TW201130977A - Mesenchymal stem cells (MSCs) isolated from mobilized peripheral blood - Google Patents

Abstract

The present invention generally relates to methods of obtaining peripheral blood-derived MSCs (PB-derived MSCs) and bone-marrow derived MSC (BM-derived MSCs) from a peripheral blood sample obtained from a human subject. In particular, the present invention relates to the surprising discovery that the mobilization of MSC into the peripheral blood is temporally regulated and that the number of circulating MSCs in human peripheral blood peaks after at least 2 days post administration of a mobilizing agent, such as G-CSF or GM-CSF to a human subject. Accordingly, in some embodiments, the present invention relates to methods of obtaining PB-derived and BM-derived MSC from peripheral blood from a human subject in vivo and ex vivo using mobilizing agents, such as G-CSF or GM-CSF, where administration of G-CSF and/or GM-CSF are optimized to mobilize MSCs and enrich the number of MSCs in the peripheral blood as apposed to other stem cell populations, such as HSCs.

Description

201130977 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to stem cells, in particular, methods and compositions for separating the optimal timing of human mesenchymal stem cells (MSCs) from mobilized peripheral blood. And isolated MSCs are then used, such as cryopreservation and/or for the treatment of individuals in need thereof, such as in autologous regenerative medicine. [Prior Art] Mesenchymal stem cells (MSC) are versatile in the field of cell therapy. cell. MSCs can differentiate into at least three downstream mesenchymal cell lineages (i.e., osteoblasts, chondroblasts, and adipocytes). To date, unique MSC markers have not been identified. Thus, these cells are identified using morphological and functional criteria. See Horwitz E et al., "Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement," Cytotherapy 7:393 (2005);

Dominici M et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement," Cytotherapy 8:315 (2006). Because MSCs can differentiate into many cell types, this technology encompasses methods for differentiated MSCs for cell-based therapies, regenerative medicine, and reconstituted medicine. A typical method for preparing MSC compositions consists of isolating MSCs based on the plastic adhesion properties of MSCs and subsequently culturing them. This method is relatively easy when MSCs are isolated from human body fluids during fetal development. However, obtaining MSCs from human body fluids is much more complicated and reproducible much lower. 152716.doc 201130977 Typically, MSCs are isolated from adult bone marrow, fat, cartilage, and muscle. Pittenger F et al., "Multilineage potential of adult human mesenchymal stem cells," Science 284: 143-147 (1999); Zuk P et al., "Multilineage cells from human adipose tissue: implications for cell-based therapies," Tissue Eng. 7:211-228 (2001); and Young H et al., "Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors," Anat. Rec. 264: 51-62 (2001). It can also be derived from human newborn tissue, such as Wharton's jelly (Wang Η et al., "Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord," Stem Cells 22: 1330-1337 (2004)), humans. Placenta (Fukuchi Y et al., "Human placenta-derived cells have mesenchymal stem/progenitor cell potential," Stem Cells 22: 649-658 (2004)); and cord blood (Erices A et al., "Mesenchymal progenitor cells in human umbilical Cord blood,"Br. J. Haematol. 109:235-242 (2000)) and human fetal tissue (Campagnoli C et al., "Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow , Blood 98: 2396-2402 (2001)) Separation of MSCs. This technique is limited in that sufficient MSC cannot be isolated for subsequent differentiation and use. Invasive procedures required to isolate even a limited number of 152716.doc 201130977 MSCs, such as bone marrow biopsy, present a significant risk to the donor, where appropriate donors are available. It is also difficult to maintain the isolated MSCs in a long-term culture state and to maintain such cultures free from contamination by bacteria or viruses. It has previously been reported that Msc can be isolated from peripheral blood that is not mobilized by humans. Kassis I et al. reported that "Is〇lati〇n 〇f mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads, using fibrin beads from G_csf d Bone Marrow Transplant. 37:967-976 (2GG6)〇Kassis I et al. reported that MSCs were obtained from mobilized peripheral blood and, to a lesser extent, from non-mobilized peripheral blood. It has been reported that by administering granulocyte communities Stimulus factor (Gr_l〇cyte_c〇l〇ny is 1)

Factor, G-CSF) mobilizes Msc to peripheral blood for at least 5 days. However, this method appears to be defective and poorly reproducible because only a small percentage of the magic is separated' and the growth and expansion potential of these Mscs is limited. Therefore, it is necessary to find an improved method for obtaining MSCs from peripheral blood that allows for the preparation of enriched MSC populations. The citation of the above documents is not intended to be a commitment to any of the foregoing matters as related prior art. All representations of all statements or content on the date of these documents are based on the information available to the applicant and are not an admission as to the correctness of the period or content of such documents. SUMMARY OF THE INVENTION The present invention is generally directed to the discovery of the optimal time sequence for collecting scorpion stem cell populations in the peripheral blood of the sputum. In particular, the inventors have discovered that in the administration of mobilizing agents (the

Optimal timing for collecting and isolating mesenchymal stem cells (MSCs) from mobilized peripheral blood such as, but not limited to, GM-CSF and G-CSF 152716.doc 201130977 or AM3100 (Plerixafor) . In other words, the inventors have surprisingly discovered a method of achieving superior MSC production by isolating the mobilized MSC at the optimum point in time when the MSC content in the circulated peripheral blood is maximized after administration of the mobilizer. The present inventors have surprisingly found that 'the mobilization of MSCs into peripheral blood can be modulated in time' and after circulating a mobilizing agent (such as G-CSF or GM-CSF) to human subjects, circulating MSCs in human peripheral blood The number of peaks is different from other mobilized stem cell populations (such as HSC). Thus, the inventors have demonstrated that the regime of using G-CSF or GM-CSF to optimize mobilization of MSCs into peripheral blood differs from the regimen typically used to mobilize other stem cells, such as CD3 4 + HSC, into peripheral blood. Thus, the present invention relates to the discovery that different stem cell populations are mobilized from bone marrow in a time-controlled manner. Specifically, the inventors have discovered that at different times after administration of a particular stem cell mobilizer, the bone marrow releases or produces a different stem cell population' and therefore needs to coordinate the timing of collecting or isolating a stem cell population from the peripheral blood mobilized. To optimize the separation of specific related stem cell populations. For example, the inventors have discovered that administration of a stem cell mobilizer to an individual will mobilize stem cells at different rates 'different durations and different amounts at different times' and thus the inventors have discovered that they are to be collected from the peripheral blood of the mobilization. The relevant stem cells will determine the coordinated dosing regimen and collect stem cells from the peripheral blood of the mobilization. For example, the inventors have discovered that peripheral blood mobilization factors (such as GM-CSF and/or G-CSF) mobilize different stem cell populations (such as hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), and other stems in a unique time pattern. 152716.doc 201130977 Cell populations, such as very small embryo-like (VSEL) stem cells ("VSEL·")) enter the peripheral blood. Surprisingly, unlike the conventional use of GM-CSF and/or G-CSF (with a period of at least 5 days and collecting HSC on day 6), the inventors have unexpectedly discovered that GM-CSF and/or can be administered. Or about 2 days after G-CSF, a high concentration of MSC is achieved in the mobilized blood. Accordingly, the inventors have discovered a method of obtaining optimal MSc yield in which MSCs are collected or isolated from the mobilized peripheral blood for at least 1 day, but up to 4 days after administration of the mobilizer. In one embodiment, 'administering the mobilizer for about 2 days' and on the last day of administration of the mobilizer or collecting MSCs in the second sputum, there are other mobilizers with different time responses' and they can be used for administration in the administration The MSC is then mobilized for at least 1 day and less than 4 days. The mobilization of stem cells at a conventional dosage (eg, typically 5 days, about 5-1 〇pg/kg per day), such as G-CSF or GM-CSF, is expensive and has undesirable side effects, including extensive bone pain, Headache, fatigue, and potentially spontaneous splenic rupture' have an unknown side effect on normal donors, including increased risk of hematopoietic malignancies and myeloid leukemia (AML) (see Casheen et al., Bone Marrow Transplant, 2007, 39; 577-588) . In addition, conventional transfer protocols are unpredictable, resulting in lower yields and typically take up to about 4 weeks of G-CSF or GM-CSF treatment to collect enough material for transplantation. After treatment with growth factors, HSC CD34+ cells reached a maximum of 2-4% in the blood. Usually obtained from bone marrow MSC » previously reported] Vise is present in peripheral blood and mobilized peripheral blood at very low levels (see Wexler et al., British J. 1527I6.doc 201130977)

