pathways that eventually produce functionally differentiated tissues such as bone, cartilage, tendon, muscle, dermis, bone marrow stroma, and other mesenchymal connective tissues. For example, a detailed example of the differentiation pathway of bone-forming cells appears in Figure 2. The lineage map implies the existence of individual control elements that recruit MSCs into the osteogenic lineage, promote preosteoblastic replication and direct step-by-step differentiation to the osteocyte end stage. Substantial research supporting the idea that each step of this complex path is controlled by different bioactive factors is reported. A similar lineage diagram has been developed for the differentiation of chondrocytes and Figure 5 is provided. Again, the progression of each lineage step is under the control of unique bioactive factors including, without limitation, the family of proteins orfogenetics of bone. Each modulator of the ion difference process, whether in bone, cartilage, muscle or in any other mesenchymal tissue, can affect the rate of lineage progression and / or can specifically affect individual steps along the pathway. That is, if a cell is dedicated from the beginning to a specific lineage, is in a biosynthetically active state, or progresses towards a final stage phenotype, it will depend on the variety and timing of bioactive factors in the local environment. The bone and cartilage (ie, osteochondrogenic potential) lineage potentials of fresh and expanded human mesenchymal cells were determined using two different in vivo assays in nude mice. One trial involved the subcutaneous implantation of porous calcium phosphate ceramics loaded with mesenchymal cells of cultured origin; the other trial involved the peritoneal implantation of diffusion chambers inoculated with mesenchymal cells of cultured origin. Whole bone marrow and aspirated fractions separated by Percol gradient were also analyzed in these in vivo assays. The histological evaluation showed bone and cartilage formation in the ceramics implanted with the cultured mesenchymal cells of origin derived from the head of the femur and the iliac crest. Ceramics loaded with mesenchymal cells of human origin with 5 million cells / ml formed bone within the pores, while ceramics loaded with mesenchymal cells of human origin with 10 million cells / ml formed cartilage within the pores. While bone marrow has not been shown to form bone when placed in a composite graft with ceramics at a subcutaneous site in the nude mouse, a quantity of bone produced is substantially less than what can be observed when mesenchymal cells of origin are employed marrow derivatives expanded in culture. These results indicate that under certain conditions, the mesenchymal cells of expanded origin in culture have the ability to differentiate into bone and cartilage when they are incubated as a graft in porous calcium phosphate ceramics. The environmental factors that influence the mesenchymal cells of origin to differentiate into bone or cartilage cells pays, in part, to be the direct access layer of the mesenchymal cells of origin to growth factors and nutrients supplied by the vasculature in ceramics of porous calcium phosphate; cells closely associated with the vasculature differentiate into bone cells while cells isolated from the vasculature differentiate into cartilage cells. The exclusion of vasculature from the pores of ceramics loaded with mesenchymal cells of concentrated human origin prevents osteogenic differentiation and provides conditions that allow chondrogenesis. As a result, the mesenchymal cells of expanded origin in culture and isolated can be used under certain specific conditions and / or under the influence of certain factors to differentiate and produce the desired cellular phenotype required for the repair or regeneration of connective tissue and / or for the implant of various prosthetic devices. For example, the use of ceramic porous cubes filled with mesenchymal cells of human origin expanded by culture, the formation of bone within the pores of the ceramics has been generated after subcutaneous incubations in immunocompatible hosts. In a recent study (13), rat marrow in a composite graft with porous ceramic was used to fill a segmented defect 1 in the rat femur. It was shown that the bone filled the pores of the ceramic and anchored the ceramic-marrow graft on the host bone. Factors that stimulate osteogenesis (ie, osteoinductive factors) from mesenchy cells to human-derived ossicles isolated in accordance with the present invention are present in various classes of molecules, including the following: morphine-bone proteins, such as for example BMP-2 (14) and BMP-3 (15); growth factors, such as, for example, basic fibroblast growth factor (bFGF); glucocorticoids, such as dexamethasone (36); and prostaglandins, such as prostaglandin El (22). In addition, ascorbic acid and its analogs, such as ascorbic acid -2-phytate (17) and glycerol phosphates, such as beta-glycerophosphate (18), are effective addition factors for advanced differentiation, even when they alone do not induce osteogenic differentiation. Factors that have a chondroinductive activity on human MSCs are also present in several classes of molecules, including the following: compounds within the superfamily of transforming Beta growth factor (TGF-beta) such as (i) TGF- betal (19), (ii) Inhibin A (20), (iii) stimulating chondrogenic activity factor (CSA) (21) and (iv) bone morphogenic proteins, such as BMP-4 (22); collagenous extracellular matrix molecules, including type I collagen, particularly in gel form (23); and analogues of vitamin A, such as retmoic acid (24). Factors that have an estragenic induction activity on human MSCs are also present in vain classes of molecules, especially interleukins, co or for example IL-1 alpha (25) and IL-2 (26). Factors that have a myogenic induction activity on human MSCs are also present in various classes of molecules, especially cytidine analogues, such as for example 5-azacytidine and 5-aza-2'-desocytidine. The effect of these modulating factors on human MSCs is presented here for the first time. It is not considered to be a complete list of the modulatory factors paten ially useful for inducing the ion difference in a particular line, but rather to illustrate the variety of compounds that have a useful biological activity for the purpose of promoting step-by-step progression. of the differentiation of mesenchymal cells of human origin to islets. EXAMPLE 1 INTE-OSTEOGENIC DIFFERENTIATION OF MSCs I VITRO. The objective of the experiments described in this example was to demonstrate that the origin mesenchymal cells (MSCs) were directed along the osteogenic lineage pathway in vitro by providing appropriate bioactive factors in the tissue culture medium. This set of experiments illustrates only one example of how MSCs can be directed throughout the osteogenic lineage. INITIAL STUDY Human MSCs were harvested and isolated from bone marrow as described above. These cells were expanded by culture in a DMEM-LG medium containing 10% of pre-selected fetal bovine serum (Complete Medium). It was replaced with fresh Complete Medium every 3-4 days until the cultures were close to confluence, at which time the cells were released from the dishes with trypsin, and seeded again in new dishes at a confluence of approximately 40% ( 400,000 cells per 100 mm plate). These MSCs placed back on the plate were allowed to settle overnight, after which the complete medium was replaced by a medium composed of DMEM-LG, 10% fetal bovine serum, and either 100 nM dexamethasone alone or 100 nM of dexamethasone with 50 μM of ascorbic acid-2-phosphate, and 10 mM of beta-glycerphosphate (Osteogenic Supplement). The Osteogenic Supplement was replaced every 3 days. The cells were examined daily to determine the morphological changes. The selected dishes were then analyzed to determine the cellular surface alkaline phosphatase (AP) activity, a marker for cells that have entered the osteogenic lineage. It is these cells that were subsequently responsible for the synthesis of the osteoid matrix. Biochemical and histochemical reagents of standard enzymes were used to demonstrate the activity of this cellular surface protein. Additional samples were evaluated to determine the presence of mineralized extracellular matrix nodules that correlate with continued phenotypic differentiation and expression of a mature population of osteoblasts. Precipitation of silver nitrate in calcium phosphate crystals was achieved with the bone nodule through the standard Von Kossa staining technique.