Haematology, 2003; 121; 368-374; "Adult Bone Marrow is a rich source of human mesenchymal "stem" cells but umbilical cord and mobilized adult blood are not"). In addition, the report has stated that not all mobilized blood can produce MSCs (see Kassis et al., Bone Marrow Transplantation, 2006; 37; 967-976). In addition, even if the MSC is collected from non-mobilized peripheral blood, it cannot be collected for future autologous transplantation, even if culture is expanded, because MSC has aging properties and loses its pluripotency potential. The number of previous passages was limited. Thus, the present invention relates to the surprising discovery that, contrary to previous reports, the optimal time for MSC to be at the highest yield in the circulation can be obtained from the mobilized peripheral blood when the MSC is obtained or collected from the circulated peripheral blood. A sufficient amount of MSC for therapeutic use. Another aspect of the present invention relates to the discovery that MSCs that migrate from bone marrow to peripheral blood during the process known as "mobilization" also proliferate in peripheral blood. Thus, a mobilizing agent (such as GM-CSF and G-CSF or an agonist thereof) as disclosed herein can (1) be administered to an individual to enhance mobilization of MSCs from the bone marrow into the peripheral blood to increase peripheral blood 趱 (趱 source 磔 MSC ( The number of "BM-derived MSCs" referred to herein, and/or (ii) ex vivo contact of the peripheral blood of the individual with a mobilizing agent (such as GM-CSF and G-CSF) to mobilize and enhance MSCs in the periphery. The proliferation in the blood increases the number of eyebrows in the peripheral blood from the "PB-derived MSC" herein. Other mobilizers for use in the practice of the invention include those selected from the group consisting of interleukin-17, AMD3100, cyclophosphamide (C), docetaxel, and (DXT). 152716. Doc 201130977 Accordingly, one aspect of the present invention relates to the administration of GM-CSF and/or G-CSF to an individual to enhance in vivo mobilization of MSC; and BM-derived MSC and PB-derived mobilization in peripheral blood of an individual The separation of MSCs is optimized. Accordingly, the present invention relates to a method for obtaining peripheral blood-derived human mesenchymal stem cells from a sputum, comprising: a.  Administering to the individual an effective amount of G-CSF or GM-CSF for 4 days or less; b.  A human mesenchymal stem cell population is obtained from a peripheral blood sample of the individual, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and surface markers of CD44+, CD90+, CD105+, CD29+, and CD73+ cells are present. Positive; and c.  Human mesenchymal stem cells are isolated relative to other somatic stem cells in the peripheral blood. The invention further relates to a method for increasing a population of human mesenchymal stem cells in peripheral blood of an individual, comprising: a_ administering an effective amount of G-CSF or GM-CSF to the periphery of the individual for 4 days or less; b· A human mesenchymal stem cell population is obtained from a peripheral blood sample of the individual, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and are surface markers for CD44+, CD90+, CD105+, CD29+, and CD73+ cells. Positive; and c. Human mesenchymal stem cells are isolated relative to other adult stem cells in the peripheral blood. In a particular embodiment of the invention, an effective amount of G-152716 is administered to the individual. Doc •10· 201130977 CSF or GM-CSF for 3 days or less, for 2 days or less, or for 1 day or less. In another embodiment of the invention, the individual is a healthy individual. Another aspect of the present invention relates to contacting peripheral blood of an individual with GM-CSF and/or G-CSF in vitro; and optimizing separation of mobilized PB-derived MSCs from peripheral blood of the individual. Accordingly, the present invention is directed to a method of obtaining human mesenchymal stem cells from an individual comprising: a.  Causing a peripheral blood sample from the individual to contact an effective amount of g-CSF or GM-CSF to increase the number of human mesenchymal stem cells in the peripheral blood sample; b.  Human mesenchymal stem cell population obtained from peripheral blood samples, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and positive for CD44+, CD90+, CD105+, CD29+, and CD73+ cell surface markers; .  Human mesenchymal stem cells are isolated relative to other adult stem cells in the peripheral blood. The invention also relates to a method of obtaining human mesenchymal stem cells from an individual, comprising: a. The peripheral blood sample is contacted with an effective amount of g-CSF or GM-CSF in vitro to increase the number of human mesenchymal stem cells in the peripheral blood sample; b_ separating the human mesenchymal stem cell population from the peripheral blood sample, wherein the human human The stem cells were negative for CD34-, CD45-cell surface markers and positive for CD44+, CD90+, CD105+, CD29+ and CD73+ cell surface markers. 152716. Doc • 11· 201130977 In one embodiment of the invention, a peripheral blood sample can be obtained from an individual via apheresis. The peripheral blood sample can be a cultured peripheral blood sample. In another embodiment of the invention, the peripheral blood sample can be a fresh peripheral blood sample. The stem cell population derived from peripheral blood can be expanded by culture. In addition, human mesenchymal stem cells derived from peripheral blood can be cryopreserved. In one embodiment of the invention, the human mesenchymal stem cell population is obtained from a peripheral blood sample using a size-based separation method. The human mesenchymal stem cell population may also use a negative selection method that excludes non-mesenchymal stem cells. Peripheral blood samples were obtained from which a human mesenchymal stem cell population was isolated and collected from samples containing mesenchymal stem cell populations and non-mesenchymal stem cell populations. Human mesenchymal stem cell populations can also be obtained from peripheral blood using any combination of the following methods: positive selection based on cell surface markers, negative selection based on cell surface markers, positive selection based on size, negative selection based on size . The invention also relates to a pure lineage cell line comprising a substantially pure human mesenchymal stem cell population isolated using the methods of the invention. The pure cell line can be placed in a container containing a suitable medium. Further, a container containing a pure cell line suitable for the medium and the human mesenchymal stem cell population can be cryopreserved. Thus, in contrast to the conventional use of G-CSF or GM-CSF for in vivo mobilization, the inventors provide a key advantage of significantly reducing the risk to the individual and the cost associated with the procedure, even providing a means of collecting cells. Timing optimization is the method of finding the time at which the concentration of cells (e.g., MSCs in the blood) is maximized, thereby increasing the yield of mobilized cells and reducing non-MSC contamination. 1527l6. Doc •12· 201130977 Another aspect of the invention relates to the mobilization of MSCs from peripheral blood, wherein the peripheral blood of the individual is contacted with a mobilizing agent such as GM-CSF and G-CSF, and the mobilized peripheral gold Peripheral MSCs were collected and isolated in vitro. In some embodiments, peripheral blood is freshly separated from an individual, such as a human individual. In some embodiments, the peripheral blood is from an enterprise library or has been stored in a blood bank. In some embodiments, peripheral blood has been stored or preserved. In some embodiments, the peripheral blood is menstrual blood, as disclosed in U.S. Patent Application Serial No. 2008/0241, the entire disclosure of which is incorporated herein by reference. Another aspect of the invention relates to the separation of mesenchymal stem cells (MSCs) at a time point in the peripheral blood from the time when the number of MSCs mobilized from bone marrow (e.g., BM-derived MSC) or from peripheral blood (e.g., PB-derived MSC) peaks. . In some embodiments, BM-derived MSCs and PB-derived MSCs can be used to generate pure human mesenchymal progenitor cell lines, and more particularly, in one embodiment, provide a utilization of BM-derived MSCs and PB-derived MSCs. Methods of producing individual (e.g., individual-specific or personalized) pure lineage MSC strains obtained from the peripheral blood of an individual in vivo or ex vivo. As discussed herein, the inventors have demonstrated that in vivo administration of low doses of GM-CSF and G-CSF (e.g., compared to conventional uses of GM-CSF and G-CSF as mobilizers, lower doses) is obtained. The peak number of MSCs in peripheral blood and the high yield of mobilized PB-derived and BM-derived MSCs present in peripheral blood of human subjects are optimal. Accordingly, the inventors have demonstrated that low doses of GM-CSF and G-CSF or agonists thereof can be used in vivo to optimize peripheral blood collection MSCs from mobilization for use in individuals (eg, human peripheral blood) MSCs prepare compositions for cell therapy. 152716. Doc-13-201130977 In some embodiments, the methods described herein comprise obtaining bone marrow-derived MSCs or peripheral blood-derived MSCs from an individual. All BM-derived MSCs or PB-derived MSCs or subpopulations thereof can then be administered to an individual, such as the same individual from which the MSC originated (eg, all〇genic transplantation) or, in some cases, administered Two individuals (eg, non-allogeneic transplants) (the second individual with an HLA type match). In some embodiments, the method also includes separating BM-derived MSCs and/or PB-derived MSCs from the blood, for example, using bloodpheresis or leuk〇pheresis. In some embodiments, the isolated BM-derived MSC or PB-derived MSC can be administered to an individual in need thereof, wherein the individual is the same individual as the MSC-derived individual (eg, allograft)' or in some cases A second individual, such as a non-allograft, is matched to a second individual (eg, an HLA type). In some embodiments, the individual in need has a condition selected from the group consisting of cancer and autoimmune diseases or requires regenerative therapy. In another aspect, the methods described herein comprise obtaining peripheral blood from an individual to mobilize peripheral blood with an effective amount (eg, an amount sufficient to increase the number of PB-derived MSCs in the peripheral blood ex vivo) as described herein. The agent (such as g_cj§f or GM-CSF) is contacted. In some embodiments, the pB-derived msc in the peripheral blood can be isolated, enriched, or purified prior to administration to the individual. As the case may be, in some embodiments the treated peripheral blood is then administered to an individual, such as the same individual or a second individual, such as a second individual that is HLA type matched. One aspect of the present invention relates to a composition β obtained by isolating mesenchymal stem cells (MSC) from peripheral blood mobilized and culturing such compositions using GM-CSF and G-CSF as a mobilizing agent. 152716. Doc -14- 201130977 The present invention relates to the use of a human mesenchymal stem cell population or a differentiated progeny group thereof for the treatment of a disease or condition in an individual in need thereof, wherein the human mesenchymal stem cell population is isolated using any of the above technical means, And wherein the individual is administered to the individual in need thereof, or the differentiated progeny group thereof, to achieve autologous regenerative therapy. In one embodiment, the invention relates to a method of treating a human subject wherein the number of MSCs is increased' and Msc is collected and used for cell transplantation. The method of the present invention employs Msc mobilizers as described in the patents and publications, including GM-CSF and G-CSF, which are incorporated herein by reference. Accordingly, the invention provides a method of treating a disease or condition in an individual with autologous mesenchymal stem cells (MSC), comprising: a.  Utilizing a MSC population of peripheral blood samples, wherein the peripheral blood sample is obtained from an individual in which the mobilizer has been administered for 4 days or less, and wherein the Msc population is an Msc population enriched relative to other stem cells in the surrounding enterprise; And b.  The MSC population is administered to the individual to treat the disease or condition. In one embodiment of the invention, the MSC can be enriched by a forward selection method or by elutriation. The enriched MSC population may comprise at least 1% MSC. The enriched MSC population can be a substantially pure MSC population. The enriched Msc population can comprise non-M S C cells. Alternatively, the MSC population can be a population of cells that have been cryopreserved. In addition, the MSC cell population can be expanded in vitro prior to administration to an individual. One aspect of the invention relates to the addition of a MSC population in a peripheral blood sample 152716. Doc • 15. 201130977, wherein the peripheral blood sample is present in an individual, such as a human individual. Another aspect of the invention is a method of enriching a BM-derived MSC and/or a PB-derived MSC population in a peripheral blood sample from an individual in vivo or ex vivo. In some embodiments, the in vivo method comprises isolating a population of human mesenchymal stem cells from peripheral blood of an individual collecting mesenchymal stem cells by the following steps: (1) administering to the individual at least one mobilizing agent (such as G_CSF or gm-CSF) ), for 4 days or less; (Η) obtaining peripheral blood samples from the individual; (iii) isolating the MSC population from other cells in the peripheral blood of the individual, and (iv) culturing the MSC as appropriate. The culture and isolation steps can also reverse the sequence, i.e., PB-derived MSCs or BM-derived MSCs as disclosed herein can be isolated from peripheral blood and subsequently cultured for amplification. Methods of isolation include leukocyte hemolysis, density gradient fractionation, immune selection, and differential adhesion separation. The medium may be a chemically defined serum-free medium or may be a "complete medium" such as DMEM or DMEM containing 1 g of serum. A suitable chemically defined serum-free medium is described in U.S. Patent No. 5,486,359 issued to Jun. 5, 1995, which is incorporated herein by reference. in. In some embodiments, BM-derived MSCs and/or PB-derived MSCs from an individual are expanded or cultured ex vivo prior to storage (e.g., cryopreservation). In some embodiments, the substantially pure human MSC population is derived from a human subject in which at least one mobilizing agent (eg, G-CSF and/or GM-CSF) has been administered for 4 days or less. A substantially pure heterologous population comprising BM-derived human MSCs and PB-derived human MSCs. 152716. Doc -16· 201130977 In some embodiments, a substantially pure human MSC population of a human individual who has been administered with at least one mobilizer (eg, g_csf and/or GM-CSF) for 4 days or less comprises BM source. A substantially pure population of human MSCs. In an alternative embodiment, a substantially pure human MSC population of a human individual from which at least one mobilizer (eg, g_csf & / or gm-CSF) has been administered for 4 days or less comprises PB-derived human MSCs. Essentially pure group. Another aspect of the invention is a method for ex vivo enrichment of a PB-derived MSC population in a peripheral blood sample of an individual. In some embodiments, the ex vivo method comprises isolating a human mesenchymal stem cell population from peripheral blood of an individual enriched mesenchymal stem cell by the step of: circumventing a peripheral blood sample previously obtained from the individual with at least one mobilizing agent (such as G-CSF or GM-CSF) exposure is for 4 days or less; the MSC population is separated from other cells in the peripheral blood sample, and MSCs are cultured as appropriate. In some embodiments, a PB-derived MSC obtained from a peripheral blood sample previously obtained from an individual can be deposited. In some embodiments, the deposit is cryopreserved. In some embodiments, the PB-derived MSC is expanded or cultured in vitro prior to storage (e.g., cryopreservation). Another aspect of the invention is additionally directed to a method of ex vivo isolation of PB-derived MSCs or BM-derived MSCs obtained by a method as disclosed herein, wherein the method comprises (1) administering to the individual at least one mobilizing agent (such as G-CSF or GM-CSF) for 4 days or less; (ii) obtaining peripheral blood samples from the individual; (iii) separating the MSC population from other cells in the peripheral blood of the individual, and optionally (iv) Cultivating MSC; and (v) preserving isolated PB-derived MSCs or BM-derived MSCs. The preservation is preferably cryopreservation. Another aspect of the invention pertains to obtaining 152716 by the method as disclosed herein. Doc -17-201130977 A substantially pure population treatment of a subject in need of treatment with a substantially pure population of PB-derived MSCs and/or BM-derived MSCs, the method comprising administering to the individual substantially pure PB-derived MSCs or BM-derived MSCs group. In some embodiments, the PB-derived MSC or BM-derived MSC has been freshly isolated by methods as disclosed herein, or in some embodiments, PB-derived MSCs or BM-derived MSCs have been known to those skilled in the art. It is usually cryopreserved using known methods. In some embodiments, a substantially pure population of PB-derived MSCs and/or BM-derived MSCs may be required to produce new tissue by, for example, systemic infusion or local implantation, such as by incision or arthroscopic procedures. The part is administered. Cells can be preserved prior to re-injection. One aspect of the invention relates to a method of obtaining peripheral blood-derived human mesenchymal stem cells from an individual comprising: (a) administering to the individual an effective amount of G-CSF or GM-CSF for 4 days or less. (b) obtaining human mesenchymal stem cell populations from peripheral blood samples obtained from the individual, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and for CD44+, CD90+ 'CD105+, CD29+, and CD73+ The cell surface marker is positive. In some embodiments, the invention relates to a method of increasing a human mesenchymal stem cell population in a peripheral blood of an individual comprising: (a) administering to the individual an effective amount of G-CSF or GM-CSF for a period of 4 days or less 4 days; (b) obtaining human mesenchymal stem cell populations from peripheral blood samples obtained from the individual, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and for CD44+, CD90+, CD105+, CD29+ and The CD73+ cell surface marker is positive, and (c) the human mesenchymal stem cells are isolated relative to the adult stem cells in the peripheral blood. 152716. Doc -18· 201130977 Another aspect of the invention relates to a method of obtaining human mesenchymal stem cells from an individual comprising: contacting a peripheral blood sample from the individual with an effective amount of G-CSF or GM-CSF to increase the periphery The number of human mesenchymal stem cells in the blood sample; (b) the human mesenchymal stem cell population obtained from the peripheral blood sample, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and for CD44+, CD90+, CD105+ , CD29+ and CD73 + cell surface markers are positive; and (c) isolation of human mesenchymal stem cells relative to adult stem cells in peripheral blood. In some embodiments the method comprises administering to the individual a mobilizing agent (such as an effective amount of G-CSF or GM-CSF, or AM3100) for a period of 3 days or less. In some embodiments, an effective amount of a mobilizing agent (such as G-CSF or GM-CSF or AM3100) is administered to an individual for a period of 2 days or less, or for 1 day or less. In some embodiments, an effective amount of a mobilizing agent is administered to a healthy individual. In some embodiments, the human mesenchymal stem cell population is obtained from a peripheral blood sample using a size-based separation method such as panning. In some embodiments, the 'human mesenchymal stem cell population is obtained from a peripheral sample using a negative selection method that excludes non-mesenchymal stem cells, wherein the human mesenchyme is isolated and collected from a sample comprising a mesenchymal stem cell population and a non-stromal stem cell population. Stem cell population. In some embodiments, the human mesenchymal stem cell population is obtained from peripheral blood using any combination of the following methods: positive selection based on cell surface markers, negative selection based on cell surface markers, positive selection based on size, based on size Negative choice. Another aspect of the invention relates to obtaining human mesenchymal stem cells from an individual 152716. Doc • 19·201130977 Method comprising: contacting a peripheral blood sample with an effective amount of G-CSF or GM-CSF in vitro to increase the number of human mesenchymal stem cells in a peripheral blood sample; (b) separating humans from peripheral blood samples a mesenchymal stem cell population, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and positive for CD44+, CD90+, CD105+, CD29+, and CD73+ cell surface markers, and (c) relative to peripheral blood Adult mesenchymal stem cells are isolated from adult stem cells. In some embodiments, the MSC is obtained from the mobilized peripheral blood sample being cultured. In some embodiments, the MSC is obtained from a peripheral blood sample that is a fresh peripheral blood sample. In some embodiments, the MSC is obtained from a population of human mesenchymal stem cells that have been cultured and expanded from peripheral blood samples. In some embodiments, the human mesenchymal stem cell population derived from the peripheral blood sample is preserved at an ultra-low temperature. In some embodiments, the individual is a human individual. Another aspect of the invention pertains to a pure lineage cell line comprising a population of substantially pure human mesenchymal stem cells isolated using the method of any of the above aspects. Another aspect of the invention pertains to a container comprising a pure lineage of a human mesenchymal stem cell population suitable for the culture medium and obtained by the methods disclosed herein. In some embodiments, a pure cell line suitable for culture and human mesenchymal stem cell populations is cryopreserved. Another aspect of the invention pertains to cryopreservation of a human mesenchymal stem cell population obtained by any of the methods disclosed herein. Another aspect of the invention relates to the use of a human mesenchymal stem cell population or a differentiated progeny population thereof for treating a disease or condition in a subject in need thereof, wherein the human mesenchymal stem cell population is used in any of 152,716. Doc • 20· 201130977 The method of the technical protocol is isolated, and the individual w-type mesenchymal stem cell population or its differentiated progeny group is required to achieve autologous regenerative therapy. A further aspect of the invention relates to a method for treating a disease or condition in an individual with autologous mesenchymal stem cells (MSC), comprising: (4) using a MSC population of peripheral blood samples, wherein the peripheral blood sample is self-mobilizing Obtained with an individual for 4 days or less, and wherein the Msc population is an enriched MSC population relative to other stem cell populations in the blood; and (b) administering the Msc population to the individual to treat the disease or condition . In some embodiments, the MSC is enriched by a forward selection method. In an alternative embodiment, the Msc is enriched by panning. In some embodiments the 'MSC population has been cryopreserved. In some embodiments, the enriched MSC population comprises non-MSC cells, or in some embodiments the 'enriched MSC population comprises at least 10% MSC or substantially pure ^8 (: population). In some embodiments, The MSC population has been expanded in vitro prior to administration to the individual. In some embodiments, the individual is a human subject. The invention further provides methods of obtaining peripheral blood-derived human stem cells from an individual comprising: a.  Administering to the individual an effective amount of the mobilizer for a period of 4 days or less; b.  Obtaining a human stem cell population from a peripheral blood sample of the individual; and c.  Human stem cells are isolated relative to other adult stem cells in the peripheral blood. In a particular embodiment of the invention, the stem cells are mesenchymal stem cells or "very small embryonic stem cells (VSELs)". Further, the mobilizer may be G-CSF or GM-CSF. Alternatively, the mobilizing agent is selected from the group consisting of interleukin-7, AMD3 100, cyclophosphamide (Cy), European paclitaxel, and (DXT) 152716. Doc -21- 201130977 Group ο [Embodiment] Mesenchymal stem cells (MSC) are non-hematopoietic stem cells usually obtained from Bone. The presence of MSCs in peripheral blood is extremely low and is collected from peripheral blood resulting in insufficient amounts for therapeutic use, even if it is expanded in vitro. The inventors have discovered that MSCs can be obtained from the peripheral blood of the mobilization. Specifically, 'self-adjustable agents (such as, but not limited to, G-CSF, GM-CSF, AMD3 100, etc.) have been administered and collected by individuals for 4 days or less (such as about 2 days). Blood Separation and Panning Collection] In some embodiments, 'human MSCs isolated from the peripheral blood of the mobilization can be cultured and/or cryopreserved for future use, for example, where Ms can be used. Used alone or in combination with other cells in regenerative therapy for non-therapeutic applications such as wound healing and bone repair and other orthopedic indications. The inventors have surprisingly discovered that it is possible to modulate the mobilization of MSCs into peripheral blood over time and to circulate in human peripheral blood at least one day after administration of a mobilizer (such as G-CSF or GM-CSF) to a human subject. The number of MSCs peaks. Thus, the inventors have demonstrated that the use of G_CSF or GM-CSF to optimize mobilization of MSCs into peripheral blood differs from the regimen typically used to mobilize other stem cells, such as CD34+HSC, into peripheral blood. The inventors have surprisingly discovered that short-term (eg, at least 2 days, but less than 4 days) after administration of G-CSF or GM-CSF to a human subject will result in the highest yield of MSC in the mobilized peripheral blood, It is not customary to use a regimen that gives individuals 5 days of G-CSF or GM-CSF. Thus, the inventors have determined that 'for a particular application to be applied' a stem cell mobilizer (such as G_152716. Doc •22· 201130977 CSF or GM-CSF) The program needs to be optimized by: G-CSF or GM-CSF under different conditions and then generally known and familiar to those skilled in the art The methods disclosed herein monitor the output of a desired subset of stem cells (such as MSCs, pebbles, hematopoietic cells) in peripheral blood. The time limit for transfer of a mobilizer (such as G-CSF or GM-CSF) to optimally recover or induce a specific cell population in the blood should be monitored, as different cell types will occur sequentially after administration of the mobilizer. Another aspect of the invention pertains to the use of MSCs derived from individual mobilized peripheral blood for personalized medical applications, such as autologous therapeutic uses' and for personalization assays to assess personal diet, drugs The effects of genetics, neurochemicals, and lifestyle on MSC function and vitality. 4 _ For convenience, some of the terms used in this document, the description, the examples, and the accompanying claims are hereby incorporated. Unless otherwise stated or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise stated or apparent from the context, the following terms and phrases do not exclude the meaning of the term or phrase in the art to which it belongs. The definitions are provided to help describe a particular embodiment and are not intended to limit the claimed invention, as the scope of the invention is limited only by the scope of the claims. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise claimed. The term "mesenchymal stem cells" is also referred to herein as r MSC", which refers to a body tissue that is capable of differentiating into more than one specific type of interstitial or connective tissue (ie, supporting specialized elements; eg, fat connective tissue, bone connective tissue) Group 152716. Doc -23· 201130977 omnipotent stem cells of woven, matrix connective tissue, cartilage connective tissue, elastic connective tissue and fibrous connective tissue. Human mesenchymal stem cells (hMSC) react with certain monoclonal antibodies called SH2, SH3 and SH4. Sex. (See U.S. Patent No. 5,486,359, incorporated herein by reference in its entirety). MSCs can be distinguished from HSCs based on their immunospecific profiles, and wherein Msc is SH2+/CD14 and human HSC is SH27CD14+. For identification purposes, human MSCs can be identified based on: (i) phenotypic expression of CD34., CD45., CD90+, CD105+, and CD44+, (ii) functional phenotype, including CFA as disclosed in the Examples herein. The ability to form a colony forming unit in assays and to differentiate into tissues that support specialized elements including, but not limited to, chondrocytes, cartilage, and fat cells. Other markers represented by MSC are known in the art and include, but are not limited to, CD71, CD73, Stro-1, CD166, and CD271. In some embodiments, the MSC is lin. The term "very small embryonic stem cells" is also referred to herein as "VSEL stem cells" and refers to omnipotent stem cells. In some embodiments, the VSEL stem cells ("VSEL") are human VSELs and can be characterized as lin·, CD45·, and CD34+. In some embodiments, the VSELs are human VSELs and can be characterized as lin·, CD45·, and CD133+. In some embodiments, the VSELs are human VSELs and can be characterized as lin-, CD45, and CXCR4+. In some embodiments, the VSEL is a human VSEL and can be characterized as lin·, CD45·, CXCR4+, CD133+, and CD34+ » human VSELs exhibit at least one of SSEA-4, Oct-4, Rex-1, and Nanog and have The nucleus of the narrow margin is surrounded by a large nucleus and contains embryonic amorphous chromatin. VSEL also has high-end granzyme activity 152,716. Doc -24- 201130977 Sex. In some embodiments, the VSEL is a human VSEL and can be characterized as lin·, CD45-, CXCR4+, CD133+, Oct 4+, SSEA4+, and CD34+. In some embodiments, human VSELs can be less native and can be characterized as lin·, CD45-, CXCR4+, CD133·, and CD34+. In some embodiments, human VSELs can be enriched with universal embryonic transcription factors, such as Oct-4, Sox2, and Nanog. In some embodiments, the human VSEL can have a diameter of 4-5 μιη ' 4-6 μηι, 4-7 μηι, 5-6 μηι, 5,8 μηι, 6-9 μπι, or 7-10 μπι. The term "peripheral blood-derived MSC" or "PB-MSC" as used herein refers to an MSC that is only mobilized from peripheral blood and may include amplification/proliferation of MSCs in peripheral blood. In some embodiments, the number of circulating PB-MSCs in the peripheral blood can be increased by contacting the peripheral blood with the mobilizing agent in vivo or ex vivo according to methods as disclosed herein. The term "bone marrow-derived MSC" or "BM-MSC" as used herein refers to MSCs that are mobilized from bone marrow to peripheral blood and may include MSCs that have migrated from BM. In some embodiments, the BM-MSCs in the peripheral blood include MSCs that have proliferated in the bone marrow prior to migrating to the peripheral blood, or MSCs that have proliferated in peripheral blood after migration from the bone marrow. In some embodiments, the number of circulating BM-MSCs in the peripheral blood can be increased by contacting the peripheral blood with the mobilizing agent in vivo according to the methods as disclosed herein. Also known as "HSC", the term "hematopoietic stem cells" refers to the ability to differentiate into any particular type of hematopoietic cells or blood cells (such as red blood cells, lymphocytes, giant cells, and megakaryocytes), especially found in the bone marrow and in peripheral blood. All stem or progenitor cells. HSC is currently considered to have a special 152716 for hematopoietic cells. Doc -25« 201130977 Some monoclonal antibodies (such as CD34) of the opposite sex are reactive. Also known as "HSC", the term "hematopoietic cells" refers to all types of hematopoietic cells in the process of self-renewal of gold-derived stem cells into (and including) mature functional blood cells through immature precursor cells of various blood lines, such as Those familiar with this technology know. The term "mobilization" as used herein refers to the process by which cells leave the bone marrow and enter the bloodstream. The mobilization can be achieved by a combination of a chemoattractant (e.g., a cytokine) and a pool of stem cell pools or groups that are present in the surrounding tissue and in the stem cell niche of the bone. An "effective amount" is an amount sufficient to achieve a significant increase in the number and/or frequency of MSCs in the peripheral blood. An effective amount can be administered by one or more administrations, administrations or administrations. The therapeutically effective amount of the mobilizing agent will depend on the selected mobilizing agent (e.g., G_csf or GM-CSF). A mobilizing agent such as G_CSF or Gm-CSF may be administered one or more times per day to one or more times per week; including once every other day. Those skilled in the art will appreciate that certain factors may affect the dosage and timing required to effectively treat an individual, including, but not limited to, prior treatment, overall health and/or age of the individual, and other conditions present. Further, treating a subject with a therapeutically effective amount of G-CSi^^GM-CSF as described herein can include an early treatment or a series of treatments. The term "interstitial cells" or "interstitial" is used interchangeably herein and in some cases refers to spindle-shaped or stellate cells found between young embryonic ectodermal and endoderm; most interstitial cells Originated from the established mesoderm, but in the head region, it is also developed by the neural ridge or neural tube ectoderm. Interstitial cells have versatile ability, specifically, in the embryonic body 152716. Doc •26- 201130977 Embryonic mesenchymal cells can develop into any type of connective tissue or supporting tissue, smooth muscle, vascular endothelium and blood cells at different locations. As used herein, the term "stem cell" is used broadly and includes conventional stem cells, tendon cells, progenitor cells, reserve cells, and the like. The term "stem cell" or "progenitor cell" is used interchangeably herein and refers to an undifferentiated cell that is capable of proliferating and producing more progenitor cells that are capable of producing a plurality of mother cells, which in turn can produce Differentiated or differentiated daughter cells. The daughter cells themselves can be induced to proliferate and produce offspring, which then differentiate into one or more mature cell types, while also allowing - or multiple cells to retain parental developmental potential. The term "stem cell" refers to a cell that has the ability or potential to differentiate into a more specific or differentiated phenotype under specific conditions, and that retains the ability to proliferate without substantial differentiation in some cases. In one embodiment, the term progenitor or stem cell line refers to a generalized mother cell 'the progeny (progeny) are usually differentiated by differentiation in different directions, for example to obtain completely individual features, such as progressive differentiation of embryonic cells and tissues. The same happens in the middle. Cells differentiate into a complex process that usually occurs through multiple cell divisions. The differentiated cells may be derived from pluripotent cells, and the pluripotent cells themselves are derived from pluripotent cells' and the like. Although each of these pluripotent cells can be considered a stem cell, the range of cell types that each can produce can vary significantly. Some differentiated cells also have the ability to produce cells with greater developmental potential. This ability can be natural or can be artificially induced when processed with various factors. In many biological situations, stem cells also have "pluripotent" because they produce offspring of more than one unique cell type, but this is not required for "stem-ness." Self-renewal is another definition of stem cells. Doc •27· 201130977 Classic part, and it is important as used in this document. In theory, self-renewal can occur by either of two main mechanisms. Stem cells can be asymmetrically split, with one progeny retaining the trunk state and the other progeny exhibiting some unique other specific functions and phenotypes. Alternatively, some of the stem cells in the population can be symmetrically split into two stem cells, thereby generally maintaining some of the stem cells in the population, while other cells in the population only produce differentiated progeny. Cells that formally start in the form of stem cells are likely to progress toward a differentiated phenotype, but then "reverse" and re-express the stem cell phenotype, a process that is commonly referred to as "dedifferentiation." Exemplary stem cells include embryonic stem cells, adult stem cells, omnipotent stem cells, neural stem cells, hepatic stem cells, muscle stem cells, muscle precursor stem cells, endothelial progenitor cells, bone marrow stem cells, cartilage stem cells, lymphoid stem cells, mesenchymal stem cells, hematopoietic stem cells, central nervous cells. System stem cells, peripheral nervous system stem cells, and similar stem cells. Description of stem cells (including methods for isolating and culturing stem cells) can be found especially in Embryonic

Stem Cells, Methods and Protocols, Turksen, ed., Humana Press, 2002; Weisman et al, Annu. Rev. Cell. Dev. Biol. 17:387 403; Pittinger et al, Science, 284: 143 47, 1999; Animal Cell Culture , Masters, ed., Oxford University Press, 2000; Jackson et al, PNAS 96(25): 14482 86, 1999; Zuk et al., Tissue Engineering, 7:211 228, 2001 ("Zuk et al."); Atala et al. In particular, Chapters 33, 41; and U.S. Patent Nos. 5,559,022, 5,672,346 and 5,827,735. Descriptions of stromal cells (including methods for isolating stromal cells) are found in particular in Prockop, Science, 276: 71 -28 152 716. doc 2011 30 977 74, 1997; Theise et al, Hepatology, 31: 235 40, 2000; Current Protocols in Cell Biology, edited by Bonifacino et al., John Wiley & Sons, 2000 (including updates up to March 2002); and U.S. Patent No. 4,963,489. The term "progenitor cell" is used herein to refer to a cell that is more prototypically phenotypically relative to a cell that can be produced by differentiation (eg, at a stage or process that is earlier than the fully differentiated cell). Progenitor cells also have significant or extremely high proliferative potential. Depending on the developmental pathway and the environment in which the cells are developed and differentiated, progenitor cells can produce a variety of uniquely differentiated cell types or single differentiated cell types. As indicated above, cells can be classified into different grades or species according to the general definition of "stem cells." There are "totipotent", "pluripotent" and "multipotent" stem cells. The term "all-round" means that stem cells can produce any tissue or cell type in the body. "Universal" stem cells produce any type of cell in the body other than germline cells. Stem cells that produce a smaller or limited number of different cell types are commonly referred to as "pluripotent." Thus, totipotent cells differentiate into pluripotent cells that produce most, but not all, tissues necessary for fetal development. The unipotent cells are further differentiated into pluripotent cells that specifically produce cells with specific functions. For example, pluripotent hematopoietic stem cells produce red blood cells, white blood cells, and platelets in the blood. The term "universal" as used herein refers to a cell type that cells can differentiate into all three germ cell layers (endoderm, mesoderm, and ectoderm) under different conditions. The main feature of omnipotent cells is their ability to differentiate into 152716.doc -29· 201130977 There are 3 germ layers (using, for example, a nude mouse teratoma formation assay). The versatility is also confirmed by the expression of embryonic stem (ES) cell markers, but a better test of omnipotence is the ability to demonstrate differentiation into cells of each of the three germ layers. In some embodiments, the omnipotent cells are undifferentiated cells. The term "universal" or "pluripotent state" as used herein refers to a cell that is capable of differentiating into all three embryonic germ layers: endoderm (digestive tract tissue), mesoderm (including blood, muscle and blood vessels), and ectoderm ( Such as skin and nerves' and usually have the potential for long-term (for example, more than 1 year or more than 30 passages) in vitro. The term "multi-energy" refers to the ability of cells to differentiate into some when used with "pluripotent cells". (but not all) cells derived from all three germ layers. Therefore, pluripotent cells are partially differentiated cells. Pluripotent cells are well known in the art, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. Multi-meaning means that stem cells can form many types of cells in a given lineage, rather than cells of other lineages. For example, pluripotent blood stem cells can form many different types of blood cells (red blood cells, white blood cells, blood platelets, etc.), but they cannot form neurons. The term "versatility" refers to the degree of versatiability (versatiHty) of cells that is less than omnipotent and omnipotent. The term "totipotency" refers to the degree of differentiation of a cell that describes the ability to form all cells in an adult as well as extraembryonic tissues, including the placenta. As with early dividing cells (blastomere), fertilized eggs (zygotes) are pluripotent. The term "isolated cells" as used herein refers to 152716.doc • 30-201130977 cells or progeny of such cells that have been removed from an individual, wherein the cells were originally discovered in the individual. The cells have been cultured in vitro, for example, in the presence of other cells, as appropriate. The cells produced by the methods disclosed herein (eg, isolated PB-derived MSCs or isolated BM-derived MSCs) are then introduced into the second individual or re-introduced into the isolated (eg, allograft) cells, as appropriate ( Or the individual from which the cell from which it is derived. The term "isolated population" as used herein with respect to an isolated population of cells refers to a population of cells that have been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, the isolated population is a substantially pure population of cells compared to a heterogeneous population in which the cells have been isolated or enriched. In some embodiments, the isolated population is a reprogrammed cell population that is substantially reprogrammed by a heterogeneous population of cells comprising reprogrammed cells and cells from which the reprogrammed cells are derived. Pure population The term "substantially pure" with respect to a particular cell population means that the cell population has a purity of at least about 75%, preferably at least about 85°, for the cells that make up the total cell population. More preferably, it is at least about 90% and optimally at least about 95%. In other words, the use of a PB-derived MSC or a BM-derived MSC population isolated by the methods disclosed herein refers to a pB-derived MSC or BM. The source MSC population contains less than about 2%, more preferably less than about 15%/〇, 10%, 8%, 7%, optimally less than about 5〇/〇, 4〇/〇, 3%, 2%, 1 Cells of % or less than 1 〇/〇 are not p B-derived M s C or BM-derived MSCs as defined herein. In some embodiments, the invention encompasses a method of amplifying a BM-derived MSC or a BM-derived MSC population, wherein the amplified sputum-derived MSC or BM-derived MSC population is the source of the priming MSqBM source 152716. Doc -31 _ 201130977 on the pure group. 2: As used herein, "proliferation" means that the number of cells in a population is increased (growth) by means of a fine knife 4. Cell proliferation is generally understood to be caused by coordinated activation of multiple signaling pathways in response to the environment, including growth factors and other fractions (4). Cell proliferation can also be promoted by the action of intracellular or extracellular signals and mechanisms that block or negatively affect cell proliferation. The term "regeneration" means the regrowth of a cell population, organ or tissue after a disease or injury. The terms "enriched" or "enriched" are used interchangeably herein and mean that the yield (fraction) of a cell of a certain type is increased by at least 10% compared to the fraction of cells of that type in the starting culture or preparation. /〇. The terms "update" or "self-renewal" or "proliferation" are used interchangeably herein and refer to the process by which a cell produces more copies of the cell itself (e. g., replication). In some embodiments, the reprogrammed cells can self-renew by dividing into the same undifferentiated cells (e.g., omnipotent or non-specific cell types) over a long period of time and/or months to years. In some cases, proliferation refers to the reprogramming of cells that are amplified by repeated splitting of individual cells into two identical daughter cells. The term "lineage" as used herein refers to a term describing a cell having a common cell or a cell having a common developmental fat (e.g., a cell derived from the same PB-derived MSC or BM-derived MSC or a progeny thereof). As used herein, the term "pure cell line" refers to a cell lineage that is maintained in culture and has the potential for unlimited reproduction. The pure cell line may be a stem cell strain (for example, a PB-derived MSC or a BM-derived MSC cell strain) or a source derived from the pb source 152716.doc •32·201130977 MSC or BM-derived MSC, and when the pure cell line is used for inclusion In the case of a pure cell line of PB-derived MSC or BM-derived MSC, the term refers to PB-derived MSC or BM-derived MSC that has been cultured under in vitro conditions that allow proliferation for several months to several years without differentiation. . Such pure stem cell lines (e. g., PB-derived MSCs or BM-derived MSCs) may have the potential to differentiate along the stem cell lineage from the original stem cells. In the case of cell development, the adjectives "differentiation" or "positive differentiation" are related terms. A "differentiated cell" is a cell that has been further developed along the developmental pathway (compared to the cell to which it is compared). Thus, stem cells can differentiate into lineage-restricted precursor cells (such as mesoderm stem cells), which in turn can differentiate into other types of pro-driver cells (such as cardiomyocyte precursors) that further develop along the pathway and then differentiate into terminally differentiated cells, It plays a characteristic role in a certain tissue type and may or may not retain the ability to further proliferate. The term "differentiation" as used herein means the formation of cells expressing markers which are known to be associated with more specialized and closer cells that become terminally differentiated cells that are unable to differentiate further. The path along which the cells have not yet been determined from the direction of differentiation is increasingly determined to be a cell of a particular cell type and eventually develops into a terminally differentiated cell called progressive differentiation or progressive typing. More specific (e.g., cells that have begun to progress along the path of progressive differentiation) but have not yet undergone end-stage disease are referred to as partial differentiation. Differentiating into cells thereby presenting a specific phenotype, such as obtaining one or more developmental processes that differ from the characteristics or functions of other cell types. In some cases, a differentiated phenotype refers to the cellular phenotype at the maturity endpoint in a developmental pathway (so-called terminally differentiated cells). In many 152716.doc -33- 201130977 * _ jin has) tissue t, the differentiation process is linked to the withdrawal of the cell cycle. At the end of the month, the differentiated cells lose or greatly limit their ability to proliferate. In addition, it should be noted that in the context of this specification, the term "differentiation" or "knife-in" refers to a cell that is more specific in its fate or function than its pre-developmental point, including cells that are terminally differentiated, although A cell that has not been differentiated but is more specialized than its previous point in the month of the month. The degree of developmental differentiation of cells from unshaped cells (e.g., stem cells) is increasingly determined to be a cell of a specific differentiated cell type 5L and eventually developed into a terminally differentiated cell called a progressive differentiation or progressive sputum type. Cells that are "differentiated" relative to sputum cells have one or more phenotypic differences relative to progenitor cells. Phenotypic differences include, but are not limited to, morphological differences and differences in gene expression and biological activity, including not only the presence or absence of a manifested marker, but also differences in the amount of labeling and differences in the co-expression patterns of a set of markers. The term "differentiation" as used herein refers to the development of cells from the native stage to cells of more mature (i.e., less native) cells. The term "directed differentiation" as used herein refers to the forced differentiation of cells from undifferentiated cell types (eg, more native cells) into more mature cell types (ie, less primitive cells) via genetic and/or environmental manipulation^ In some embodiments, reprogrammed cells are disclosed as directed herein to differentiate into specific cell types, such as neuronal cell types, myocyte types, and the like. The term "medium" as referred to herein is a medium (e.g., "medium") that maintains a tissue or cell population or a population of culture cells that contains nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following: 152716.doc -34 - 201130977 combination: salts, buffers, amino acids, glucose or other sugars, antibiotic blood or blood π substitutes, and other components, such as Growth factors, etc. Cell culture media for use in a particular cell type are well known to those skilled in the art. A "phenotype" refers to the definition of one or many of the total biological characteristics of a cell or organism, regardless of the actual genotype, under a specific set of environmental conditions and factors. As used herein, "label" describes the characteristics and/or phenotype of a cell. Markers can be used to select cells that contain related features. The label will vary with the particular cell. Marked as a characteristic (whether morphological, functional or biochemical (enzymatic) characteristic) that is specific for a cell type or for that cell type. Such labels are preferably proteins, and more preferably have epitopes for antibodies or other binding molecules useful in the art. However, the label may be composed of any molecule found in the cell including, but not limited to, proteins (peptides and polymorphs), lipids, polysaccharides, nucleic acids, and steroids, examples of which may include, but are not limited to, shapes, Size, and ratio of nuclear to cytoplasm. Examples of functional features or traits include, but are not limited to, the ability to adhere to a particular receptor, the ability to incorporate or exclude a particular dye, the ability to migrate under specific conditions, and the ability to differentiate along a particular lineage. The marker can be detected by any method available to those skilled in the art. The term "contacting" as used herein with respect to contacting a peripheral blood sample in vivo may comprise administering to the individual via a suitable route of administration a mobilizing agent, optionally contained in the composition, such that the compound contacts the peripheral blood sample in vivo. The term "contacting" as used herein with respect to ex vivo contact with a peripheral blood sample can include administering a mobilizing agent (as appropriate in the composition) to a peripheral blood sample 152716.doc-35-201130977 to allow the mobilizing agent to be in contact with the body. The peripheral blood sample. As used herein, the terms 'administering,' "introducing," and "transplanting" are used interchangeably and refer to a method or pathway by which a human PB-derived MSC or BM-derived MSC is at least partially localized at a desired site. The peripheral blood-derived MSC or bone marrow-derived MSC is placed in the individual. Human PB-derived MSCs or BM-derived MSCs can be administered by any suitable route that can be delivered to a desired location in an individual, wherein at least a portion of the human PB-derived MSCs or BM-derived MSCs are still viable after the cells are administered to the individual. The survival period can be as short as several hours (for example, 24 hours) to several days, up to several years. As used herein, the term "donor" refers to the individual from which the organ, tissue or cell to be transplanted is collected. As used herein, the term "recipient" refers to an individual who will receive a transplanted organ, tissue or cell. The term "transplantation" as used herein refers to the process by which free (unattached) cells, tissues or organs are integrated into a tissue after implantation into an individual. The term "allograft" refers to a transplanted cell, tissue or organ derived from a different animal of the same species. The term "Xen〇graft/Xen〇transplant" as used herein refers to a transplanted cell, tissue or organ derived from an animal of a different species. In some embodiments, the xenograft is a graft from a species of a species that is surgically implanted into a different species, genus, or family. For example, a graft implanted into a human body is a xenograft. 152716.doc •36· 201130977 The term “xenograft” refers to the process by which a living cell, tissue or organ is transplanted from one species to another, such as from a pig to a human. The terms "subject" and "individual" are used interchangeable herein and refer to an animal from which PB-derived MSCs as disclosed herein can be isolated and collected according to the methods and compositions described herein. 'For example, humans, and individuals may be transplanted as appropriate (eg, pB-derived MSC or BM-derived MSC-implantable individuals) for, for example, treatment, including prophylaxis. Treating diseases, illness. For a disease state peculiar to a particular animal, such as a human individual, the term "individual" refers to a particular animal. The terms "non-animal" and non-human mammals are used interchangeably herein and include mammals such as rats, mice, 4, sheep, cats, dogs, cows, pigs, and non-human primates. The term "individual" also covers any vertebrate, including (but not limited to) 商 于, commercial animals, reptiles, amphibians and fish. However, "individual ig» SL· . is a mammal such as a human, or other