The results indicate that after only three days of exposure to dexamethasone, the MSCs in culture had already begun to express alkaline phosphatase on their surface. By day six of the culture, approximately 80% of the cells were positive for alkaline phosphatase. The general organization of the culture dish had changed from almost confluent spirals of fibroblast-like cells on day 1 to numerous areas of polygonal cells stacked on top of one another. By day 9, several small bi-refractive extracellular matrix nodules were associated with these foci of polygonal cells in layers. These areas were positively stained by the von ossa method for minerals. Control culture fed only with Complete Medium never developed these mineralized bone nodules, and only rarely did they contain cells positive for alkaline phosphatase. In contrast, the MSCs treated with the osteogenic complement uniformly acquired an alkaline phosphatase activity and synthesized mineralized extracellular matrix nodules in the dish. Although not in itself, the presence of ascorbic acid-2-phosphate and beta-glycerophosphate in the complete Osteogenic complement additionally supports the maturation of the extracellular matrix and mineral deposit, respectively. Figure 3 graphically demonstrates the increase in alkaline phosphatase enzyme activity as a function of time in culture. On day 8 and after, substantially greater enzyme activity is observed in cells exposed to osteogenic complement (OS) than in cells cultured with control medium. Together these studies demonstrate that MSCs can rapidly and uniformly stimulate differentiation along the in vitro osteogenic lineage. Furthermore, MSCs are not only recruited in the initial stages within the lineage, evidenced by the expression of alkaline phosphatase, but MSCs progress through the lineage to become mature osteoblasts that secrete and mineralize an extracellular matrix similar to bone. Additional evidence of this comes from the observation that when chicken MSCs are treated with osteogenic complement, progress through the stages of the osteogenic lineage presented in Figure 2 as determined by staining of manoclonal antibodies against stage-specific cellular surface antigens. SUBSEQUENT STUDY Using published techniques, the MSCs were purified from three different patients (ages 26 to 47 years), expanded by culture (27), and planted overnight in 48-well culture dishes at a confluence of 20% in DMEM-LG with 10% FBS from selected batches. The base means for comparison were DMEM-LG, BGLb, alphaMEM, and DMEM / F-1 (1: 1). Triggered cultures for each assay were cultured for 10% FBS in the absence or presence of "osteogenic supplements" (OS) (100 nM dexamethasone, 50 μm ascorbic acid-2-phosphate, and 10 M beta-glycerophosphate (28). The media were changed for three days.Each set of cultures was assayed for cell number by means of the c-istalin violet assay, cell surface alkaline phosphatase (AP) by histochemistry as well as mineralized pellet formation by Van oss stain. The alkaline phosphatase enzyme activity was calculated by incubating live cultures with 5 mM p-nor trofeni l phosphatase in 50 M Tris, 150 mM NaCl, pH 9.0 and the quantification of the colorimetric reaction by scanning the samples to 405 n in an ELISA plate reader The activity of the enzyme alkaline phosphatase was expressed in nanomoles of product / minute / 1000 cells.The percentage of cells positive for alkaline phosphatase in cad The well was determined from the stained cultures, and the number of mineralized pellets per well was counted. Tests were performed for 4 days during the 16-day culture period. The t test of two paired samples was performed on selected samples. The data in Figure 4 represent a patient even when similar results were obtained from all the samples. The MSCs uniformly fixed on the plates assumed their characteristic spindle-shaped morphology and proliferated to reach confluence within a period of 8 days. During this period and especially afterwards, the cells treated with OS developed a cuboidal morphology as their number was increased. density, forming multiple layers. For the sake of clarity, only selected aspects of the parameters described above were graphically represented in Figure 4. All samples cultured in BGJb + OS died within three days, while cultures in BGJb survived during the protocol period. For this reason, all BGJb data were omitted from the graphs. Outgoing traits of this study demonstrate a substantially higher proliferation in alphaMEM compared to DMEM / F-12 or DMEM alone (ie, p <; 0.01 and p < 0.05 on day 16). The addition of OS to alfaMEM cultures inhibited proliferation on days 8 and 12 (p <0.04 and p <0.03), but not on day 16 (P> 0.05). alphaMEM + OS also stimulated a significant proportion of cells to express alkaline phosphatase on their surface compared to MSCs maintained in DMEM (p 0.02 on day 8. p <0.01 on day 16). However, no significant difference was observed in the percentage of alkaline phosphatase cells between alphaMEM with and without OS (p,> 0.02 on day 8, p,> 0.05 on day 16). It is noteworthy to observe that alfaMEM + OS induces a higher alkaline phosphatase activity than any other medium during the culture period, including alphaMEM or DMEM (it is to go, p ≤ 0.004 and p.O0O2 on day 16). However, there is no difference in alkaline phosphatase activity between alf MEM and DMEM + 0S during the study period (ie, p ", 0.2 on day 16.) Among all the tested media, the number of mineralized nodules for day 16 it was higher in DMEM + 0S (p <0.02 compared to DMEM) .This research shows that human MSCs expanded in purified culture can be induced in the osteogenic lineage in vi tro, thus establishing a model for differentiation of human osteoblast At the beginning of the culture period (day 8) only alphaMEM + OS induced a substantial osteoblastic re-route of MSCs (> 50%), as observed by the alkaline phosphatase surface cell staining. however, all cultures except DMEM contained more than 60% alkaline phosphatase-stained cells.In all media studied, the addition of OS provides a higher alkaline phosphatase activity than After 4 days, even though a large percentage of cells in most media were stained with alkaline phosphatase by day 16, the substantial differences in the alkaline phosphatase activity assay probably reflects the amount of enzyme on the cell surface, and consequently the degree of progression in *? 1 osteoblastic lineage. At least, OS can up-regulate the expression of this surface marker of ost oblitestic cells. It is interesting to note that despite a lower alkaline phosphatase activity, the DMEM + OS cells generated more myelinated nodules of alfaMEM + OS. This observation may suggest that within the 16-day culture period, DMEM- + 0S supports a greater osteogenic ion difference of MSCs than alphaMEM + OS. It is possible that, given more time, alfaMEM + OS would cause a few even more mineralized than DMEM + OS. The differences in the media favor the maintenance of the MSC phenotype (DMEM) as evidenced by specific immunization for MSC, or maximum re-treatment and induction in the osteogenic lineage (alphaMEM + OS), observed by the percentage of cells positive for Alkaline phosphatase and alkaline phosphatase activity are inherently interesting and warrant further examination. The use of several onclonal and polyclonal antibodies against matrix components and specific cells during this inductive phenomenon is currently being performed, and will provide additional information as to the nature of the in vitro difference-ion process.

EXAMPLE 2 THE GFNERATION OF MONOCLONAL ANTIBODIES AGAINST CELLS HUMAN OSTEOGENIC REVIEWED THE EMBRYOUS FORMATION OF BONES IN VIVO AND THE ASSIGNMENT OF ORIGINAL CELLS MESENQUI PURIFIED ATOSES In Vitro It has been established that pragepitoras cells (tifjsenqui matoses derived from the bone marrow can differentiate into osteoblasts.) In addition, these mesenchymal cells of origin (MSCs) also cause e3 emergence of cartilage, tendon, ligament, muscle and other tissues, however, knowledge of the steps involved in the dedication and differentiation of MSCs throughout these various lineages has been limited, in part due to the lack of specific probes for cells at various stages within the osteogenic pathway or other differentiation pathways Since the anoclonal antibodies are useful probes to study differentiation, they immunize the mice with intact preparations of live cells of MSCs derived from human bone marrow induced in the osteogenic lineage in vitro. We studied 3 colonies of hybridomas against purified MSCs, MSCs undergoing osteogenic differentiation and frozen sections of embnomatic human limbs where long bones are being developed around cartilage rudiment. Is ? sifted screening favors the selection of ia =. antibodies that reacted with MSCs to differentiate in vitro and human osteogenic cells m alive. Using this approach, we have generated monoclonal antibodies against specific surface antigens for lineage stages in astrogenic cells derived from MSCs of human marrow. Using published techniques, MSCs were purified from 5 different patients (ages 28-46), expanded by culture (29), and cultured in DMEM-LG with 10% FBS and "osteogenic supplements" (100 nM dexaraetapasopa, 50 μM of ascorbic acid-2-phosphate, and 10 mM of beta-glycerophosphate (28). The s 3 and 6 of culíivo, the initial part during the expression of alkaline phosphatase, and before the formation of nodules. In addition, the cells were released from the dishes with 5 M EGTA, approximately 4 million cells were combined for days 3 and 6 for each of 5 weekly immunizations in Balbc / J mice. monoclonal hybridomas were produced, and culture supernatants were screened by semi-efficient ISAA against purified MSCs, and MSCs cultured for 3 to 6 days with osteogenic complements.In short, they were placed in MSCs dishes in 96-well culinary dishes. , exposed to supplements or stegogenic, and then reacted with culture supernatants followed by goat anti-mouse IgG conjugated with rum sour peroxidase. The secondary antibody was rinsed, and a-pheni lend iamine substrate was added to the dishes. The binding of primary mouse monoclonal antibodies was evaluated by the quantified calorimetric reaction by means of well exploration at 490 nm in an ELISA plate reader. Colonies of interest were selected based on differential binding to control MSCs and osteogenic cells derived from MSCs. Selected colonies were screened originally by immunofluorescence in frozen sections not fixed in human embryonic members. Hybridoma colonies of interest were cloned and inoculation and inoculation were performed. Additional proteins in various normal tissues and derivatives are derived from human, rat, rabbit, chicken and bovine sources. About 10,000 colonies of hybridomas were screened by the modified ELISA protocol described above. Based on differential binding with purified MSCs, or MSCs grown for 3 and 6 days with osteogenic supplements, 224 colonies were selected for immunofluorescent screening against human and brionic members of 55 to 60 days. The majority of these 224 colonies reacted with either multiple tissue types present in the developing limb, or were not detected in the developing bone. To date, 9 colonies have been identified that demonstrate a specific immunoreactivity on cells of the osteogenic lineage. The patterns of reactivity vary; some hybridoma supernatants react with a large population of cells within the asterogenic collar and periosteum that contain the osteoprogenitor, while others react only with cells that appear to be actively involved in matrix synthesis. Two hybridoma colonies appear to react with osteogenic cells as well as hypertrophic chondrocytes. The results are summarized in Table 1. Table 1 L les ine MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS 3 3 3 3 3 3 3 control 20E8 0 1 8 1 133CC99 0 0 1 3 5D9 0 1 2 18H4 0 3 5 18D4 0 2 4 10F1 0 0 2 13B12 0 4 2 Table 1 shows the immunoreactivity of colonies of selected hybridomas against untreated MSCs, or MSCs cultured with Osteogenic Complement (OS) for 3 or 6 days. The figures reflect the relative amount of antibody bound in the ELISA assay described above.