Milk animals, such as the horses, feeding the quilt, I, ... ^ € such as dogs, squad, horses and similar movements # + cows, sheep, pigs and similar animals)

Covered in the term individual. W The term "tissue" refers to a group or layer resembling a shell-like cell, and a hill performs some special functions. The source or characteristic characteristics of the peppers of the peppers and the scorpion. The term "agent" as used herein means any such as, but not limited to, small molecules, sputum sub-substances, Pharmacy I can be used to sip a cup of syrup. The agent "T is any chemical, f synthetic and naturally occurring protein and non-protein entities: in = not limited to" «祖 in some embodiments, 152716.doc •37- 201130977 Pharmacy is a nucleic acid; nucleic acid analog; protein; antibody; peptide; aptamer; oligomer of nucleic acid, amino acid or carbohydrate, including but not limited to protein, oligonucleotide, ribonuclease , DNase, glycoprotein, siRNA, lipoprotein, aptamer and modified forms and combinations thereof. In certain embodiments, the agent is a small molecule having a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic or heterocyclic moieties' including maCrolide, 丨ept〇mycin and related natural products or the like. Things. The compounds are known to have a desired activity and/or property, or a pool of different compounds. As used herein, the term "small molecule" refers to a chemical agent, which may include, but is not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, Aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams per mole (for example including heteroorganic compounds and organometallic compounds), organics having a molecular weight of less than about 5,000 agram per mole Inorganic compound, organic or inorganic compound having a molecular weight of less than about 1, (10) gramm/mole, an organic or inorganic compound having a molecular weight of less than about 5 gram/mole, and salts, esters and pharmaceutically acceptable salts of such compounds Other forms. § "Disease" or "condition" is used interchangeably herein and refers to any change in the state of the body or organs that interrupts or interferes with the function of the lamp and/or causes symptoms of the patient or the person who is in contact with the individual. , such as discomfort, dysfunction, pain, or even death. A disease or condition can also mean heat, discomfort, disorders, confusion, abnormalities. Nausea, illness, discomfort, microspasm or disease. 1527l6.doc -38 - 201130977 The term "pathology" as used herein refers to a condition causing a disease or condition, such as a change in the structure and function of a cell, tissue or organ. For example, 'pathology can be related to a specific nucleic acid sequence or a "pathological nucleic acid", which refers to a nucleic acid sequence that completely or partially causes pathology, for example, 5' pathological nucleic acid can be encoded to have a specific pathology or pathology. A nucleic acid sequence of a gene associated with a mutation or polymorphism. The pathology may be associated with the performance of pathological proteins or pathological polypeptides that completely or partially cause pathology associated with a particular disease or condition. In another embodiment, the pathology is associated, for example, with other factors, such as ischemia and the like. As used herein, the term "treating" includes alleviating or alleviating at least one of the adverse effects or symptoms of a condition, disease or condition. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, usually injection, and includes (but is not limited to) intravenous, intramuscular, intraarterial, intrathecal, intraventricular. , intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, cerebrospinal, and intrasternal injections and infusions. As used herein, the phrase "systemic administration" or "peripheral administration" means PB-derived MSC or BM-derived MSC or its differentiated progeny and/or its progeny and/or compounds and/or other substances to Administration into the body of the animal and thereby undergoing metabolism and other similar processes (eg, subcutaneous or intravenous administration) rather than directly into the individual. The phrase "pharmaceutically acceptable" is used herein to mean that it is suitable for contact with humans and animal tissues in a reasonable medical judgment (4) without excessive toxicity, irritation, allergic reaction or other problems or complications and reasonable benefits/ Risk 152716.doc •39- 201130977 The proportion of their compounds, substances, compositions and/or dosage forms. The phrase "pharmaceutically acceptable carrier" as used herein means pharmaceutically acceptable in connection with the delivery or delivery of a target agent to another organ or part of the body in an organ or part of the body. A substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The term "drug" or "compound" as used herein refers to a chemical entity or biological product, or a combination of chemical entities or biological products, administered to an individual to treat or prevent or control a disease or condition. Chemical entities or biological products are preferably, but not necessarily, low molecular weight compounds, but may also be larger compounds such as nucleic acids, amino acids, or oligomers of carbohydrates including, but not limited to, proteins, oligos Glycosylates, ribonucleases, DNases, glycoproteins, siRNAs, lipoproteins, aptamers, and modified forms and combinations thereof. The term "transplantation" as used herein refers to the administration of new cells (eg, PB-derived MSCs or BM-derived MSCs or their differentiated progeny) or tissues (such as differentiated cells produced by pb-derived MSCs or BM-derived MSCs) or The organ is introduced into the host (i.e., the recipient of the graft or the individual of the graft). The term "modulation j" is used in accordance with its usage in the art, for example to cause or promote qualitative or quantitative changes, changes or modifications of related processes, paths or phenomena. This change may be, without limitation, a process, path or phenomenon. An increase, decrease, or change in the relative strength or activity of a different component or branch. A "regulator" is an agent that causes or promotes a change, change, or modification in the nature or quantity of a related process, pathway, or phenomenon. I52716.doc -40· 201130977 The term "reduction", "reduction" or "inhibition" is used herein to mean a statistically significant reduction. However, for the avoidance of doubt, "decreasing; or "reducing" or "suppressing" means reducing the reference content to two wrists, for example, reducing at least about 鸠, or at least about 鸠, or to / 娜, or at least about 乡, or at least about 6Q%, or at least %% / as for the spring. Or up to and including 1% reduction (e.g., comparable, °, no content), or any decrease from 1 to 100% compared to the reference content. 1 in 10 - the terms "increase" or "enhancement" or "activation" are generally used herein to mean "the stomach is increased by a statistically significant amount; to avoid any doubt, the term "doing" or "35" or "activation" By means of an increase of at least 0, for example at least about 20%, or at least about 30%, or at least about peach, or at least (four)%, or at least about 峨, or at least about Cong, or at least = 〇%, compared to the reference content. Or at least about 90% or up to and including just % increase or as compared to > the test content is between 1 (M00% of any increase, or at least about 2 times the reference content' or at least about 3 times Or at least about 4 times, or at least about $ times or at least about H) times or any increase between 2 times and 1G times or more. J is "statistically significant" and "significant" means statistically significant and is less than two standard deviations (2SD) lower than the standard or lower concentration of the marker. This ΓΓ refers to statistical evidence that confirms the difference. It is defined as the decision to exclude the null hypothesis when the illusion is false - the truth is true. The term "substantially" as used herein means 6 〇 /. Or preferably at least about or at least about 嶋' or at least 约娜, to 152716.doc •41- 201130977 less than about 95%, at least about 97% or at least about 99% or more, or between 70% The ratio of any integer between 1 and 〇〇%. As used in the specification and the appended claims, the singular forms " By way of example, reference to "the method" includes one or more methods and/or types of steps which are described herein and/or which are apparent to those skilled in the art in reading the invention, and so forth. The above detailed description and the following examples are to be construed as illustrative and not restrictive of the invention. Retouching will be apparent to those skilled in the art. In addition, all patents, specific disclosures, and publications identified herein are hereby expressly incorporated by reference for the purposes of the disclosure of the disclosure of the disclosure of ^ These publications are for disclosure only, prior to the filing date of the request. In this regard, it is not to be understood that the inventors have the right to advance the present invention for the prior invention or for any other reason. All representations of all statements or content in the sequel to these documents are based on the information available to the applicant and do not constitute any recognition as to the correctness of the date or content of such documents. MSC mobilizers, in aspects of the invention, provide a method of treatment comprising administering to a mammal (eg, a human) an effective amount of a mobilizing agent, such as a certain form of G CSF or GM-CSF, and its medicinal Acceptable salts are used to enhance (i), stimulate MSC production/release, as measured by phenotypic, physical or chemical properties or by any other feature used by the skilled artisan to identify MSC-type cells. In one embodiment, the peak number of circulating MSCs is obtained after about 2 injections per day of administration of a mobilizer such as GM-CSF or G-CSF. The peak of circulating MSCs in the peripheral blood decreased after at least 4 daily injections. Any combination of interstitial or interstitial agents can be administered to an individual at a defined time to optimize mobilization of the MSC into the peripheral blood. In some embodiments, the MSC mobilizing agent is GM-CSF or G-CSF (granulocyte community stimulating factor). In some embodiments, the individual may be administered a combination of other agents, including, but not limited to, G-CSF, GM-CSF, Flt-3 ligand, stem cell factor (eg, but not limited to) a single mobilizer or a combination of such mobilizers ( SCF), dexamethazone, CXCR4 receptor inhibitor, interleukin-1 (IL-1), interleukin-3 (IL-3), and Diniplestim (IL-3) Agonist), Leridistim (IL-3 agonist-G-CSF fungus), progenipoietin-l (Flt-3 ligand-G-CSF) Chimeric molecule), peg-filgrastim (NEULASTATM), interleukin-8 (IL-8), PIXY-321 (GM-CSF/IL-3 fusion protein), macrophage Cellular inflammatory proteins, stem cell factor (SCF), thrombopoietin, and growth-related oncogenes. Other mobilizing agents include, for example, but are not limited to, CXCR4 inhibitors such as AMD3100, ALX40-4C, T22, T134, T140, and TAK-779, which are disclosed in U.S. Patent No. 7,169,750, hereby incorporated hereinby entirety in. In some embodiments, such as G-CSF or GM-CSF, or interleukin-17 (IL-17), AMD3100, cyclophosphamide (Cy), and European paclitaxel (DXT) and 152716.doc • 43- 201130977 The combined mobilizer can be administered to the individual or ex vivo to the peripheral blood sample to stimulate the PB-derived MSC. In some embodiments, a mobilizing agent such as G-CSF or GM-CSF may be administered to an individual or ex vivo to a peripheral blood sample in combination with other therapeutically or nutritionally active ingredients, such as antibiotics. , vitamins, herbal extracts, anti-inflammatory agents, glucose, antipyretics, analgesics, granulocyte-macrophage community stimulating factor (GM_CSF), interleukin-1 (IL1), interleukin-3 (IL-3) , interleukin-8 (IL-8), PIXY-321 (GM-CSF/IL-3 fusion protein), macrophage inflammatory protein, stem cell factor, thrombopoietin and analogs thereof. A mobilized peripheral blood stem cell cluster (PBSC) produced using a mobilizer such as G-CSF or GM-CSF can be transplanted into, for example, a cancer patient. Such clusters may be the total population of purified HSCs obtained from mobilized white blood cells or from mobilized peripheral blood. Hsc is able to reorganize the hematopoietic system of organisms, which requires self-renewal of HSCs and differentiation into cells of various hematopoietic lineages. The cD34 is labeled as unique to the HSC. Other cells mobilized using such mobilizers include polymorphonuclear leukocytes (cells that mediate inflammation and pathogen clearance), mononuclear white blood cells (lymphocytes and monocytes), and red blood cell progenitor cells (red blood cells). The mobilization of CD34+ HSC is a rapid amplification clinical technique for obtaining materials for autologous or allogeneic hematopoietic transplantation. In addition, the mobilization of polymorphonuclear leukocytes is an important aid to heavy chemotherapy to maintain an innate defense mechanism. Currently, both methods rely on expensive growth factors (❻/" or GM_CSF) that cause bone pain and have unknown side effects to normal donors. It takes about 4 weeks to process. To collect enough material for transplantation. After growth factor treatment, CD34+ cells reach a maximum of 2-4% in blood. G-CSF: In some embodiments, for G- in a method as disclosed herein CSF can be prepared using recombinant methods and variants of G-CSF can be used. The term G-CSF or G-CSF variant according to the invention encompasses all naturally occurring G-CSF variants, as well as recombinant DNA derived therefrom The technically modified G-CSF protein, in particular, a fusion protein of other protein sequences in addition to the G-CSF moiety. In this sense, a G-CSF mutant protein having an N-terminal Met residue at position 1 is particularly preferred (mutein). It is suitable for expression in prokaryotic cells. Recombinant G-CSF variants which are prepared according to WOA-91/11520 and which are free of guanidine thioglycol are also suitable, which is incorporated herein by reference in its entirety. G-CSF variant Comprising one or more amino acids may be deleted or replaced by other amino acids of their G-CSF molecule having the essential nature of G-CSF, a large number of specific words can mobilize bone marrow cells reservations. Suitable G-CSF muteins are described, for example, in EP 0,456,200, the disclosure of which is incorporated herein in its entirety. In some embodiments, any commercially available form of G-CSF known to those skilled in the art can be used, such as, but not limited to, Neupogen® (F. Hoffmann-La Roche Ltd), as disclosed herein. ., Basel, Switzerland) and its variants. GM-CSF: According to the present invention, GM-CSF (granule 152716.doc -45-201130977 cell-macrophage colony stimulating factor) prepared by a recombinant method and a variant thereof can be used. The term GM-CSF or GM-CSF variant according to the invention encompasses all naturally occurring GM-CSF variants, as well as GM-CSF proteins derived therefrom modified by recombinant DNA techniques, especially in addition to the GM-CSF moiety. A fusion protein containing other protein sequences. In this sense, a GM-CSF mutein having an N-terminal Met residue at position 1 is particularly preferred, which is suitable for expression in prokaryotic cells. Also suitable are recombinant GM-CSF variants which are free of methionine prepared according to WOA-91/11520, which is incorporated herein by reference in its entirety. The term "GM-CSF variant" is understood to include those G-CSF molecules which may be deficient in one or more amino acids or substituted by other amino acids, which have the basic properties of GM-CSF, specifically capable of mobilizing A large number of preserved bone marrow cells. Suitable GM-CSF muteins are described, for example, in U.S. Patent No. 0,456,200, the disclosure of which is incorporated herein in its entirety. Among the four "granulocyte-macrophage" CSFs, GM-CSF was first isolated and characterized. GM-CSF has been shown to induce proliferation of murine bone marrow-derived or spleen-derived hematopoietic cells containing granulocytes and macrophage progenitor cells, resulting in a community of granulocyte- and macrophage precursors. In this regard, GM-CSF appears to have a common biological property with the subsequently characterized IL-3. However, recent studies have shown that GM-CSF acts on "more advanced" pluripotent cells compared to IL-3. In addition, GM-CSF appears to stimulate the proliferation of red blood cell lines and megakaryocyte precursors to be less active than IL-3. However, similar to IL-3, GM-CSF has been shown to be active against mature cells of the granulocyte and macrophage lineages. Without wishing to be bound by theory, GM-CSF (granulocyte-macrophage colony stimulating factor) acts directly and selectively on granulocyte/macrophage progenitor cells 152716.doc -46- 201130977 to stimulate belonging to these lineages In vitro growth and differentiation of cells (e.g., neutrophils, eosinophils, macrophages) have also demonstrated such pleiotropic activities of recombinant GM-CSF. In addition to regulating the proliferation and differentiation of progenitor/precursor cells of the myeloid lineage, GM-CSF has also been shown to activate the function of mature bone marrow cell types. For example, GM-CSF has been found to induce tumorigenic activity of macrophages against malignant melanoma cell line A375. IFNy can also act as a macrophage activating factor, but in contrast to GM-CSF, it requires another secondary stimulator (such as bacterial LPS) to induce tumoricidal activity. In addition, GM-CSF activates giant scorpion cells to inhibit Krebs cones. Trypanosoma cruzi (a single-celled parasite that is responsible for the onset of Chagas disease or American trypanosomiasis) replicates and enhances the respiratory oxidation process. Furthermore, replication of HIV-1 in human monocyte cell line U937 has been shown to be moderately inhibited by GM-CSF and is more effectively inhibited by the combination of GM-CSF and IFNY. In neutrophils and eosinophils, GM-CSF stimulates many functions. In particular, GM-CSF enhances the phagocytosis of neutrophils to bacteria and yeast. Purified recombinant human GM-CSF has also been shown to enhance the cytotoxic activity of neutrophils and eosinophils against antibody-coated target cells. It has been reported that when mice are repeatedly intraperitoneally injected with recombinant murine GM-CSF, the number and functional activity of peritoneal giant sputum cells, granulocytes (neutrophils and eosinophils) are rapidly and continuously increased and circulating monocytes are The number has also increased. (GM-CSF usually takes about two weeks to work). After injection of recombinant human gm-CSF into AIDS patients and non-human primates, it has also been observed 152716.doc •47- 201130977 A significant increase in the number of neutrophils, eosinophils and monocytes was detected. . However, there may be complications associated with GM_CSF therapy. Metcalf and colleagues have shown that transgenic mice containing constitutively expressed murine gm csf* are caused by activated giant cell infiltration in various tissues (including lens, retina, and striated muscle) shortly after birth. Pathological lesions. (Activated macrophages are known to produce many inflammatory mediators that can induce tissue damage, including cytokines such as TNFa and IL-1.) 相反 In contrast to the growth stimulating effects of GM-CSF, GM-CSF acts as a differentiation factor. Its effects on, for example, mature macrophages and neutrophils can be seen as a result of their differentiation-inducing ability. A way to limit tumor cell proliferation is to link growth factor-driven self-renewal with growth factor-induced differentiation. In other words, the more "differentiated" tumor cells, the smaller their proliferation ability. In this regard, GM-CSF has been shown to induce differentiation of myeloid leukemia cell line HL6 and inhibit self-renewal. However, in several other studies, GM_CSF stimulated HL60 cell proliferation. Differentiation can be monitored by measuring the performance of various plasma membrane associated antigens such as CD14 (monocyte/macrophage marker) and cd57 (NK cell marker). In some embodiments, any commercially available form of GM-CSF known to those skilled in the art, such as, but not limited to, Leucomax® (Schering Pl〇 Ugh-Sandoz) may be used in the methods and compositions disclosed herein. Pharma Ltd"