These investigations indicate the presence of specific cell surface differentiation markers for human osteogenic lineage stages similar to those detailed for osteogenic cells of birds (31). The staining of the osteogenic cells in the developing member supports the viewpoint according to which the MSCs cultured with Osteogenic Supplements become "authentic" osteoblasts in culture. Osteagenic differentiation in vitro is therefore confirmed by molecular probes that extend beyond the traditional criteria of alkaline phosphase expression and mineralized nodule formation. The correlation of in vitro detailed observations with in vivo analysis of antigen expression will be useful in further studies of osteogenesis. The characterization of the specific elements of tissue culture, that is, bioactive factors, that promote the progression of cells through the steps of osteogenic lineage will be crucial. The identification of the surface of osteogenic cells, and / or extracellular matrix antigens should provide additional knowledge of the physiology of bone cells. These and other monoclonal antibodies currently under investigation will prove their usefulness in future studies on MSC differentiation. EXAMPLE 3 C0NDR06E DIFFERENTIATION ICA INDUCED IN VITRO MSCs The objective of the experiment described in this example was to demonstrate that mesenchymal stem cells (MSCs) were directed along the chondrogenic lineage pathway in vitro by providing appropriate bioactive factors in the tissue culture medium. This set of experiments represents only one example of how MSCs can be directed along the chondrogenic lineage. The human MSCs were harvested and isolated from bone marrow according to the above described. The cells were expanded by culture on DMEM-LG medium containing 10% of preselected fetal bovine serum (Complete Medium). It was replaced with fresh medium every 3-4 days until the cultures are near confluence, at this time the cells were released from the dishes with trypsin, and seeded again in new dishes with a confluence of approximately 50% (500,000 cells per 100 mm dish). These MSCs placed back into plates were allowed to filter overnight, after which the Complete Medium was replaced by DMEM-LG with 10% fetal bovine serum, and 5 mg / ml partially purified bone morphogenic protein ( Condrsgenic Supplement), provided by Dr. Marshall R. Urist. This Chondrogenic Complement was replaced every 3 days. The cells were examined daily to determine morphological changes. The selected dishes were then analyzed immunohistochemically to determine CSPG-M, a marker for cells that have entered the csndragénico lineage. These cells are then managed for the synthesis of type II collagen matrix of the cartilage. Standard immunohistochemistry reagents were used to demonstrate the presence of this extracellular matrix protein. Additional samples were evaluated to determine the presence of nodules marked with Toluidine Blue that correlated them with the continuous differentiation and phenotypic expression of a mature population of chondrocytes. Van ossa staining to determine the presence of mineralized nitrites of hypertrophic chondrocytes was negative. The results indicated that after only 3 days of exposure to the Condragic Supplement, MSCs in culture had already begun expressing CSPG-M in their extracellular matrix. The approximate organization of the culture dish had changed from spirals of fibroblast-like cells on day 1 to numerous foci of polygonal or round cells in multiple layers surrounded by a thin layer of fibroblastic cells resembling a perichondium. The extracellular matrix of these nodules was strongly immunoreactive for type II collagen. Control cultures fed only with Complete Medium never developed these cartilage nodules. Taken together, these studies demonstrate that MSCs have been stimulated to differentiate along the in vitro in vitro caryogenic lineage. In addition, the MSCs were not only recruited in the initial stages within the lineage, evidenced by the expression of CSPG-M, but the MSCs progressed along the lineage to become mature chondrocytes that secreted extracellular matrix rich in type II collagen. . To date, the terminat differentiation of chondrocytes derived from MSCs, evidenced by hypertrophic cells in a calcified matrix, has not been observed in vitro. This finding reflects the need to design a Condrogenic Complement specifically focused on the promotion of this step of terminal differentiation. It is interesting to note that Pacifici. and its collaborators (32) have developed a medium containing retinoic acid that stimulates the terminal differentiation of chicken chondrocytes. The addition to the Complete Medium constituting the Condrogenic Complement in the previous example is only one of the known factors that stimulate the differentiation of the chondrogenic cells or their proliferation in vitro. EXAMPLE 4 INDUCED DIFFERENTIATION OF ESTROMAL CELLS OF MEDULA FROM MSCs I ITRO The object of the experiment described in this example was to show that MSCs derived from human marrow were directed along the lineage of introgenic oestrus estrus in vitro by supplying bioactive factors appropriate in the culture medium. The MSCs derived from the human marrow were isolated from the bone marrow and expanded in culture in accordance with what has been described above. To demonstrate the ability of human MSCs to be induced along the cell line stromal 1 cell line, the cytokine specific expression was measured as a differentiation marker. The MSCs were cultured under conditions that favor the proliferation of MSCs without differentiation using medium consisting of DMEM-LG containing preselected 10% fetal bovine serum (Complete Medium) or conditions that favor the expression and differentiation in the steric phenotype 1 of marrow using a medium comprising Complete Medium plus 10 U / ml of interleukin-lalfa (IL-lalfa) (Stromaxin Supplement (SS)). The conditioned culture media of these tissue culture populations were analyzed for the presence of cytokines using commercial sandwich ELISA biaensays (R & amp; amp; amp;; D Systems). The cytokines that were tested are the cytokines known to be secreted by stromal.es cells and that influence hematopoiesis. These include interleukin-3 (IL-3), interleukin-6 (IL-6), grapulocyte colony stimulation factor (G-CSF), granulocyte-colony stimulation factor-macrophage (GM-CSF), factor of origin cells (SCF), leukemia inhibitory factors (LIF) as well as transforming factors factor-beta-2 (TGF-beta2). In each case, second passage MSCs were placed in 35 mm culture dishes in a density of approximately 30% confluence (30,000 cells per 35 mm dish). After allowing the cells to attach overnight, the culture media were removed and replaced by Complete Medium or Complete Medium plus Stromagenic Complement. Figure 6 illustrates the expression of the cytokines of human MSCs under the two plate placement conditions. In the absence of IL-lalfa, the MSCs expressed G-CSF, GM-CSF, LIF and SCF at very low levels, but expressed IL-6 in high abundance. In comparison, after 3 days of stimulation with IL-lalfa, dramatically higher levels of cytokines were detected for all previous species. The MSCs did not express I L-3 or TGF-beta2 under either of the two culture conditions. These data show that IL-1-alpha increases the expression of MSC from a cytokine profile that has been documented to support the documentation of the hematopoietic cell of origin and that is characterized by differentiated stromal cells of the marrow.