Basel, Switzerland) and its variants. Dosing, duration, frequency, route (intraperitoneal (i.p>, subcutaneous (sc)) G-CSF or GM-CSF dose can be seen by various factors (such as medication, 152716.doc -48· 201130977 species, Depending on age or individual condition. In some embodiments, the dose of G-CSF or GM-CSF administered subcutaneously (SC) is about 100 to 700 pg of G-CSF or GM-CSF per day for at least 2 days, or G-CSF or GM-CSF is administered at least once a day for at least 3 days and up to 4 days. Different doses of G-CSF or GM-CSF include, but are not limited to, at least about 100 pg per day, or at least about 200 pg, or at least About 300 pg, or at least about 400 pg, or at least about 500 pg, or at least about 600 pg, or at least about 700 pg, or more than 700 pg of G-CSF or GM-CSF. In some embodiments, the dose does not exceed each day. 1000 pg for 2 days. In some embodiments, the dose of G-CSF or GM-CSF is about 450 pg per day for at least 1 day but less than 4 days. In some embodiments, G-CSF or GM-CSF The dose is about 400 pg per day or about 500 pg per day for at least 1 day but less than 4 days. In some embodiments, the duration of administration of G-CSF is less than 5 And because these doses have been associated with severe side effects, preferably less than 1 〇pg/kg per day, or less than 7.5 pg/kg per day, such side effects include increased spleen length, headache, nausea, fatigue, and bone pain. And in some cases including spontaneous spleen rupture (see review below: Cashen et al, Bone marrow transplantation, 2007; 39; 577-588 and Pusic et al, Current Pharmaceutical Design, 2008; 14; 1950-1961, full text The manner of reference is incorporated herein. As disclosed herein, the inventors have demonstrated that most of the mobilization of PB-derived MSCs is obtained after a subcutaneous administration of a dose of 450 pg per day for at least one day. However, it is easy to know that it is possible to increase or decrease the dose of G-CSF or GM-CSF to find the optimal dose for each use. This can be done as follows: 152716.doc -49- 201130977 g_CSF4Gmcsf and analyzes a blood sample, such as a blood sample obtained as described herein. Further, depending on the cell to be mobilized, it may be higher or lower than the optimal concentration of 45 gg per day per (Or at other intervals) g_csf infusion or GM-CSF, rather than sequentially increasing the dose on each of said. It is also possible to check the different frequencies and durations of G-CSF or GM-CSF. Suitable dosage ranges for G-CSF or GM-CSF will vary depending on such considerations, but in general, the compound will be administered in a dosage range of about 1 每天, or at least about 200 pg, or at least about 300, or at least About 4 〇〇, or at least about 500, or at least about 600 pg, or at least about 7 ton, or more than 7 〇〇 pg of G_CSF or GM-CSF. The dose may be higher when the compound is administered orally or transdermally, as compared to, for example, intravenous administration. For administration of g_CSF4GM_csf to an individual as disclosed herein, G_CSF or GM_CSF can be formulated for administration to an individual animal, such as a human, using a universal demodulation technique well known in the art. Formulations suitable for a particular mode of administration can be found in Remingt〇n , s Pharmaceuticai Sciences, latest edition, Mack Publishing Company, Easton, Pa. In some embodiments, G_CSF4GM-CSF is administered to an individual such as a human using a standard mode of administration wherein the injectable solution is preferred. Water is preferably used as an injection medium, which includes adjuvants commonly found in injectable solutions, such as stabilizers, solubilizers, and buffers. For example, such adjuvants are tartrate and citrate buffers; ethanol; complexing agents such as ethylenediaminetetraacetic acid and its non-toxic salts; high molecular weight polymers for controlling viscosity, such as liquid polyethylene oxide. The liquid vehicle for injecting the solution must be sterile and preferably filled into the ampoule. 152716.doc .50· 201130977 G-CSF or GM-CSF is preferably administered by injection, preferably by intravenous injection, but also by subcutaneous or intraperitoneal injection and the like, such as humans. Other parenteral routes of administration include intramuscular and intra-articular injections. For intravenous or parenteral administration, G_CSF or GM_CSF may be formulated into a suitable liquid form if necessary. G_CSF4GM_CSF can be administered to an individual, such as a human subject, in a liposome or other suitable carrier. The solution is made isotonic with respect to intravenous injection using standard formulations such as Henk's solution (Hank, s s〇luti〇n). In addition to injection, other routes of administration may be used. In some embodiments, the G_CSF*GM_CSF to be administered to an individual, such as a human individual, may be formulated as a lozenge, capsule, syrup, powder, or other form suitable for oral administration. g_CSF4GM_csf can be administered via the mucosa using a suppository or an intranasal spray by using a suitable excipient. Transdermal administration can also be achieved by using a suitable penetrant and controlling the release rate. In addition to intravenous infusion, different routes of administration of G-CSF or GM-CSF are also effective. For example, a "depot" of G_CSF or gm·CSF placed subcutaneously or intraperitoneally can provide a long-term continuous infusion and is a convenient and efficient method of providing g_CSF or GM-CSF. If G-CSF or GM-CSF is administered subcutaneously or intraperitoneally, for example, continuous rounds can be achieved and the individual monitored over time. Alternatively, G-CSF or GM-CSF can be administered orally. The present invention encompasses the administration of G-CSF or GM-CSF by such means and by all other means as would be appreciated by those skilled in the art. Different G-CSF or GM-CSF dosing regimens (including different g_csf or GM-CSF doses, G-CSF or GM-CSF route of administration and duration) will be adjusted to 152716.doc • 51· 201130977 to move different stem cell populations, such as MSC. As discussed above, the inventors have surprisingly found that the optimization of the transfer of ^8 (: (}) ^ _ (: Ting and (3_ (: § 17 dosing scheme is different from the conventional use for mobilizing HSC Thus, an optimization scheme can be applied for a particular application: g_csf or CSF is administered under different conditions. Then the output of a subset of the desired stem cells (such as MSCs or hematopoietic cells) in the blood is monitored according to the methods as disclosed herein. As indicated in the examples herein, different cell types will occur sequentially during the period after administration of the mobilizer, so the time to optimize recovery or induce a specific cell population in the blood after the infusion should be monitored. Selected formulations and administration The route should be suitable for the individual individual, the nature of the individual's condition to be treated, and generally the judgment of the attending physician. In some embodiments, 'G-CSF or GM-CSF can be administered in a single dose, administered over time. (eg, intravenous or transdermal administration) or multiple administrations to an individual, such as a human individual. In addition to direct administration to the body, G-CSF or GM-CSF can be used in an ex vivo treatment regimen to extract blood from the periphery of the individual. Separation of MSC It can be used to prepare MSC cultures which can optionally be cryopreserved and stored and subsequently used for individual cell-based regenerative therapies when MSCs are required. In some embodiments, this ex vivo production of MSC is available. Separating and collecting autologous MSCs. These autologous MSCs are collected from peripheral blood or, in some cases, from allografts of matching donors. In vitro or in combination with other agents, such as megalin cell inflammatory proteins. The concentration of the mobilizer in the peripheral blood is a problem of conventional optimization. A large number of granulocytes in the spleen can be observed by administration of G-CSF or GM-CSF '152716.doc -52·201130977 and the spleen weight is significantly increased, according to Bungart et al, Brit j 76, 174-179, 1990, this can be attributed to stem cell mobilization or MSC mobilization. In some embodiments, peripheral blood samples for use in methods as disclosed herein are from a mammalian individual such as a human Individually obtained. In some embodiments, a human subject has previously administered a mobilizing agent to increase the yield or number of circulating stem cells in the peripheral blood. Any mobilizing agent can be used, for example, according to the art. A commonly known method, and includes, for example, but not limited to, the methods disclosed in U.S. Patent Application No. 6,261,549, and U.S. Patent Application Serial No. 2009/0155225, the disclosure of each of In some embodiments, a mobilizing agent such as G-CSF or GM-CSF can be administered to an individual or ex vivo to a peripheral blood sample in combination with any one or more other mobilizing agents, such as selected from The following composition of the agents: interleukin-17 (IL-17), AMD3100, cyclophosphamide (Cy) and European paclitaxel (DXT) and combinations thereof. In some embodiments, a mobilizing agent such as G-CSF or GM-CSF can be administered to an individual or ex vivo to a peripheral blood sample, such as an antibiotic, in combination with other therapeutically or nutritionally active ingredients. Vitamins, herbal extracts, anti-inflammatory agents, glucose, antipyretics, analgesics, granulocyte-maize cell community stimulating factor (GM-CSF), interleukin-1 (IL-1), interleukin-3 (IL) -3), interleukin-8 (IL-8), PIXY-321 (GM-CSF/IL-3 fusion protein), megakaryocytic inflammatory protein, stem cell factor, thrombopoietin, and the like. Any individual can perform MSC mobilization using the methods disclosed herein, and can mobilize and collect MSCs from peripheral blood of any individual (such as a human individual, including adult or non-neonatal 152716.doc • 53·201130977 children). The MSC can be collected - or multiple collection steps or collection cycles. For example, from the individual's mobilization week, blood collection (eg, using blood separation) Μ% at least 2 times, at least 3 or to / 5- people "collected from each kilogram of body weight during each collection step The total number of nucleated cells may be - million (ΐχΐ()6) or more than one million (e.g., 1〇7, 1><1〇8,1><1〇9,1><<> 1〇1〇, 1><1〇11,1><1〇12, 1X10:: 1X101:, I, 15, 1, 1X10-, 1X101«, 1〇1X10). In a preferred embodiment, the number of cells in the single batch collection of segregation products (which are used for the separation of Μ% from non-MSCs) may be equal to or greater than ΐχΐ〇丨 depending on the weight and age of the donor individual. 5 total nucleated cells (TNC), or at least about 1χ1〇Μ, ΐχΐ〇13, ΐχΐ〇丨2, lxio6, lxio5 lxlO1 丨, 1x10 丨. , 1χ10, 1χ1〇8, 1χ1〇7 Total nucleated cells. Visual form and MSCM that can be obtained from any human individual donor using methods as disclosed herein, preferably at a human adult donor at an "adult" or mature j age (unless otherwise used in a particular context to achieve a different meaning) Otherwise, the term "adult" as used herein refers to and includes both adult and non-neonatal) and/or a certain minimum weight from which dry fines & By way of example, MSCs may be collected when the individual is in the range of 10 to 200 kg (according to an embodiment of the invention) or any range within this range, such as within 2 Torr to 4 〇 kg. Alternatively or in addition, according to an embodiment of the invention, the individual may be required to have a range of 2-80 years old (eg 2_1〇, 1〇_15, 1218, 16 2(), 20-26, 26-30, 30-35, An age within 30-40, 40-45, 40-50, 55-60, 60-65, 60_70, and 70-80 years old. 152716.doc •54· 201130977 Isolation of MSCs from other stem cell populations in peripheral gold The method of obtaining and isolating PB-derived MSCs and BM-derived MSCs from peripheral blood is well known in the art and includes leukocyte separation. , partial separation of density gradients, immune selection, and differential adhesion separation. Substantially pure MSC populations can be collected in a culture medium such as a chemically defined serum-free medium or a complete medium (such as DMEM or DMEM containing 1 g serum). A suitable chemically defined serum-free medium is described in U.S. Patent No. 08, 464, 359, filed on Jun. These patents are incorporated herein by reference. One aspect of the invention is the separation of MSCs from the peripheral blood mobilized using centrifugal panning. Without wishing to be bound by theory, in a panning process, a peripheral blood sample is introduced into a funnel-shaped separation chamber located on a rotary centrifuge. A liquid panning buffer stream or a low density stream is then introduced into the chamber containing the peripheral blood sample. When the flow rate of the liquid panning buffer solution through the chamber is increased (usually in a stepwise manner), the liquid will migrate to the eluting boundary of the chamber with smaller size and slower sedimentation cells, and the larger cell migration will settle faster. The indoor area to the balance of centrifugal force and sedimentation (drag) force. Thus, when peripheral blood (e.g., mobilized peripheral blood) comprises a plurality of different stem cell populations, one aspect of the present invention relates to the use of panning to separate such different populations of stem cells present in peripheral blood into different populations. Wherein smaller stem cells and larger fractions are isolated. In some aspects of the invention, 'any panning device can be used to obtain PB-derived MSCs and BM-derived MSCs and other peripheral blood. Stem cell population 152716.doc -55- 201130977 Isolation. In some embodiments, the panning device uses a separation product (in which platelets and blood cells are returned to the individual donor), and the remaining peripheral blood product is a separation product that is panned at a certain flow rate to peripheral blood. The sample is partially separated into a fraction comprising a MSC population. In some embodiments, commercially available panning devices (such as the ELUTRA® centrifuge manufactured by Gambro BCT, Inc.) can be used to separate MSCs from peripheral blood » ELUTRA CELL SEPARATION SYSTEM® Using the size and density, the circulated peripheral blood is separated into a portion containing MSC, so that cell enrichment, depletion, concentration, and washing can be performed in a closed functional system. ELUTRA CELL SEPARATION SYSTEM® is capable of enriching stem cell populations directly from leukocyte detachment products without the need for antibodies or pretreatment in less than 1 hour. The Elutra Cell Separation System® uses a countercurrent centrifugal panning method in which fluid flows through the cell layer in a centrifugal field to separate the cell population. In some embodiments, other panning devices (including but not limited to) COBE® Spectra separation systems, Trima® systems, and Trima Accel® systems, also manufactured by Gambro BTC Inc., and others for separating blood components Commercially available panning devices can be used to separate MSCs from peripheral blood. In some embodiments, the MSC population can be obtained from a mobilized peripheral blood sample using a cell separator, such as a COBE® Spectra separation system. The COBE® SPECTRATM centrifuges are described in U.S. Patent Nos. 4,425,172, 4,708, 712, and 6, 022, 306, incorporated herein by reference. In this embodiment, the mobilized peripheral blood sample (or the isolated product obtained from an individual who has administered the mobilizer according to the methods disclosed herein) is introduced into a cell separator (such as C0BE). In the system), and optionally add anticoagulant solution to the blood to prevent it from clotting during the process. The blood/anticoagulant mixture is circulated through a centrifuge to separate peripheral blood samples into MSC populations and to separate mononuclear cells from other blood components and plasma. The system draws the separated MSC into a collection bag for storage, while other blood sets and blood is returned to the patient. All tubing sets and needles used are sterile' so there is no risk of disease transmission. Other blood separation devices may be used, such as those described in the following: U.S. Patent No. 5,722,926, issued March 3, 1998, and U.S. Patent No. 5,951,877, issued on September 14, 1999; U.S. Patent No. 6,053,856, issued on Apr. 25, the entire disclosure of U.S. Patent No. 6,34,842, issued Jan. 1, 2002; U.S. Patent No. 7, 198, 848, U.S. Patent No. 6, 222, 362, U.S. Patent No. 6, 589, 526, U.S. Patent Application Serial No. PCT/A No. The entire disclosure of each of the patent applications is incorporated herein by reference. The inventors have discovered that it is desirable to adjust the hematocrit range used for bloodpheresis to enhance the properties of the collected cell population. For example, in one embodiment, the hematocrit range is selected to increase the ratio of VSELs. In another embodiment, the hematocrit range is selected to enhance cryopreservation of the collected VSELs', e.g., by minimizing granulocyte collection. In another embodiment, the hematocrit range is selected to ensure simultaneous collection of multiple stem cell populations, such as VSELs and MSCs. In one embodiment, the hematocrit range is selected to be 2-3%. In another embodiment, the hematocrit range is selected to be 3_152716.doc • 57- 201130977 4%.

Identification of MSCs As disclosed herein, MSCs can be identified by FAC sorting, and immunoselection and phenotypic analysis using light microscopy and CFU-F assays. By way of example only, Wright-Giemsa stained cells in cytospin preparations in culture plates or cells isolated from EDTA may be subjected to light microscopy. Histochemical staining can be used for Sudan Black, Peric Acid-Schiff (PAS), a-naphthyl butyrate esterase, acid phosphatase and alkaline phosphatase. The diagnostic kit (Sigma) is performed by standard protocols. For immunofluorescence studies, MSCs can be treated with a fixation/permeation reagent (Cytoperm®, Serotech Ltd., Oxford, England) and labeled with a suitable free monoclonal antibody or FITC-conjugated monoclonal antibody (as discussed herein) and The Nikon microscope was Detected under 50 nm UV illumination using a mercury gas lamp. For flow cytometric analysis, MSCs in EDTA-released MSCs or isolates can be stained with either monoclonal antibodies or FITC- or PE-conjugated monoclonal antibodies. Non-specific isotype matched antibodies can be used to determine background fluorescence. MSCs can be analyzed on a FACScan flow cytometer (Becton Dickinson, BD) and can be accessed using the FAC Scan Lysis II (BD) study software according to procedures generally known in the art. The following monoclonal antibodies can be used to detect MSC: anti-CD-45-FITC, anti-CD14-PE, anti-CD34-PE (Becton Dickinson); anti-collagen I, anti-collagen III and fibronectin (Sigma) ); anti-152716.doc * 58 - 201130977 Collagen VI (Gibco BRL, Grand Island, NY, USA); anti-ICAM-1 (CD54) and anti-VCAM-1 (CD 106) (R&D Systems, Minneapolis, Minn .,USA). Control mice IgG 1-PE, IgG 1-FITC, IgG 2a-PE and F(ab')2-PE (Becton Dickinson). Monoclonal antibodies SH-2 (IgG 1) and SH-3. (IgG 2b) (Case Western Reserve University, Cleveland, Ohio, USA or Osiris Therapeutics, Inc. (Baltimore, Md., USA)). These antibodies recognize antigens on the surface of MSC cells but do not react with BM-derived HSCs and the cell surface of osteoblasts or bone cells. The total cell count and cell viability of any of the collected samples of the substantially pure MSC population can be tested by dye exclusion by Trypan blue. The presence of cells expressing the cell marker of a sample of substantially pure MSC population can also be analyzed. Total cell count and cell viability before, after, and on any other cell sample can be manually counted by a hemocytometer, flow cytometry, or other means suitable for obtaining cell counts, such as ViCell (Beckman Coulter) or suitable for The soft body of the cell count shown on the microscopic image was quantified. Cryopreservation In some embodiments, a substantially pure human MSC population obtained by a method as disclosed herein can be cryopreserved (e.g., frozen) at liquid nitrogen temperature and stored for extended periods of time for use during thawing. If frozen, the MSC will typically be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Human MSCs - after thawing, can be cultured and expanded in vitro in the presence of growth factors and/or feeder layers for appropriate periods of time, and subsequently implanted in individuals in need of 152716.doc -59 - 201130977. Methods for cryopreservation of stem cells, such as MSCs, are generally known to those of ordinary skill in the art and are disclosed in U.S. Patent Application Nos. 2008/0220520, 2009/0022693, 2008/0241113, and 2005/0106554, and U.S. Patent Nos. 5,759,764 and 7,112,576 and 7,604,930. For cryopreservation, the substantially pure MSC population obtained by the methods disclosed herein can be suspended in DPBS and placed on ice for at least about 15 minutes in preparation for cryopreservation. The preparation process can include adding a cryopreservation medium to the substantially pure MSC population, followed by performing a mixture on a mixture using a controlled speed freezer or other suitable refrigeration system (dump-freeze refrigeration system or cryogenic vessel (Nalgene)) Several cooling steps are performed to reduce the temperature of the substantially pure MSC population to a final temperature of about -90 °C. Suitable speed-controlled cold bed devices include, but are not limited to, Cryomed Thermo Forma Cooling Camera 7544 (Thermo Electron, Corp.), Plane Controlled Cooling Machine Kryo 10/16 (TS Scientific) ' Gordinier ' Bio-Cool- -FTS Systems ' and Asymptote EF600, BIOSTOR CBS 2100 series. An ultra-low temperature storage medium comprising a medium and DMSO can be prepared. Approximately 3 ml of DPBS can be added to a container (such as a 50 ml conical tube). About 1 ml of human serum albumin (HSA) can be added to about 3 ml of DPBS, followed by cooling on ice for about 10 minutes. About 1 ml of cooled 99% DMSO was added to HSA and DPBS to prepare a final cryopreservation medium. The cryopreservation medium and the substantially pure MSC population sample can then be placed on ice for about 15 minutes, followed by the addition of cryopreservation medium to the cell sample. Aliquots of cryopreservation medium were added to the cell samples using a batch process. For example, a first-class 152716.doc • 60. 201130977 portion of a suspension of approximately 100 μΐ of substantially pure MSC population is combined with approximately 3 ml of DPBS, 1 ml of HSA, and approximately 1 ml of 99% DMSO. Approximately 2 aliquots of approximately 200 μM MSC cell suspension are combined with approximately 6 ml DPBS, 2 ml HSA, and approximately 2 ml 99 〇/0 DMSO. Approximately 5 aliquots of the cell sample can be combined with about 15 in DPBS, about 5 ml HSA, and about 5 ml 99% DMSO. Approximately 10 aliquots of the cell sample may be combined with about 30 ml of DPBS, about 10 ml of HSA, and about 10 ml of 99% DMSO. In an alternative embodiment, other cryogenic storage media can be used. For example, cryopreservation media containing cryopreservatives (such as CryoStor CS10 or CS5 (Bi〇life), embryo cryopreservation media supplemented with propylene glycol and sucrose (Vitr〇life), or SAGE media (c〇〇per Surgical) ) can be used to maintain high cell viability results after thawing. Glycerin may be used with other cryopreservatives (such as DMSO) or may be used alone in a medium containing a suitable protein at a concentration of about 1%. The cryopreservation medium can be added to a substantially pure Msc cell population sample. The cryopreservation medium can be added dropwise to the DPBS in which the substantially pure Msc population is suspended until the total volume of the suspended hematopoietic stem cells and cryopreservation medium reaches the final desired volume of the cryopreserved cell mixture. The final cryopreserved cell medium can be transferred to a 2<5 mi bar coded cryo-roller (Cry® quat) where the qC sample is stored in a 5 mi vial cap I. Use a pipette to remove approximately μ1 of the sample and add the aliquot to the Q. lid. After filling the cap, add 4.8 ml of the remaining sample to the 5 (6) vial. Samples should be sent for infectious disease analysis and other analysis. The cold bead vials should be placed in a CRYOMED cold-squeezing device and cooled to a temperature of about 152716.doc -61 - 201130977 at -85 °C. Samples cryopreserved should be transferred to a reservoir (~ to beat) for storage in liquid nitrogen vapor at -150t or below -uot. Any sample that is positive for an infectious disease test should be quarantined. Any sample that is negative for an infectious disease test can be transferred to a different permanent reservoir. The step of lowering the temperature can be programmed in a controlled speed freezer: first, the mixture of cryopreservative and menstrual blood stem cells is cooled. The menstrual blood stem cells combined with the cryopreservation can be cooled at a controlled rate to be finally stored in the freezer. The controlled rate cooling is designed to maintain cell viability. Cry〇_Med Freezer (Thermo Electron Corp.), Liquid Nitrogen Cylinder and Portable Cry〇_

The Med chiller can be used to control the temperature drop for final storage in the freezer. The cells can be cooled at a controlled rate in a frozen vial or freezer bag to a temperature of about -90 °C. For a substantially pure MSC population sample collected in a freezer bag, the cells can be subjected to the following rate-controlled cooling profile: wait at about 4 t), 丨〇 (>c to -6.0 ° C (sample) per minute per Minute 25 〇〇c drops to _5〇〇t (chamber), drops to _M.0eC (chamber) at 10.0C per minute, at 1.〇 per minute. (: down to -45.0 C (chamber) ), to 1 〇〇〇c per minute to _9 〇〇 <> c (chamber), and end (sample at -85.0 ° C or below _85. (Tc under). For collection A substantially pure MSC population sample in a frozen vial, the cells can withstand the following rate-controlled cooling profile: waiting at 4 (rc, 1 〇 per minute) to -3.0 C (chamber), per minute (7) ^ falls to _2〇〇t>c (chamber), drops to -40.0 ° C (chamber) at 1.0 C per minute, drops to -90.0 ° C (chamber) at 1 ° C per minute, And at the end. Once the mixture of cryopreservative and menstrual blood stem cells is at about _75 workers 152716.doc -62·201130977 or below -85 °C, the cryopreservation vials are transferred to a very cold storage unit and at about ^ ^ or below _13Γ (: temperature of liquid nitrogen Stored in steam, or vials can be stored in liquid nitrogen. For example, s 'suitable extremely cold storage devices include (but are not limited to) ln2 cold bundler MVE 1830 (Chart Industries) « can also be flowed Cytometry analyzes fresh samples of substantially pure MSC populations and thawing samples of previously purely cryopreserved substantially pure MSC populations for analysis of cell surface markers, cell viability, and other cellular characteristics. The substance can also be analyzed after cell lysis according to the following protocol Fresh samples from the pure MSC population. The cryopreserved MSC must be resolved according to the method described in this article. In the case that MSC cell samples must be thawed, the substantially pure Msc population may need to be after thawing or immediately after culturing the cells. Flow cytometry analysis is performed to assess passage of a certain cell. The cryopreserved sample can be stirred in a 37 C water bath without allowing the cells to completely thaw. The cells can be transferred to about 1 ml of the cooled wash medium and inverted by inversion. Mix and mix. The sample can be centrifuged at about 2000 rpm for about 7 minutes. The supernatant can be removed and the cells resuspended in about 100 μL. (25% HSA, DNAse, heparin and HBSS w/ Ca+ and Mg+). The resuspended cells can then be centrifuged in a Blood Bank Serofuge for about 1 minute. The supernatant can be decanted and the cells re-exposed. Suspension in about 1.2 ml of Sheath fluid and full rotation. Medium to long term storage of all or a portion of human MSCs (such as medium or long term storage in cell banks) known by the methods disclosed herein is also in the present invention. Within the scope of the. The medium to long term storage of the cells in the cell bank is disclosed in U.S. Patent Application Serial No. 2003/005433, filed on Jan. Into this article. At the end of the treatment, the human Msc as disclosed herein can be loaded into a delivery device such as a syringe for placement into a recipient individual by any means known to those of ordinary skill. In some embodiments, a human MSC obtained by a method as disclosed herein can be packaged in a suitable container in the presence of a suitable medium, as appropriate. In some embodiments, the package further comprises written instructions for the desired purpose, such as methods of implanting human MSCs into an individual to ameliorate or treat the disease (and, where appropriate, storage methods and/or thawing methods (if cryopreserved) )), or a written instruction for a treatment plan for regenerative medicine or therapy. One aspect as disclosed herein relates to a method of treating an individual in need of an increase in the number of stem cells, such as MSCs. In some embodiments, the individual is scheduled or intended to donate stem cells such as MSCs for, for example, heterologous or autologous transplantation. In general, the methods comprise administering to a subject in need of such treatment or having determined that such treatment is administered a therapeutically effective amount of a mobilizing agent, such as gm_csf or G-CSF, as described herein. Administration of a therapeutically effective amount of a mobilizing agent (such as G-CSF or GM-CSF) as described herein for the treatment of such individuals for a period of at least 2 days or at least 3 days but up to 4 days will result in the number of MSCs in the peripheral blood and / or frequency increases. In some embodiments, the administration will increase the number of MSCs in the peripheral blood by about 1 〇 _ 500 times. Methods for measuring such increases are known in the art and are disclosed herein, and see, for example, Neben et al, Blood. 1993; 81(7): 1960-7; Ashihara et al. Exp. Hematol. 2000; 152716.doc •64- 201130977