EXAMPLE 5 INDUCED MIOGENIC DIFFERENTIATION OF IN VITRO MSCs The purpose of the study described in this example was to demonstrate that 5-azacit idina induces the mesenchymal cells of origin (MSCs) to differentiate along the myogenic lineage. The compound, 5-azacytidine (5-aza-CR; Sigma Chemical Co., St. Louis, MO), an analogue of cytidine, causes the hypomulation of a certain amount of cytokine in DNA which may be involved in the Activation of specific genes for phenotype. Mouse embryonic cell lines, C3H / 10T1 / 2 C18 and Swiss 3T3, after exposure to 5-aza-CR, were converted into 3 different lineages of esoperic, myoblast, adipocyte and chondrocyte cells (33-34). In part, it seems that the mechanism by which 5-aza-CR activates isgenic genes involves MyoDl (35-36). With the above mentioned in mind, we have exposed MSCs derived from rat bone marrow exposed to 5-aza-CR and we have focused our analysis towards its conversion into myogenic phenotypes. Femurs and tibias of male Fisher rats (Charles River, Indianapolis, IN) with an average body weight of 100 g were collected and the adherent soft tissues were removed. Several cells and marrow isolates came from rats 250 g. A meticulous dissection of the long bones to remove the soft tissues was carried out to ensure that the iogenic precursors were not carried in the marrow preparation. Regarding this point, iogenic cells were never observed in untreated MSC cultures. Both ends of the bones were cut from the diaphysis with scissors for bones. The bone marrow caps were removed hydrostatically from the bones by inserting size 18 needles fixed onto 10 ml syringes filled with the Complete Medium consisting of DMEM containing selected lots of 10% fetal bovine serum (FCS; IR Scientific Inc., Woodland, CA), 5% horse serum (HS, Hazleton Biologics Inc., Lenexa, KS), and antibiotics (Gibco Laboratories, penicillin G, 100 U / ml, streptomycin, 100 μg / ml, amphotericin B, 0.25 μg / ml). The needles were inserted into the distal ends of the femur and proximal ends of the tibias and the marrow caps were removed from the opposite ends. The marrow caps were dissociated by sequential passage through needles of size 18, size 20, and size 22 and these scattered cells were centrifuged and suspended again twice in Full Medium. After the cells were counted in a hemsci tómetrs, 50 million cells in 7-10 ml of complete medium were introduced in 100 mm petri dishes. Three days later, the medium was changed and the non-adherent cells were discarded. The medium was completely replaced every 3 days. Approximately 10 days after sowing, the dishes became almost confluent and the adherent cells were released from the dishes with 0.25% trypsin in lmM of sodium EDTA (Gibca Laboratories, Grand Island, NY), divided 1: 3, and planted in fresh dishes. After these passed cells once became almost confluent, they were harvested and used for the experiments described below. We refer to these cells as MSCs derived from rat marrow. In total, 8 MSC preparations derived from marrow from separate rats were used in this study. The cells were routinely cultured in Complete Medium at a temperature of 37 ° C in a humidified atmosphere of 5% C02. The MSCs passed twice were seeded in 35 mm dishes at three cell densities, 500, 5,000 and 50,000 cells / dish. Beginning 24 hours after sowing, these cultures were treated for 24 hours with a Myogenic Medium consisting of complete medium containing several concentrations of 5-aza-CR. After the cultures were washed twice with a balanced salt solution of Tyrode (Sigma Chemical Co.), the medium was changed to complete medium without the addition of 5-aza-CR and subsequently changed twice a week until the end of the experiment., 40 days after treatment. As described in detail in the results, several culture conditions were tested to try to optimize the effects of 5-aza-CR, especially to optimize iogenesis. The rat-marrow MSCs passed twice were seeded in 35 mm dishes in 5,000 cells / pl and treated with four concentrations (0.1 μM, 0.3 μM, 1 μM and 10 μM) of 5-aza-2'-deacetyl t idina (5-aza-dCR; Sigma Chemical Co.) in the same manner as described above for 5-aza-CR. At various times during the treatment, the morphology of the cultures was observed. The live cultures were examined every day with a phase contrast microscope (Olympus Optical Co., Ltd., Tokyo, Japan), eventually some of the cultures were fixed for histology to good immunohistochemistry. Muscle cells were first identified morphologically in phase contrast by the presence of myotubes muí t inucleadas, and subsequently immunohistochemically by the presence of the specific pratein for skeletal muscles, myocin. The contraction of the putative muscle cells was stimulated by a 1 mM drop of acetylcholine (Sigma Chemical Co.) in a balanced salt solution of Tyrode. For immunochemistry, the cultured cells were fixed with methanol at -20 ° C (Fisher Scientific Co., Fair Lawn, NJ) for 10 minutes and incubated with a mouse monoclonal antibody on skeletal fast cross rat myosin (Sigma Chemical Co., ascites fluid, 1/400 dilution) in PBS (Phosphate buffered saline, pH 7.4) containing 0.1% BSA (bovine serum albumin, Sigma Chemical Co.). The second antibody was sheep anti-mouse IgG conjugated with biotin (Organon Teknika Corp., West Chester, PA, 1/50 dilution) followed by treatment with avidin conjugated with Texan red (Organon Teknika Corp, 1 / 4,000 dilution) . All incubations were for 30 minutes at room temperature, each preceded by blocking for 5 minutes with PBS containing 1% BSA, followed by two 5 minute washes in PBS. The cells were mounted on Fluoromount-G. { Fisher Biotech, Pittsburgh, PA) and observed with an Olympus microscope (BH-2) equipped for fluorescence and photographed with a Kodak TMAX 400 film. The second passage rat bone marrow MSCs were placed in 96-well plates at dilution of limitation of a cell / well; the cells were placed in dishes in medium consisting of 50% complete medium and 50% conditioned medium, which was obtained from rat bone marrow cells near the confluence cultured in complete medium for 2 days. From a total of 384 wells, 50 colonies were detected; these colonies were subcultured, maintained and eventually four survived. These 4 clones were treated with 5-aza-CR in accordance with the aforementioned and qualified in terms of their myogenic and adipacitic morphologies. The first passage rat bone marrow cells were exposed to 10 μm 5-aza-CR for 24 hours and placed in 96-well dishes in imitation dilution of one cell / pair as before. The number of clones that showed morphologies of myogenic cell, multiple nuclei or adipocyte (positive for Sudan black) was determined. To compare the conversion capacity of bone marrow MSCs in several mesodermal genotypes with the capacity of two pure hydroblasts, rat brain fibroblasts were exposed either to 5-aza-CR or 5-aza-CdR. Whole brains of 3 male Fisher rats were collected from the inside of the skull and cut into small pieces with a sharp scalpel. These pieces were transferred to a 50 ml conical centrifuge tube, centrifuged at 500 xg for 10 minutes, re-suspended in 10 ml of a balanced salt solution of Tyrode, and heated with a Dounce loose-set homogenizer. The homogenate was incubated with 0.1% collagenase (CLS2, 247 U / mg, Worthington Biochemical Co., Freehold, NJ) at 37 ° C for 3 hours, during that period it was vortexed for 30 seconds every 30 minutes. After treatment, the liberated cells were passed through a Ni tex filter, 110 μm, centrifuged, resuspended in 10 ml of low glucose DMEM-LG (Gibco Laboratories) containing 10% FCS, and cultured in 3 dishes of 100 mm culture at 37 ° C in a C02 incubator. The medium was changed twice a week and the cells were cultured until the dishes reached confluence. Third-passage rat brain fibroblasts were seeded on 35-mm dishes at a density of 50,000 cells / dish and treated with 1 μm, 3 μ or 10 μM of 5-aza-CR or 0.1 μM, 0.3 μM or 1 μM μM of 5-aza-CdR in the same manner as the MSCs of rat marrow. After 24 hours, the medium was changed to DMEM-LG containing 10% FCS, 5% HS and 50 nM hydrocortisone without addition of 5-aza-CR or 5-aza-CdR and subsequently changed 2 times by week until the end of the experiment. Myogenic cells derived from MSCs from rat bone marrow were compared with normal fetal rat myogenic cells, since there is a substantial database for the latter. Muscle cells were dissociated from the muscles of the hind paws of 17-day Fisher rat fetuses with 0.2% trypsin (Sigma Chemical Cs.) In Tyrode calcium and magnesium excency for 35 minutes at 37 ° C with occasional shaking. After passing through a Nitex filter of 110 μm, the concentration of fibroblasts was reduced by incubation of cell suspensions for 30 minutes in Falcon plastic dish, which results in the preferential fixation of the fibroblasts. A suspension of 500,000 unique cells that were not fixed on the uncoated plate was placed on a plate in a 35 mm plastic culture dish coated with collagen (1.5 ml of 0.14% gelatin, JT Baker Chemical Co., Phi 11 ipsberg , NJ) containing 2 ml of 70% DMEM, 10% HS and 1% non-essential amino acids (Gibco Laboratories). The cells were cultured at 37 ° C in a humidified atmosphere of 5% C02. Cultures of MSCs derived from rat bone marrow (5,000 cells / 35 mm dish) were exposed at various concentrations of 5-aza-CR (0, 1, 3, 10, 20, and 50 μM) 24 hours after sowing of the cells in culture dishes. The medium containing 5-aza-CR was removed after a 24-hour exposure period and replaced with a medium without 5-aza-CR. Seven days after this exposure, long nucleated nuclei cells were observed in some of the dishes treated with more than 3 μM of 5-aza-CR (Figure 7A >;; the cells in these cultures were found in approximately 80% confluence. The number of such cells with multiple nuclei increased with isolated colonies or groups, and reached a maximum (9 colonies in 10 dishes of 35 mm) 2 weeks after the initial treatment.

The number of such cells decreased (6 colonies in 10 dishes of 35 mm) 5 weeks after treatment; 7 disappeared probably due to their contraction and detachment of the dish and 4 new colonies disappeared during this period of time; a substantial proportion of the multicell cells remained for up to 40 days after the initial exposure, which was the longest observation period. The morphology of multiple nuclei cells, observed by phase contrast microscopy of live cultures (Figure 7A) was similar to the morphology of rat muscle in culture. It does not observe any discernible striae, as routinely observed in embryonic chicken myogenic cells in culture, even when myotubes derived from myogenic cells obtained from normal fetal rat members also did not show striae (Figure 7B). Therefore neither the myotubes derived from MSCs nor the myotubes obtained from normal rat embryos showed stretch marks under the conditions used in these studies. Waves of spontaneous contractions or crossing of some of these multiple nuclei cells were observed when live cultures were seen. The contraction of these cells could also be stimulated by placing a drop of acetylcholine solution in these cells, which additionally indicates that these cells are myogenic.

To further confirm the identity of these multigene cells, skeletal muscle antibodies specific for iokine were presented to a fixed preparation of these cultures. Figure 8 shows a positively labeled one with an antimicrobial antibody. Again, cross streaks were not observed. We also had motubos 2 weeks and 5 weeks after treatment with 5-aza-CR with an antimiocin antibody. Myotubes 2 weeks after treatment were stained strongly positive (Figure 9A and 9B), even though these 5 weeks after treatment were lightly stained (Figure 9C and 9D). The effect of 5-aza-CR appears to be dependent on the concentration presented to MSCs. No iotube was found in dishes treated with 0 to 1 μM of 5-aza-CR, but in dishes treated with 4-50 μM of 5-aza-CR, myotubes were observed with a comparable incidence (Table 2). TABLE 2 NUMBER OF GROUPS OF MYOTUBES 0 WELL ADSP0CST0S FOUND BY CROP FOR MSCs EXPOSED TO DIFFERENT CONCENTRATIONS OF 5-AZA-CR Concentration of Adipocyte Myotubes SI * (5-aza-CR) 0 μM 0/12 3/12 27% 1 μM 0/12 19/12 21% 3 μM 3/12 16/12 15% 10 μM 4/9 19/9 12% 20 μM 2/5 9/5 7% 50 μM 2/5 8/5 6% Secondary crops of rat bone marrow cells were placed in dishes with 5,000 cells per dish of 35 mm, treated with the located concentration of 5-aza-CR and observed 14 days after treatment. The numbers for the incidence of myotubes and adipocytes indicate the total number of phenotypically discernible groups observed and the total number of culture dishes examined. To measure the Survival Index (SI *), in the presence of 5-aza-CR, MSCs were seeded at a density of 200 cells / 35 mm dish and treated with 5-aza-CR 24 hours after placement in plate. After 14 days, the colonies containing more than 10 cells were counted, and this number was multiplied by 100% and divided by 200 to generate the percentage. When the cells were treated with higher concentrations of 5-aza-CR, the group of cells in the dish decreased, and 10 μM appeared to be the most effective concentration in terms of the maximum number of myogenic cells and the survival of cells (efficiency of cells). placement on plate of Table 2). Therefore, all subsequent experiments were performed with 10 M of 5-aza-CR.

To examine the effect of 5-aza-2'-deoxy idine (5-aza-dCR), an analogous deoxy of 5-aza-CR, rat bone marrow MSCs were treated with 0.3 μM, 1 μM, and 10 μM. μM of 5-aza-dCR in the same manner as 5-aza-CR. Of the concentrations tested. 0.3 μM of 5-aza-CdR provided the highest incidence of biogenic conversion and the observed incidence was much higher than in the case of cells exposed to 10 μM of 5-aza-CR (Table 3).

NUMBER OF GROUPS OF MYOTUBES FOUND BY CULTIVATION FOR MSCs EXPOSED TO DIFFERENT CONCENTRATIONS OF 5-AZA-CdR AND 5-AZA-CR Cytidine analogue: Myotube entry SI * 5-aza-CdR 0.1 μM 10/10 16% 5-aza- CdR 0.3 μM 24/10 10% 5-aza-CdR 1.0 μM 3/10 3% 5-aza-CdR 10 μM 1/10 1% 5-aza-CR 10 μM 7/10 14% * Survival index Secondary cultures of rat bone marrow cells were cultured in 5,000 cells per 35 mm dish, treated with the indicated concentration of 5-aza-dCR or -aza-CR, and observed for 14 days after treatment. The incidence numbers of iotubss indicate the total number of typically discernible phenotypic groups observed and the total number of culture dishes examined. To measure the survival rate in the presence of 5-aza-CdR or 5-aza-CR, MSCs were seeded in 200 cells / 35 mm dish and treated with 5-aza-dCR or 5-aza-CR 24 hours after placing on plates. After 14 days, the colonies containing more than 10 cells were counted, and this number was multiplied by 100 and divided by 200 to generate the percentage. To eliminate the possibility of contamination by adjacent muscle-derived ioblasts at the time of harvest of the bone marrow, second-pass rat bone marrow MSCs were cloned, as described herein. Four clones of indistinguishable morphologies were obtained from this procedure and were exposed to 5-aza-CR for 24 hours; for fasis, no cell in these clones presented muscle-like characteristics or was positive for immunity to muscle-specific myosin prior to exposure to 5-aza-CR. Of the 4 clones exposed to 5-aza-CR, one clone presented the distinctive morphology of the mistubos and adipocytes, which we interpreted to indicate that the non-muscle cells were converted into myoblasts or adipacites or influenced to become misblasts or adipocytes. The MSCs derived from rat first-passage bone marrow were exposed to 10 μM of 5-aza-CR for 24 hours and cloned. Out of a total of 768 wells, 136 colonies were detected. Of these 136 colonies, 7 (5%) presented an iogenic phenotype, 27 (20%) presented an adipocyte phenotype, and the other colonies did not present morphologies obviously related to discernible phenotypes. To test the effect of 5-aza-CR and 5-aza-dCR on non-MSC preparations, we exposed brain fibroblasts to these same reactants. The rat brain fibroblasts were seeded in 35 mm dishes at a density of 50,000 cells / dish and treated with 1 μM, 3 μM and 10 μM of 5-aza-CR, or 0.1 μM, _0.3 μM or 1 μM of 5-aza-dCR in the same manner as in the case of rat MSCs. Each group had 9 dishes and the cells were monitored until 14 days after the exposure. On day 7, all dishes reached confluency except in the case of the group treated with 10 μM of 5-aza-CR. No fat cell or myotube could be found in any of the dishes during the observation period. The MSCs were collected from the bone marrow of young (4 weeks old, 100 g) and adult (3 months old, 250 g) donor rats and the number of colonies of myogenic phenotype was compared after exposure to 5- aza-CR (Table 4). TABLE 4 NUMBER OF GROUPS OF MYOTUBES BY CULTIVATION OF MSCs EXPOSED TO 5-AZA-CR FCS HS HC iotubes 10% 5% + U / 5 10% 5% - 8/5 10% 0% + 2/5 10% 0% - 0/5 5% 0% + 0/5 5% 0% + 0/5 0% 5% + 0/5 0% 5% - 0/5 Secondary cultures of MSCs from rat bone marrow were placed on plates 5,000 cells per 35mm dish, treated with 5M-aza-CR and 24 hours later changed to DMEM with different levels of FCS, HS, or 50μM HC, and observed for 14 days after the termination of exposure to 5-aza-CR. The figures of the incidence of mybaubos indicate the total number of culture dishes examined. MSCs from young donor rats had colonies more myogenic than those from adult rats. Second passage cultures of MSCs from young donors exposed to 5-aza-CR produced more myogenic colonies compared to MSCs from older donors tested in cultures from first to fourth pas e.