28(3): 311-7; Pruijt et al., proc.  Natl.  Acad.  Sci.  U. S. A 1999; 96(19): 10863-8. The dosage, toxicity and therapeutic efficacy of a mobilizer such as G-CSF or GM-CSF can be determined by standard medical procedures such as standard medicine for the determination of LD50 (50% lethal dose in the population) and ED50 (50% therapeutically effective dose in the population). The program is determined, for example, in a cell culture or an experimental animal. The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index and can be expressed as the ratio LD50/ED50. A mobilizer (such as G-CSF or GM-CSF) exhibiting a high therapeutic index is preferred. Although compounds exhibiting toxic side effects can be used, care should be taken to design a delivery system that targets these compounds to the site of the affected tissue. Minimize potential damage to uninfected cells and thereby reduce side effects. Information obtained from cell culture assays and animal studies can be used to determine the range of doses for human use. The dosage of such compounds is preferably in the range of circulating concentrations including ED50 with little or no toxicity. Depending on the dosage form employed and the route of administration employed, the dosage may vary within this range. The therapeutically effective dose of any of the compounds used in the methods of the invention can be initially estimated using cell culture assays. (4) It can be determined in the animal model to obtain the circulating gold concentration range 'which includes IC5 as determined in cell culture (that is, the test compound 丨 农度 which achieves the half maximum inhibition of symptoms). This information can be used for the determination of the application. The dose in humans. The amount in plasma can be measured by, for example, Southern Liquid Chromatography. According to another aspect of the invention, the MSC is mobilized as a single-agent from a certain tissue to/before other clinical procedures. Another method in tissue. These cells include non-hematopoietic positive f cells and in their life cycle to 152,716. Doc •65· 201130977 The potential to experience in vivo mobilization/migration during one stage of differentiation. According to another aspect of the present invention, there is provided a method of mobilizing an MSC during collection and during collection of an organ to be used for organ transplantation by infusion of an effective amount of a mobilizing agent such as GM-CSF or G-CSF. In some embodiments, the invention also provides methods of transferring MSCs from ex vivo or peripheral blood samples that have been collected from an individual using an ex vivo mobilizer such as GM-CSF or G-CSF. This includes, for example, in vivo perfusion of in vivo peripheral blood from a donor of a legitimate death organ prior to collection of PB-derived or BM-derived MSCs from a Zhao. In one embodiment, 'administering an effective amount of a mobilizing agent, such as G_CSF or GM-CSF', to the individual to mobilize the bm-derived MSC in the peripheral blood of the individual » In another embodiment, 'administering to the individual' An effective amount of a mobilizing agent, such as G-CSF or GM-CSF, to mobilize the pB-derived MSC in the peripheral blood of the individual » in another embodiment 'to make an effective amount of a mobilizing agent (such as g_csf or GM-CSF) Contacting a peripheral blood sample of the individual to mobilize the PB-derived MSC in the peripheral blood ex vivo. The "effective amount" is an amount sufficient to achieve a significant increase in the number and/or frequency of msc in the peripheral blood. An effective amount can be administered by one or more administrations, administrations, or administrations. The therapeutically effective amount of the mobilizing agent will depend on the chosen mobilizing agent (e.g., G-CSF or GM-CSF). A mobilizing agent such as G-CSF or GM-CSF may be administered one or more times per day to one or more times per week; including every other day. Those skilled in the art will appreciate that certain factors may affect the dosage and timing necessary to effectively treat an individual, including, but not limited to, prior treatment, overall health and/or age of the individual, and other conditions present. Furthermore, treating a subject with a therapeutically effective amount of G-CSF or GM-CSF as described herein can include a single treatment or a series of treatments. 152716. Doc-66-201130977 In some embodiments, the methods of treatment described herein comprise administering another mobilizing agent, for example, an agent selected from the group consisting of, but not limited to, the following: Interleukin-17 (IL-17; Journal Of Immunol.  2001; 167: 2081-2086), AMD3100 (Flomenberg et al., Acta Haematol.  2005; 114(4): 198-205), cyclophosphamide (Cy), European paclitaxel and (DXT) (Ojeifo et al., Experimental Hematology 2000; 28: 451-459). In some embodiments, the method comprises administering to the individual a PB-derived MSC or a BM-derived MSC or a differentiated progeny thereof, eg, reintroducing the cell into the same individual or transplanting the cell into a second individual (eg, an HLA-type match) Two individuals). In some embodiments, an individual that can be effectively treated with PB-derived MSCs or BM-derived MSCs includes any individual that can be typically treated with a bone marrow or stem cell transplant, such as having a cancer, such as a neuroblastoma (produced) Individuals in immature neurons that primarily affect cancer in infants and children, myelodysplasia, myelofibrosis, breast cancer, renal cell carcinoma, or multiple myeloma. For example, PB-derived MSCs or BM-derived MSCs can be transplanted into an individual with a cancer that is resistant to radiation therapy or chemotherapy, for example to restore high dose chemotherapy and/or radiation for the treatment of cancer. Stem cells destroyed by therapy. In some embodiments, the MSC can then be centrifuged in a blood bank serum centrifuge for about 1 minute. The supernatant can be decanted and the cells resuspended at about 1. 2 ml of sputum and fully swirled. 152716. Doc-67-201130977 Medium to long term storage All or a portion of the MSC population obtained by the methods disclosed herein or a substantially pure MSC population (such as intermediate or long term storage in a cell bank) is also within the scope of the invention. The medium to long term storage of the cells in the cell bank is disclosed in U.S. Patent Application Serial No. 2003/005433, the entire disclosure of which is incorporated herein by reference. At the end of the treatment, the MSC population or substantially pure MSC population as disclosed herein can be loaded into a delivery device such as a syringe for placement into the recipient individual by any means known to those of ordinary skill. In some embodiments, a population of MSCs obtained using the methods disclosed herein can be suspended in a short-term storage medium suitable for infusion into an individual, such as the medium disclosed in U.S. Patent No. 5,955,257, the disclosure of in. In some embodiments, MSCs collected and enriched using methods as disclosed herein can be stored (eg, for a donor (eg, allogeneic) or another body for future treatment) or directly transplanted, eg, to facilitate post-operative use Heal. In one aspect of the invention, the plurality of MSCs collected by the methods disclosed herein can be further sorted into at least two subpopulations which can be cryopreserved separately or together (e.g., contained in the same vial). In some embodiments at least two MSC subpopulations can be two different MSC subpopulations. For example, 14|5(: can be sorted into at least 2 cell subpopulations such as, but not limited to, (1) a stem cell population or a MSC rich group and (2) a non-stem cell population or an MSC exclusion group. It is also contemplated that two subpopulations (i.e., above (1) and (2)) may be cryopreserved together. In one embodiment, the sorting of MSCs into subpopulations may be positive or negative sorting or a combination thereof. In some embodiments, cell surface protein markers can be used for positive selection or 152716. Doc -68- 201130977 Select or remove stem cells negatively. Examples of such markers are well known in the art and are shown in Table 1: Table 1 Cell type markers Hematopoietic stem cells (HSC) CD34+, CD45+, CXCR4+ Endothelial progenitor cells CD34+, CD45+, CD133+, KDR+ Very small embryo-like cells (VSEL CD34+, CD133+, CXCR4+, SSEA4+ mesenchymal stem cells (MSC) CD34·, CD45_, CD90+, CD105+, CD106+, CD44+ In some embodiments, a MSC population or a substantially pure MSC population obtained by the methods disclosed herein can be visualized The condition is packaged in a suitable container in the presence of a suitable medium. In some embodiments, the package further comprises written instructions for the desired purpose, such as methods of implanting a MSC or a substantially pure MSC population into an individual to ameliorate or treat the disease (and, where appropriate, storage methods and/or thawing methods) (If cryopreserved), or a written instruction for a treatment plan for regenerative medicine or therapy. MSC for use in short cell therapy: In one embodiment of the invention, MSCs collected from peripheral blood mobilized by an individual as described herein require such cell therapy (e.g., autologous cell therapy) in the individual. It can be introduced or transplanted back into the individual. In some embodiments, MSCs can be used alone or in combination with other cells for untreated therapeutic applications in regenerative therapies, such as wound healing, cardiac repair and bone repair, and other bridge-shaped indications, as disclosed herein in the Examples. In some embodiments, the peripheral blood is mobilized from the individual by a method as disclosed herein 152716. Doc-69-201130977 The collection of MSCs can be used to repair or treat eye diseases such as retinopathy or macular degeneration, as discussed in Vossmerbaeumer et al., Cytotherapy, 2009; 11; 177-188, which is incorporated herein by reference in its entirety. in. In some embodiments, MSCs collected from peripheral blood mobilized by an individual as disclosed herein can differentiate into any number of different cell types originating from mesoderm, including adipocytes, osteoblasts, and chondrocytes, As discussed in Musina et al., Cell Technologies in Biology and Medicine, 2006; 2; 147-151, which is incorporated herein by reference in its entirety. Methods of implanting MSCs for the treatment of cardiovascular disorders and other disorders are well known to those of ordinary skill in the art and are disclosed in International Patent No. WO 2008/0548 19 and U.S. Patent Nos. 6,387,369, 7,514,074, 6,174,333 and 6,225,119, and U.S. Patent Application Serial No. These patents are incorporated herein by reference in their entirety in the entirety of the entire disclosure. In one embodiment of the invention, MSCs obtained from individual mobilized peripheral blood can be used in personalized medical applications, such as autologous therapeutic uses, and in personalized assays to assess personal diet, pharmacogenetics, nerves The impact of chemicals and lifestyles on MSC function and vitality. A MSC composition comprising some or all of the MSCs collected from peripheral blood of an individual by methods as disclosed herein can be used to repair, treat or ameliorate various aesthetic or functional conditions (eg, defects) via augmentation of damaged tissue^ MSCs collected from peripheral blood of an individual by methods as disclosed herein provide an important resource for rebuilding or supplementing damaged tissue. In one embodiment, MSCs collected from peripheral blood of an individual by methods as disclosed herein can be used in tissue engineering and regenerative medicine to replace 152716. Doc •70· 201130977 The body part of the defect, injury, disease or aging wear and tear. MSCs collected from peripheral blood of an individual by methods as disclosed herein provide a unique system in which msc can differentiate into distinct cells and produce a particular lineage or genotype of the same individual. Thus, MSCs collected from peripheral blood of an individual by the methods disclosed herein provide significant advantages of individualized stem cell therapy. Moreover, in the absence of disease or injury, such Mscs and compositions thereof collected from peripheral blood of an individual by methods as disclosed herein may be added to the soft tissue region 'splits, depressions or voids (such as For flattening to supplement soft tissue unrelated to injury. The invention also encompasses multiple and continuous administration of MSCs collected from peripheral blood of an individual by methods as disclosed herein. For stem cell-based treatment, by this document Methods of Revealing MSC populations collected from peripheral blood of an individual are preferably collected from an autologous source. The MSC composition is then prepared and isolated as described herein, and in particular, with other stem cell populations present in the peripheral blood of the mobilized Separation. In order to introduce or transplant a mononuclear cell suspension from a peripheral gold collection of an individual and/or a composition thereof into a human or animal recipient by a method as disclosed herein. The solution contains a concentrate of MSC and can be separated from other stem cells present in the peripheral blood, and the Msc is collected and resuspended in a physiologically acceptable load. Or in an excipient or diluent. Alternatively, the MSC suspension may be contained in a serum-free sterile solution (such as a cryopreservation solution). In some embodiments, an MSC-rich formulation may also be used. In some embodiments The MSC suspension can then be introduced into one or more sites of the donor tissue via, for example, injection. Doc • 71 · 201130977 The concentrated or enriched stem cells can be administered in the form of a pharmaceutically or physiologically acceptable formulation or composition containing a physiologically acceptable carrier, excipient or diluent, and An organization that deals with the recipient's organisms, including humans and non-human animals. MSCs can be prepared by resuspending the cells in a suitable liquid or solution, such as sterile saline or other physiologically acceptable injectable aqueous liquid. (Composition. The amount of each component in such compositions. It can be determined in a conventional manner by those skilled in the art. In some embodiments, 'a population of MSCs or a combination thereof collected from a peripheral blood of Zhao as disclosed herein can be administered as follows: a stem cell suspension is placed Adhering to an absorbent or adhesive material (ie, a collagen sponge matrix) and inserting the material containing the MSC into the relevant site or related site, or 'by collecting blood from the peripheral blood of the individual by methods not disclosed herein. % of the population can be administered by parenteral injection (including subcutaneous, intravenous, intramuscular, and intrasternal injection). Other modes of administration include, but are not limited to, intranasal, intrathecal, intradermal, transdermal, and intestinal. And sublingual administration. In one embodiment, the MSC population collected from the peripheral blood of the individual by the method disclosed can be administered by endoscopic surgery. Method of Revealing MSCs The composition of MSCs collected from the peripheral blood of an individual can be suspended in a sterile solution or suspension or can be resuspended in a pharmaceutically and physiologically acceptable aqueous or oily vehicle, which may contain a preventive agent. Stabilizers and substances which cause the solution or suspension to be isotonic with the body fluid (ie, blood) of the recipient. Non-limiting examples of suitable excipients include water, sodium salt buffered saline (pH 7 4), 〇15 M gasification aqueous solution, dextrose, glycerin, dilute ethanol and their analogues, and their mixtures 152,716. Doc -72· 201130977 Things. Illustrative stabilizers are polyethylene glycols, proteins, sugars, amino acids, inorganic acids, and organic acids that can be used alone or in combination. The amount or amount and the route of administration employed will depend on the individual and will be equivalent to the amount employed in a similar dosage form or indication known to those skilled in the art, in accordance with the present invention, by the method disclosed herein from the periphery of the individual. Blood-collected MSC populations can be administered to body tissues, including epithelial tissue (ie, skin, lumen, etc.), muscle tissue (ie, smooth muscle), blood, brain, and various organ tissues, such as those associated with the urinary system ( That is, bladder, urethra, ureter, kidney, etc.). According to the general treatment methods described herein, a population of MSCs collected from peripheral blood of an individual by methods as disclosed herein may comprise other cells (eg, a portion of a cell comprising a population of target stem cells) and may be administered to the individual, eg, by intravenous (ι·ν. The catheter is infused into the bloodstream of the individual (similar to any other intravenous fluid). Alternatively, however, an individualized mixture of MSC populations can be generated, such as to provide a cell therapy mixture specifically for the therapeutic needs of the individual. A comprehensive cell mixture such as obtained via a separation method can be characterized for sorting and separation into different cell populations. Cell markers (such as VSEL, MSC, and HSC markers) or tissue-specific markers can be used to phenotypically characterize a population of cells harvested from peripheral blood. Using these markers, it is possible to separate and sort based on cell type. The cell mixture is thereby converted into a population of cells which can be broadly divided into two parts: a stem cell fraction and a non-stem cell fraction. The non-stem cell fraction can be further divided into progenitor cells or fibroblast fractions as well as functional cells or fully differentiated cell fractions. Once the peripheral blood cell mixture is sorted, the MSC group and the non-stem cell parts can be cryopreserved and stored separately. In this way, it can produce 152716. Doc •73- 201130977 Born from a collection or repository of different cell groups of Zhao. Alternatively, the Msc population and non-stem cell fractions can be cryopreserved together and then sorted and separated prior to use. In some embodiments, a population of MSCs collected from peripheral blood of an individual by methods as disclosed herein can be used to produce or differentiate into any population of a certain cell type, which is derived from germ layers (ie, endoderm, mesoderm, and ectoderm) ) developed. Such cells include, but are not limited to, differentiated cells, neural progenitor or differentiated cells, glial progenitor or differentiated cells, oligodendrocyte glial progenitor or differentiated cells, dermal progenitor or differentiated cells, hepatic progenitor cells or Differentiated cells, muscle progenitor cells or differentiated cells, osteoprogenitor or differentiated cells, mesenchymal stem cells or progenitor cells, pancreatic progenitor cells or differentiated cells, progenitor cells or differentiated chondrocytes, stromal progenitor cells or differentiated cells, cultured and expanded Stem cells or progenitor cells, cultured differentiated stem or progenitor cells, or a combination thereof. Progenitor cells are also of interest, such as hematopoiesis, nerves, stroma, muscle (including smooth muscle), liver, lung, gastrointestinal and mesenchymal progenitor cells. Also contemplated are differentiated MSC populations such as differentiated into cells such as, but not limited to, osteoblasts, hepatocytes, granulocytes, chondrocytes, myocytes, adipocytes, neuronal cells, pancreatic cells, or combinations thereof and mixture. In some embodiments, a population of MSCs collected from peripheral blood of an individual by methods as disclosed herein can be combined, recombinantly or compounded into a mixture of cell therapy cells suitable for treating disease and/or tissue regeneration in a subject. The combination of a MS C population collected from a peripheral blood of Zhao and a tissue-specific progenitor cell and optionally a functional cell by a method as disclosed herein can be used, for example, to enhance the graftability of the transplanted MSC. 152716. Doc • 74· 201130977 Accordingly, in one embodiment, the present invention provides methods and products for using an autologous mixture of MSC populations collected from peripheral blood of an individual by methods as disclosed herein. This MCS population can be used alone or in combination with other suitable cells. In some embodiments, the MSC can be combined with other stem cells derived from peripheral blood, or other functionally suitable non-stem cells. Thus, individual MSCs can be used alone or selectively recombined (customized mix) for individualized autologous therapeutic applications in regenerative therapies. In other words, the autologous MSC population can be selectively combined with other cells, including stem cells, such as other non-MSC stem cells derived from peripheral blood, for autologous cell therapy. In some embodiments, the mixed MSC population can comprise