Several culture conditions were tested to try to optimize the expression of the myogenetic phenotype of cultured MSCs exposed to 5-aza-CR. The exposed cells were cultured in medium containing various concentrations of FCS, HS, basic fibroblast growth factor (bFGF) and hydroscortone isone. Table 4 shows that the medium containing 10% FCS, 5% HS and hydrocortisone appeared to be the optimal medium for MSC expression of myogenic properties. The medium containing bFGF seemed to increase the expression of the myogenic phenotype (Table 5), although this may be related to an increase in the number of myoblasts due to the division of myoblasts as opposed to the increased conversion of progenitor cells. TABLE 5 COMPARISON OF MYOTUBES INDUCED BY 5-AZA-CR BY MSCs OF YOUNG AND OLD RAT BONE MEDULA WITH EACH PASSAGE Initial number first second third quarter of young cells 50,000 / plate + bFGF 3/5 9/5 3/5 0 / 5 (100 g> 50,000 / dish -bFGF 3/5 16/15 2/5 1/5 5,000 / silver + bFGF 1/5 10/5 2/5 2/5 5,000 / dish -bFGF 3/5 13 / 15 2/5 5/5 old 50,000 / plate + bFGF 1/5 0/5 2/5 0/5 (250 g) 50,000 / plate -bFGF 0/5 0/5 0/5 0/5 5,000 / silver + bFGF 1/5 0/5 1/5 3/5 5,000 / silver -bFGF 0/5 0/5 0/5 2/5 The cells were cultured in DMEM with 10% FCS, 5% HS and 50 μM HC, with or without bFGF. The figures for the incidence of myotubes indicate the total number of phenotypically discernible colonies to well-observed groups and the total number of culture dishes examined. MSCs were obtained from young (100 g) or old (250 g) rats. In addition, MSCs derived from bone marrow were placed on plates as follows: 500 cells / dish, 5,000 cells / plate and 50,000 cells / dish and were then exposed to 5-aza-CR. At a density of 500 cells / dish, the miagenic cells were observed for the first time 20 days after treatment, and the cells became confluent 25 days after treatment; Several groups of myogenic cells were observed in 5 dishes 29 days after treatment. With a concentration of 5,000 cells / dish, the mogenic cells were observed for the first time. 7th day, and the cells became confluent 10 days after treatment; 3 groups were observed in 4 dishes 14 days after treatment. At a concentration of 50,000 cells / dish, mogenic cells were observed for the first time at 6 days, and the cells became confluent 7 days after treatment; 10 groups were observed in 5 dishes 14 days after treatment. The observations presented here indicate that MSCs from rat bone marrow have the ability to differentiate into the myogenic lineage in vitro after a brief »exposure to 5-aza-CR. The observed mogenic cells showed the characteristic multinuclear morphology of the myotubes, which contracted spontaneously, contracted when exposed to acetylcholine, and stained positively with a monoclonal antibody specific for skeletal muscle, motion, even though these myotubes never They showed apparent striae. However, normal rat mystablasts collected from fetal rat muscles did not form, in our hands, striated myotubes in culture. We have tried to exclude the possibility of contamination by delicate myogenic cells by removing the soft tissue attached to the bones at the time of harvesting the bone marrow. It is important to note that we have never seen myotubes in any culture of MSCs from rat bone marrow in hundreds of preparations, except in the case of those exposed to sufficient concentrations of 5-aza-CR. In addition, a clone of MSCs from rat bone marrow was converted into iogenic and adipocyte phenotypes after treatment with 5-aza-CR, which we interpreted as meaning that the muscle-specific pragenite cells were converted into these two phenotypes. Since the skeletal muscle has not been observed in the bone marrow, we believe that 5-aza-CR converts these marrow-derived MSCs into myogenic cells. EXAMPLE 6 EXPRESSION OF CYTOKINE BY ORIGINAL MESENCHYMATE CELLS DERIVED FROM BONE MEDULA In Vitro: EFFECTS OF IL-1ALFA AND DEXAMETHASONE The purpose of the present study was to further establish the phenotypic characteristics of cultured MSCs through the identification of a cytokine profile. We have used commercial ELISAs to identify and mediate cytokine expression levels known to be important for the regulation of cell division, differentiation or expression of a variety of mesenchymal phenotypes. We have identified MSC cytokine expression under culture conditions that, as previously reported, allow MSCs to expand mitotically without differentiation (constitutive culture expansion medium). In addition, we have tested cytokine expression by MSCs in culture medium supplemented with dexamethasone or IL-lalfa. Dexamethasone has been reported to induce the differentiation of ssteoprogens in osteoblast. In contrast, IL-lalfa, secreted into the marrow microenvironment by several cells during the inflammatory response has been reported to increase the stromal capacity of the bone marrow to support hematopoiesis and can therefore play a role in the control of the differentiation and / or expression of bone marrow stromal fibroblasts. The data from these analyzes show that cultured MSCs express a unique cytokine profile. In addition, dexamethasone IL-lalfa alters the cytokine expression profile of MSC in different ways. These data help us to understand the unique phenotypic profile of MSCs, and also identify macromolecules whose expression is regulated in terms of their development as the MSCs differentiate or modulate their phenotype towards osteogenic lineage or marrow stromal phenotype. MATERIALS AND METHODS Isolation of MSC and expansion by culture Bone marrow was obtained from 6 human donors, 3 men and 3 women of various ages (Table 6). TABLE 6 CHARACTERISTICS OF DONORS Donor No. Donor age Clinical condition Gender 1 39 NHL * M 2 58 breast cancer M 3 38 myelodysplasia M 4 3 medulloblastoma H 5 28 Hodgkin's lymphoma H 6 47 AML * H * NHL = 1 infona not from Hod in; AML = acute myelogenous leukemia Each donor was in remission of cancer and was under transplant of harvested marrow for future autologous bone marrow transplants. Ap or 10 μl of unfractionated bone marrow was obtained from the harvest and was used in the trials of this study. The MSCs were purified and cultured by a modification of previously reported methods. Briefly, bone marrow aspirates were transferred from their syringes into 50 ml conical tubes containing 25 ml of complete medium consisting of a Dulbecco-modified Ea medium supplemented with fetal bovine serum (FBS) from selected lots, up to a final volume of 10%. The tubes were aspirated in a Beckman tabletop centrifuge at 1200 rpm in a GS-6 rolling bowl rotor for 5 minutes to form the blast cells. The layer of fat that forms on the top of the samples and the supernatants were aspirated using a serological pipette and discarded. The cell pellets were resuspended to a volume of 5 ml with complete medium and then transferred to the top of 70% Percoll preformed gradients. Samples were loaded on a Sorvall GS-34 fixed-angle rotor and centrifuged in a Sorvall high-speed centrifuge at 430 x g for 15 minutes. The low density fraction of approximately 15 ml (combined density = 1.03 g / ml) was collected from each gradient and transferred to 50 ml conical tubes to which 30 ml of complete medium was added. The tubes were centrifuged at 1200 rpm to form the cells in pellets. The supernatants were discarded and the cells were suspended again in 20 ml of complete medium and counted with an ociometer after lysing the red blood cells with acetic acid at room temperature. %. The cells were adjusted to a concentration of 5 million cells per 7 ml seeded in culture dishes of 100 mm to 8 ml per dish. CULTIVATION AND PASSAGE OF DERIVED MEDULA MSCs The MSCs were cultured in complete medium at 37 ° C in a humidified atmosphere containing 95% air and 5% C02, with medium change every 3-4 days. When the primary culture dishes became almost confluent, the cells were detached with 1 mM EDTA (GIBC0) containing 0.25% trypsin for 5 minutes at 37 ° C. The enzymatic activity of trypsin was suspended by the addition of 1/2 volume of FBS. The cells were counted, divided 1: 3 and placed back into dishes in 7 ml of complete medium. These first passage cells were allowed to divide for 4-6 days until they became almost confluent. Nearly confluent first passage cells were trypsinized and re-plated in the assay formats according to what is described below. QUANTITATIVE ELISA The levels of cytokine expression were measured by MSCs using quantitative ELISA. Element pools for ELISA (R & D Systems, Minneapolis MN) were purchased with antibody specificities for the following cytokines: interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-11 (IL-) 11), granulation cell stimulation factor (G-CSF), granulocyte-macrophage colony stimulation factor (GM-CSF), macrophage colony stimulation activity (M-CSF), cell factor of origin ( SCF), leukemia inhibition factor (LIF) and transforming growth factor beta-2 (TGF-beta-2). The almost confluent first passage MSCs were again placed in 35 mm dishes at a density of 50,000 cells per dish and allowed to settle overnight. The culture conditions were then changed to one of the three test conditions: fresh complete medium; complete medium with osteogenic complement; and complete medium with stromagenic complement. The cultures were allowed to incubate in the test media for 24 or 48 hours and then the supernatants were collected., it was instantly frozen in dry ice-ethanol and stored at -70 ° C in a Reveo freezer until all the samples were prepared for analysis together. Tests were performed by applying 100 μL of culture well in wells of the dish for ELISA and by processing the dishes according to the manufacturer's instructions. Standard curves were generated using standard cytokines supplied with the sets of elements and diluted at the appropriate concentrations. In some cases (especially in the case of the IL-6 assay), the supernatants had to be substantially diluted to generate sufficiently low absorbance measurements * so that they could be accurately quantified from the standard curves. QUANTIFICATION OF THE NUMBER OF CELLS RESULTS CONDITION OF THE MEANS OF EXPANSION BY CONSTITUTIVE CULTIVATION Detectable levels of six of the nine cytokines tested were found 24 hours after exposure to expansion conditions by consi tive culture. See Figures 12A-12D and 13A-13C and Tables 7-10 below). TABLE 7 DETECTED LEVELS OF CYTOKIN (24 HOURS) Donor G-CSF 24 h GM-CSF 24 h SCF 24 h LIF 24 h Control 15 56 52 0 53 107 0 28 134 4 0 0 16 7 0 0 30 40 6 37 0 26 119 Average 10 1 35 66 Deviation 14 1 16 51 standard OS 22 0 80 11 2 O 1 61 20 3 6 0 34 44 4 1 0 17 11 4 0 22 11 6 0 0 34 87 Average 6 0 41 31 Deviation 8 0 24 30 standard Value P csn: 0S 0.5464 0.5761 0.1900 0.0274 Value P 0S: SF 0.0358 0.0054 0.4714 0.0176 IL-1 322 527 66 644 2 966 741 83 622 3 1266 413 43 1008 4 143 198 28 152 410 307 0 191 6 164 210 69 338 Average 545 399 48 493 Deviation 463 209 31 327 standard P value with: SS 0 ^ 38 0.0054 0.2434 0.0180 TABLE 8 DETECTED LEVELS OF CITOQUI A (24 HOURS) Donor M-CSF 24 h IL-11 24 h IL-6 24 h TGF-be 24h Control 200 830 7547 O 2 233 741 9887 0 3 303 659 6962 0 4 132 144 6987 0 130 509 5384 OR 6 134 343 7761 0 Average 178 438 742 0 Deviation 70 259 1467 0 standard OS 548 1714 or 545 0 338 0 550 52 1842 0 4 73 0 650 0 162 9 1111 O 6 170 0 919 O Average 308 9 1096 0 Deviation 206 21 591 or standard P value with: 0S 0. .1119 0.0038 0. 004 Value P 0S: SS 0, .0123 0. 375 0. 065 SS 1222 3583 216666 or 1 1355 4277 255555 or 2099 7351 340540 or 4 290 355 76033 or 753 1189 109473 or 6 589 1226 122666 or Average 1051 2997 186822 or Deviation 648 2620 101604 or standard P value with: SS 0.0149 0.0569 0.0074 TABLE 9 DETECTED LEVELS OF CYTOCHINE (48 HOURS) Donor G-CSF 48 h GM-CSF 48 h SCF 48 h LIF 48 h Control 2 0 112 92 2 0 0 129 123 3 0 0 41 142 4 0 0 67 45 0 0 27 28 6. 5 * 7 38 74 Average 1 0 69 84 Deviation 2 1 42 44 standard OS 1 - 7 0 98 43 2 0 0 76 22 0 29 26 4 10 0 100 40 2 0 29 0 6 0 0 17 8 Average 4 0 58 23 Deviation 4 0 38 17 standard P value with: 0S 0.3053 0.3632 0., 3901 0.0171 Value P 0S: SF P.0115 0.0027 0., 1276 .0040 SS 1 452 348 144 841 2 989 564 162 795 3 1214 291 53 866 4 343 198 28 152 410 307 0 191 6 164 210 69 338 Average 545 399 48 493 Deviation 463 209 31 327 standard P value with: SS 0, .038 0.0054 0.2434 0.0180 TABLE 10 DETECTED LEVELS OF CITOCHINE (48 HOURS) Donor M-CSF 48 h IL-11 48 h IL-6 48 h TGF-β 48 h Control 975 143.4 11707 0 2 451 905 10598 O 3 632 761 10691 or 4 337 225 4878 or 279 561 4814 or 6 7 467 5645 or Average 483 722 8056 or Deviation 282 413 3261 or standard OS 1 867 184 1230 or 2 530 0 493 or 3 655 52 1395 or 4 305 0 1090 or 361 9 1134 0 264 0 357 or Average 497 31 950 or Deviation 75 422 or standard P value with: OS 0.6513 0.0049 .0029 Value P OS.-SS 0.0114 0.0167 0.0152 SS 1 3.188 4735 3.82352 or 1416 5500 36666 or 1847 7351 349629 or 290 355 76033 or 753 1189 109473 or 6 589 1226 122666 or P average 1051 2997 186822 or Deviation 648 2620 101604 or standard P value with: SS 0.0149 0.0569 0.0074 The cytokines expressed in terms of pg / 10,000 cells in 24 or 48 hours, from the lowest to the highest were: G-CSF, SCF, LIF, M-CSF, IL-11 and IL-6. Three cytokines were not detected in the supernatants under the constitutive conditions of expansion for culture: GM-CSF, IL-3 and TGF-beta2. Large differences were observed in the average cytokine expression of each cytokine compared to the average levels of expression of other cytokines. At the extremes, the average detectable level of G-CSF expression (10 pg / 10,000 cells / 24 hours) was more than 700 times higher than the average level of IL-6 expression (7421 pg / 10,000 cells / 24 hours). CONDITIONS OF 0STE0GENIC COMPLEMENT CROPPING The addition of osteogenic supplements to the complete medium did not result in any detectable change in G-CSF, M-CSF and SCF relative to the control (Figures 12A-3.2D and 13A-13B; tables 7-10). In contrast, the OS medium significantly down regulated the expression of LIF (pic.01), IL-6 (pic.OOl) and IL-11 (p <.005) in relation to the expression of these cytokines under conditions constitutive of expansion medium by culture at 24 hours. These levels remained statistically lower than the cytokine levels under constitutive conditions of expansion medium per culture at 24 hours (Figures 12A-12D and 13A-13C, Tables 7-10). the amount of inhibition mediated by average OS varied for the three citscinas; at 24 hours the average level of cytokine expression in the OS medium in relation to the constitutive conditions of the expansion medium per culture was as follows: expression of LIF 55% +/- 54%, IL-6 16% +/- 9% and IL-U 1% +/- 3%. The large standard deviation in LIF percentage change was primarily due to measurements from a donor (donor No. 4) where the level of LIF expression was in fact higher under conditions of OS mean compared to the conditions constitutive expansion by crop (table 7). For a given donor, the percentage inhibition of a cytokine in relation to the average absolute level of inhibition of this cytokine was independent of the percentage inhibition of the other two cytokines, in relation to their average absolute inhibition levels (tables 7-10). In addition, for each of the cytokines, the percentage inhibition for a given cytokine among the individual sies in the population was independent of the initial levels of expression under constitutive conditions of expansion by culture (Figures 3.2A-12D and 13A-13C; Tables 7-3.0). CULTIVATION CONDITIONS WITH ESTR0MAGENIC COMPLEMENT The SS medium increased the expression of several cytokines by MSCs in a concentration-dependent manner. Figure 14 illustrates the 24-hour response of MSCs from second passage to the increase of IL-lalfa concentrations in terms of GM-CSF expression. There is an almost linear increase in the level of secretion of GM-CSF by MSCs, with increasing levels of IL-lalfa in the culture medium between 0.1-10.0 U / ml. Additional logarithmic increases of IL-lalfa to the culture medium result in a small additional increase in the expression of GM-CSF. These data were used to identify the concentration of IL-alpha to complement the culture media in the experiments described below. For all subsequent assays, 10 U / ml of IL-lalfa was added to the culture medium. The culture medium supplemented with 10 U / ml of IL-lalfa induced a statistically significant upregulation of the expression of G-CSF (PÍ.05), M-CSF (p <0.02), LIF (p <0.02) , IL-6 (p <0.01) and IL-11 (p <0.06) in relation to cells grown in expansion medium by constitutive culture. In addition, IL-lalfa induced GM-CSF expression that could not be detected in constitutive culture expansion medium. In contrast, IL-lalfa had no statistically significant effect on the expression of SCF in relation to the level of expression under conditions of expansion medium of constitutive culture. The increase in the response to IL-1 alpha varied according to the cytokine. IL-6 (increase of 25.1 times +/- 13.4 times) was stimulated to a greater extent, followed by LIF pair (9.2 ± 6.9 times), M-CSF (5.2 ± 1.7 times) and IL-11 (4.9 ± 3.3 times). The average increase in times for G-CSF and GM-CSF was not calculated since these cytokines were not detected in some or all of the constitutive culture expansion cultures.

COMMENTS Our continuous analyzes of MSCs in this study aimed to identify additional phenotypic characteristics, and to determine how this phenotype is altered when MSCs are exposed to regulatory molecules that cause differentiation or phenatypic modulation. In this study, we used ELISA assays to characterize the cytosine expression of MSCs under conditions of expansion of constitutive culture, and in the presence of OS or SS. MSCs express a unique profile of cytokines that include G-CSF, M-CSF, SCF, LIF, IL-6 and IL-11 under conditions of expansion of constitutive culture. They do not express GM-CSF, IL-3 and TGF-beta 2 under these conditions. OS downregulates the expression of LIF, IL-6 and IL-11, while it does not affect the expression of the other cytokines expressed under constitutive culture conditions. It was not observed that OS up-regulated the expression of any of the cytokines tested in this study. In contrast, SS upregulates the expression of G-CSF, M-CSF, LIF, IL-6 and IL-11, and induces the expression of GM-CSF which was not detected under conditions of expansion of constitutive culture. SS had no effect on the expression of SCF and was not observed to down-regulate any of the cytokines tested in this study. By means of these data, a unique cytokine expression profile has been generated that can help distinguish MSCs from other mesenchyme-bust phenotypes. The identity of the profile of the cytokines must provide clues to determine the function that these cells play in the microenvironment of the. Bone marrow which provides the inductive and regulatory information that supports hematopoiesis. In addition, alterations in this cytokine profile in response to OS and SS identify specific cytokines whose expression levels change as MSCs differentiate or modulate their phenotype in response to regulatory molecules. IL-alpha, released into the bone marrow by several cell types during inflammatory responses, induces MSCs to upregulate the expression of cytokines that support granulocytic differentiation (G-CSF and GM-CSF), monocyte ica / osteoclastic (GM-CSF, LIF, M-CSF, IL-6) and megakaryocytic (IL-11). It has been shown that IL-lalfa protects the bone marrow against radio-ablation and hamo-ablation. The up-regulation by IL-lalfa of cytokine expression by MSCs probably plays a role in the mechanisms of the protective effects of IL-lalfa. Dexamethasone, which induces MSCs to differentiate into osteblasts, attenuates the expression of monocytic / osteoclastic cytokines (LIF, IL-6) and megakaryocytes (IL-11) of support, and has no effect on the expression of cytokines that support granulocytic progenitors (G-CSF, GM-CSF). The three cytokines inhibited by dexamethasone are interesting because each one mediates their. signal through a receiver that uses gpl30 in its signaling path. LITERATURE CITED I. Caplan AI, in: 39 Si popsi? M Annual of the Society for Development Biology, edited by S. Subtelney and U Abbott, p. 3768. New York, Alan R Liss Inc, 1981. 2. Elmer et al., Teratology, 24: 215-223, 1981. 3. Hauschka SD, Dev Bial, 37: 345-368, 1974. 4. Salursh et al. ., Dev Biol, 83: 9-19, 1981. - 5. Swalla et al., Dev Biol, 116: 31-38,1986. 6. Goshima et al., Clin Orthop Rei Res, 269: 274-283, 1991. 7. Ashton et al., Clin Orthop Rei Res, 151: 294-307, 1980. 8. Bruder et al., Bone Mineral, 11: 141-151, 1990. 9. Bennett et al., J Cell Sci, 99: 131-139, 1991. 10. Benayahu et al., J Cell Physiol, 140: 1- 7, 1989. II. Nakahara et al., Exp Cell Res, 195: 492-503, 1991. 12. Dennis et al., Cell Transpl, 1: 2332, 1991. 13. Ohgushi, et al., Acta Scandia., 60: 334-339 , 1989. 14. Wang et al., Grswth Factors, 9:57, 1993. 15. Vukicevic et al., PNAS, 86: 8793, 1989. 16. Cheng et al., Endocrinology, 134: 277, 1994. 17 Tenenbaum et al., Calcif. Tissue Int., 34:76, 1982. 18. Bruder et al., Trans. Ortho Res. Soc., 16:58, 1991. 19. Leonard et al., Devl. Biol., 145: 99, 1991. 20. Chen et al., Exp. Cell Res., 206: 199, 1993. 21. Syftestad et al., Di fferentiation, 29: 230, 1985. 22. Chen et al. ., Exp. Cell Res., 195: 509, 1991. 23. Kimura et al., Biornad. Res., 5: 465, 1984. 24. Langille et al., Di fferent ion, 40:84, 1989. 25. Russell et al., Exp. Hematol., 20: 75-79, 1992. 26. Brenner et al., Brit. J. of Hae. , 77: 237-244, 1991. 27. Haynesworth, et al., Bone, 13: 8-88, 1992. 28. Grigoriadis, et al., J. Cell Biol., 106: 2139-2151, 1988, 29. Haynesworth, et al., Bone, 13: 69-80, 1992. 30. Bruder% > . Haynes? Orth, in preparation. 31. Bruder f. Caplan, Dev. Biol., 141: 319-329, 1990. 32. Pacific, et al., Exp. Cell Res., 195: 38, 1991. 33. Canstantinides et al., Nature, 267: 364-366, 1977. 34. Taylor et al., Cell, 17: 771-779, 1979. 35. Kanieczny et al., Cell, 38: 791-800, 1984. 36. Lassar, Cell, 47: 649-656, 1986.

Claims (4)

1. CLAIMS 1. A method for effecting lineage-directed induction of isolated, expanded, culture-spread mesenchymal cells of human origin, comprising contacting the mesenchymal cells of origin with an effective biactive factor to induce their differentiation into a lineage of choice. The method of claim 1 wherein the bioactive factor induces the differentiation of these cells into a selected mesenchymal bear lineage within the group consisting of an osteogenic, chondrogenic, tendonogenic, 1 igamentogenic, myogenic, stromal, medullar, adipogenic, and dermogenic lineage. 3. The method of claim 1 wherein the cells are contacted with the bisactive factor ex vivo. 4. The method of claim 3 wherein the cells are in contact with the bioactive factor in a rigid porous vessel. The method of claim 4 wherein the rigid porous container is a ceramic cube. 6. The method of claim 3 wherein the cells are in contact with the bioactive factor in a culture vessel. 7. The method of claim 6 wherein the culture vessel is formed of a material selected from the group consisting of glass and plastic. 8. The method of claim 3 wherein the cells are in contact with the biactive factor in an injectable liquid. 9. The rei indication method 8 where the liquid is suitable for intramuscular, intravenous or intraarticular injection. 10. The method of claim 1 comprising administering to an individual therein requiring a composition comprising mesenchymal cells of human origin expanded by ailated cultivars and an effective bioactive factor to induce the differentiation of such cells into cells. a lineage of choice. The method of claim 1 comprising administering the bioactive factor to an individual to whom a preparation comprising mesenchymal cells of isolated human origin has been administered. 12. A method for inducing the in vivo production of human cytokines in an individual requiring them which comprises the administration to the individual of mesenchymal cells of human origin expanded by isolated cultures and an effective bioactive factor to induce the differentiation of said cells in a mesenchymal lineage that produces cytokines in such an individual. The method of claim 12 wherein the mesenchymal cells of origin and the bioactive factor are administered together. 1.4. The rei-indication method 12 where the mesenchyimtase cell of origin and the bioactive factor are administered separately. 15. The method of claim 1 comprising the induction of osteogenic lineage and the bioactive factor is an osteoinductive factor. 16. The method of claim 15 wherein the osteainductive factor is a bone morphogenic pratein. The method of claim 16 wherein the bone morphogenic protein is selected from the group consisting of BMP-2 and BMP-3. 18. The method of claim 15 wherein the factor or theoinductive is a fibroblast growth factor. 19. The method of claim 18 wherein the fibroblast growth factor is a basic fibroblast growth factor. 20. The method of claim 15 wherein the osteoinductive factor is a glucocorticoi. 21. The method of claim 20 wherein the glucocarticoid is dexamethasone. 22. The method of claim 15 wherein the factor or einductive is a landline. 23. The method of claim 22 wherein the β; prostaglandin is prostaglandin El. 24. The method of claim 15 further comprising contacting isolated mesenchymal cells of isolated human origin with an adjunct factor for advanced differentiation. 25. The method of claim 24 wherein the adjunct factor is selected within the group that consists of ascorbic acid and its analogues and a glyraphosphate. 26. The method of claim 1 comprising the chondrogenic induction and the bioactive factor is a chondrogenic factor. 27. The method of claim 26 wherein the chondroinduct factor is a member of the transforming growth factor 13 superfamily. 28. The rei indication method 27 where the member of the transforming growth factor superfamily β is a bone morphogenic protein. 29. The rei indication method 28 where the bone morphogenic protein is BMP-4, 30. The method of claim 27 wherein the superfamily 1 ai of transformation factor β transformation member is TGF-beta 1. 31. The method of claim 27 wherein the member of the transforming growth factor superfamily β is inhibited A. 32. The method of claim 27 wherein the superfamily member of transforming growth factor β is a chondrogenic stimulation activity factor. 33. The rei indication method 26 where the chondroindu t i va factor is a component of the extracellular matrix calagenasa. 34. The method of claim 33 wherein the extracellular matrix collagenase component is collagen I. 35. The method of claim 34 wherein the collagen I is in the form of a gel. 36. The method of claim 26 wherein the chondroinductive factor is an analogue of vitamin A. 37. The method of claim 36 wherein the vitamin A analogue is retinoic acid. 38. The method of claim 1 comprising the stromagenic induction and the bioactive factor is an inducing stromal factor. 39. The method of claim 38 wherein the inducing stromal factor is an interleukin. 40. The method of claim 39 wherein the interleukin is selected from the group consisting of interleukin lalfa and interleukin-2. 41. The method of claim 1 comprising the myogenic induction and the bioactive factor is a myoinductive factor. 42. The method of claim 41 wherein the ioinductive factor is an analogue of cytidine. 43. The method of claim 42 wherein the cytidine analog is selected from the group consisting of 5-azacytidine and 5-aza-2'-deoxycytidine. 44. A composition comprising mesenchymal cells of human origin expanded by culture, isolated and an effective bioactive factor to induce the differentiation of such cells in a lineage of choice. 45. The composition of claim 14 further comprising a culture medium. 46. A composition of matter comprising the composition of claim 44 in a pharmaceutically acceptable carrier, 47. The composition of claim 46 wherein the pharmaceutically acceptable carrier is a rigid porous container. 48. The composition of claim 46 wherein the pharmaceutically acceptable carrier is a gel. 49. The composition of claim 46 wherein the pharmaceutically acceptable carrier is an injectable liquid.