from about 10% to about 90% of MSCs collected from peripheral blood of the individual by methods as disclosed herein, from about 10% to about 80%, or from about 10% to about 60%. %, or from about 10% to about 40%, or from about 10% to about 90% of MSCs collected from peripheral blood of an individual by methods as disclosed herein. In some embodiments, the MSC population may also comprise non-stem cells (eg, functional cells) and/or populations of stem cells that are also derived from peripheral blood, such as from about 5% to about 50% functional cells and/or non-MSCs. Stem cells, from about 5% to about 40% functional cells and/or non-MSC stem cells, from about 5% to about 30% functional cells and/or non-MSC stem cells, from about 5% to about 20% functional cells and/or non-MSC stem cells, Or from about 5% to about 10% functional cells and/or non-MSC stem cells. One of the above cell therapy products is suitable as an example of an autologous mixture of HSC and MSC or other functional cells of the hematopoietic system. Another example is a cell therapy product comprising an autologous MSC population mixed with other cells, such as at least one or any combination of the following cell types: HSC, PBSC, 152716. Doc -75- 201130977 Cardiac progenitor cells and cardiomyocytes as appropriate. Accordingly, in another embodiment, a method of treating a patient in need thereof, comprising administering to the individual, alone or in combination (separately or in combination), the MSC population collected from peripheral blood of the individual by a method as disclosed herein Automated Character 0 Stem Cell Banking In another aspect of the invention, a population of MSCs collected from peripheral blood of an individual by methods as disclosed herein can be stored in a cell bank to support optional health care insurance. Models that effectively protect group members from future diseases. Individual individuals can be treated and preserved from the periphery when they are in a healthy state: collect their own stem cell population for future distribution of their health care needs. Thus, in an embodiment, the MSC population collected from the peripheral blood of the individual is "stored" in a stem cell bank or a storage or storage facility, or in a place where the target stem cell population is preserved for storage by a method as disclosed herein. Future use. The storage wire can be designed in such a way that a catastrophic event such as a nuclear attack can be designed in a manner that is safe from the periphery of the individual by the method disclosed herein. In or in the cellar. In other embodiments, it may be on the side of the mountain or in outer space. Stored in the mask 4 can be embedded in (4) such as the wrong masking material. According to a further embodiment, providing a 4-step stem cell reservoir (10) comprises administering to the individual one or more mobilizing agents to increase the amount of moths in the peripheral blood of the individual. The steps are included by 152716. Doc • 76 · 201130977 Peripheral blood collects MSC populations, where the individual does not have immediate health conditions, such as no conditions in which individuals need to use their own target stem cell population for their own autologous transplantation. Step C includes preserving the population of MSCs collected from peripheral blood by a method as disclosed herein in the form of a deposited cell population. Step D includes obtaining a deposited MSC population collected by a method as disclosed herein to autologously transplant the MSC population into the individual. Reference will be made in detail to the embodiments of the invention, examples of which are illustrated in the accompanying drawings. In some embodiments of the invention, the MSC is separated from the mobilized blood using an Elutra® centrifuge manufactured by Gambro BCT, Inc. of Lakewood, Colorado. Although the embodiments of the present invention are described in connection with an Elutra® centrifuge, this reference is for illustrative purposes only and is not intended to limit the invention in any sense. It will be appreciated that embodiments of the invention may use other centrifuges including, but not limited to, also by Gambro BTC Inc. COBE® Spectra separation system, Trima® system and Trima Accel® system, as well as other panning devices for separating blood components. MSC is used for personalized medical diagnosis and prognostic testing. Another aspect of the invention relates to the use of MSCs derived from individual mobilized peripheral blood for personalized medical applications, such as autotherapeutic uses, and for personalization assays to assess personal diet, medications The effects of genetics, neurochemicals, and lifestyle on MSC function and vitality. Such assays are currently well known in the art and include high yield screening in which a compound, agent or environmental stimulator is contacted with MSC, and can measure the function of a compound, agent or environmental stimulator against MSC (eg, differentiation potential) , proliferation, survival) and vitality effects, and the results are compared with reference control samples (eg not 152716. Doc -77· 201130977 The presence of a compound) or a positive control sample 'such as the presence of BMP6 and the like in the aspect of the invention, the milk C population obtained from the individual can be used in high yield screening to assess the individual versus the agent Individual reactions (such as for specific drugs, hormone therapy, or medicine or therapy). Briefly, the MSC cell population is contacted with the relevant agent and the agent is effected by monitoring the output parameters (such as marker expression, fine tongue feature, pluripotency, and the like). The MSC can be freshly isolated using methods as disclosed herein, or in some embodiments, cultured, cryopreserved, or genetically engineered prior to use in assaying t. In some embodiments, the individual's position may be an environmental change of the pure line culture (4): for example, splitting into independent poplar and growing under unique conditions (eg, with or without virus; presence or absence of other cytokines or combinations thereof) . Importantly, because MSCs are specific to individual genetic makeup, the use of MSCs provides a tool for pharmacogenetic analysis of MSCs. For example, individual MSCs can have specific variants that result in unique pathological features. For example, because Msc is derived from an individual (e.g., autologous)', it has the desired pathology (4), e.g., a diseased lesion or a mutation and/or polymorphism that results in a poor or enhanced response to a therapeutic agent. Thus, Msc obtained by the methods as disclosed herein can be used to assess an individual's response to a particular compound, an agent's MSC, and in some cases this may be applicable to the prognosis of the disease. In this embodiment, the method of the invention can be used to screen for agents that alleviate lesions, or agents that positively affect MSC proliferation or function. In an alternative embodiment, the method of the invention can be used for screening as compared to 152716. Doc . 78 · 201130977 Other individual MSCs (eg, no mutations and/or polymorphisms) that cause different MSC responses in an individual (due to intrinsic genetic composition or specific mutations and/or polymorphisms), so these methods are available For example, assessing the effects of specific drugs and/or agents on individual Msc populations compared to other human and/or MSC cells, thus acting as a high yield screening for personalized medicine and/or pharmacogenetics. The manner in which MSCs react to a drug (specifically, a pharmacological agent) (including the timing of the reaction) is an important reflection of the physiological state of the cell. The parameters are quantifiable components of MSC, in particular, components that are accurately measured in high yield systems. The parameter can be any cellular component or cellular product, including cell surface determinants, receptors, proteins or their conformational or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids (eg, mRNA, DNA) Etc.), or derived from a portion of the cellular component, or a combination thereof. Although most parameters will provide a quantitative reading, in some cases semi-quantitative or qualitative results are acceptable. The readings may include a single measurement, or may include an average, median or deviation, and the like. A characteristic range of parameter readings for each parameter will be obtained using many of the same assays. Variability is expected and a range of values for each test parameter set will be obtained using standard statistical methods and common statistical methods for providing a single value. Compounds including candidate agents are obtained from a wide variety of sources, including pools of synthetic or natural compounds. For example, a variety of means are available for the random and directed synthesis of a wide variety of organic compounds, including biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, a collection of natural compound pools in the form of bacterial, fungal, plant and animal extracts may be utilized or readily produced. In addition, collections and compounds produced naturally or synthetically are readily accessible via 152716. Doc -79· 201130977 Conventional chemical, physical and biochemical modifications and can be used to generate combinatorial collection libraries. Oriented or random chemical modifications can be made to known pharmacological agents, such as deuteration, alkylation, acetification, aromatization, etc., to produce structural analogs. The agent is screened for the effect of the agent on the stem cell population by adding the drug (usually along with the cells lacking the agent) to at least one and typically a plurality of stem cell samples. The change in parameters that are responsive to the agent is measured and the results are evaluated by comparison to a reference culture (e.g., obtained with other agents in the presence and absence of the agent, etc.). In some embodiments, the agent is preferably added to the culture medium of the stem cells in culture in solution or in a soluble form. The agent may be added in a flow or through system in the form of a batch or continuous flow, or the compound bolus may be added one by one or incrementally to another still solution. In a flow through system, 'two fluids' are used, one of which is a physiologically neutral solution and the other is the same solution as the added test compound. The first fluid and the second fluid are sequentially passed over the stem cells' in a single solution method' the test compound bolus is added to a volumetric medium surrounding the cells. The total concentration of the components of the medium should not vary significantly with the addition of the bolus, or in the flow-through method, it should not vary significantly between the two solutions. In some embodiments, the pharmaceutical formulation does not include other components, such as preservatives, that can have a significant effect on the overall formulation. Therefore, it is preferred that the substance is substantially composed of a biologically active compound and a physiologically acceptable carrier such as water, ethanol, DMSO or the like. However, if the compound is a solventless liquid, the formulation may consist essentially of the compound itself. 152716. Doc -80- 201130977 Multiple tests can be run in parallel with different drug concentrations to obtain differential responses to various concentrations. As is known in the art, a range of concentrations produced on a 1:10 or other logarithmic scale, dilution, is typically used to determine the effective concentration of the agent. If necessary, the concentration can be further improved by the dilution of the second series. Typically, one of these concentrations acts as a negative control, i.e., at or below the level of detection of the agent or at or below the concentration of the drug that does not produce a detectable phenotypic change. The individual MSCs used for screening can be manipulated to express the desired gene product, e.g., to confer undesirable physiological and/or polymorphic phenomena in the genetic makeup of the MSC. Thus, the gene therapy is performed on the individual msc ex vivo before the individual MSC is autologously transplanted back into the individual. In some embodiments, the MSC can be genetically modified to replace the gene product or to add or inhibit the gene product. In some embodiments, genetic engineering can promote tissue regeneration, treat disease, or improve survival (i.e., prevent rejection) of MSCs after implantation in an individual. Techniques for transfecting cells are known in the art. A number of cells are used for autologous transplantation, and those skilled in the art will be able to contemplate that a wide variety of genes can transfer beneficial properties to transfected individual mesenchymal cells. In some embodiments, depending on the optic gene and the examples, the added gene may ultimately remain in the recipient MSC and all of its progeny, or may be only transiently retained. For example, a gene encoding an angiogenic factor may be transduced. Dyeing into MSCs isolated from peripheral blood. When MSCs are used to differentiate into cardiomyocytes and repair myocardium, these genes will be useful for inducing lateral vascularization. In some cases, it may be desirable to transfect cells with more than one gene. 152716. Doc -81 - 201130977 In some cases, it is necessary to secrete the gene product. In such cases, the gene product preferably contains a secretion signal sequence that promotes secretion of the protein. For example, if the desired gene product is an angiogenic protein, those skilled in the art can select an angiogenic protein with a native signal sequence, such as VEGF' or can modify the gene product using conventional genetic manipulation (see Nabel et al., ι 993). To contain such a sequence. In one embodiment of the invention, MSCs obtained from individual mobilized peripheral blood can be individually assayed for use in studying and understanding the signaling pathways of individual MSC growth and differentiation. The individual MSCs of the invention are useful for helping to develop therapeutic applications for innate and adult heart failure. The use of such individual MSCs of the present invention enables the study of specific differentiation into different lineages (e.g., osteogenic lineage, cardiac lineage) without the need for complex, time consuming animal models. In another embodiment, a MSC can be genetically modified to carry a particular disease and/or a pathological trait and phenotype of a particular disease or condition. In one embodiment, the assay comprises a plurality of individuals of the invention or their differentiated progeny. In one embodiment, the assay can be used to study the differentiation pathways of individual Msc, such as, but not limited to, differentiation along the myocardial cell lineage, the smooth muscle lineage, the endothelial lineage, and subpopulations of such lineages. In another embodiment, the assay can be used to study the pathological features of an individual Msc, such as a disease and/or genetic characteristics associated with the disease or condition. In some embodiments, the disease or condition is a wound healing condition, a bone condition, or a cardiovascular condition or disease. In some embodiments, the individual Msc has been genetically engineered to include features associated with the disease or condition. These methods of genetically engineering individual MSCs are well known to those skilled in the art and include by means of 152716. Doc • 82- 201130977 Transfection (such as, but not limited to, transfection with a viral vector) or introduction of nucleic acids into cells by other means known in the art. In some embodiments, an individual MSC can be readily manipulated in an experimental system that provides advantages in targeting lineage differentiation as well as homologous homogeneity and is capable of operating an external environment. Furthermore, because experimentally altering the human germline is ethically unacceptable, the ES cell transgene pathway cannot be used for experiments involving the manipulation of human genes. The gene targeting in the human individual Msc of the present invention allows for the important application of the system in which the system cannot be tested in vivo due to ethical issues, and can appropriately reproduce the human biology or disease process of a particular individual. In another embodiment, the individual MSCs of the invention can be used to prepare cDna-contaminated cdna pools of libraries that are relatively unexpressed in cells of other lineages. The individual MSCs of the invention can also be used to prepare antibodies specific for the signature of cardiomyocytes and their precursors. Multiple antibodies can be prepared by injecting vertebrate cells in the immunogenic form of the cells of the invention. The production of monoclonal antibodies is described in standard references such as U.S. Patent Nos. 4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology 73B:3 (1981). Specific antibody molecules can also be produced by contacting a pool of immunocompetent cells or virions with a target antigen and growing a positively selected line. See Marks et al., New Eng.  J.  Med.  335:730, 1996 and McGuiness et al., Nature Biotechnol.  Another alternative to 14:1449, 1996 ° is to reassemble random DNA fragments into the antibody coding region as described in EP Patent Application Serial No. 94,1,8 a. 1527l6. Doc •83· 201130977 Antibodies can in turn be used to identify or remediate (eg, restore phenotype) cells from a mixed cell population with the desired phenotype to achieve co-staining, such as during immunodiagnosis using tissue samples, and to make precursor cells and terminal cells The purpose of cell differentiation of differentiated individuals Msc and other lineages. In another embodiment, an individual MSC can be used to examine gene expression profiles during and after differentiation of individual MSCs of the invention. The expressed genome can be compared to other MSC subpopulations from different individual or control MSC samples known in the art. Any suitable qualitative or quantitative method known in the art for the determination of a particular mRNA can be used. mRNA can be hybridized, for example, to a microarray, in situ hybridization in a tissue section, by reverse transcriptase-PCR, or The northern blot of p〇iy a+ mRNA is detected. Those skilled in the art can readily use these methods to determine the molecular size or molecular weight difference of mRNA transcripts between two samples. Any method suitable for detecting and comparing the amount of mRNA expression in a sample can be used in conjunction with the methods of the invention. For example, the amount of mRNA expression in a sample can be determined by generating a pool of expressed sequence tags (ESTs) from the sample. An enumeration of relative representations of ESTs in the pool can be used to estimate the relative degree of expression of the gene transcripts within the starting sample. The results of the EST analysis of the test sample can then be compared to the EST analysis of the reference sample to determine the selected polynucleotide (specifically, a polynucleotide corresponding to one or more of the differentially expressed genes described herein) The relative amount of performance. Alternatively, gene expression in test samples can be performed using the Gene Expression Continuous Analysis (SAGE) method (Velculescu et al., Science (1995) 270: 484) into I52716. Doc -84 - 201130977 Hit. Briefly, SAGE involves the isolation of short unique sequence tags from specific locations within each transcript. Sequence tags can be linked, colonized, and sequenced. The frequency of a particular transcript within the starting sample is reflected by the number of times the associated sequence label meets the sequence group. The gene expression in the test sample can also be analyzed using the differential representation (DD) method in DD, and the fragment defined by the specific sequence delimiter (eg, restriction enzyme site) is used as the unique identifier of the gene, coupled to the length of the fragment or Information about the location of the fragment within the expressed gene. The relative degree of expression of the expressed genes in the sample can then be estimated based on the relative performance of the fragments associated with the gene within the pool of all possible fragments. Methods and compositions for entering RDD are well known in the art, see, for example, U.S. Patent No. 5,776,683; and U.S. Patent No. 5,807,6. Alternatively, the system is based on nuclear interactions.  A specific hybridization assay detects gene expression in the sample. The nucleus acid or cDNA can be used to selectively identify or capture a DNA or RNA having a specific sequence composition, and qualitatively or quantitatively determine the amount of rna or cDNA that hybridizes to a known capture sequence to provide a specific message within the pool of cell information in the sample. Information on the relative performance. Hybridization assays can be designed to allow for hundreds to thousands of screenings by using, for example, array-based techniques with high density formats (including filters, microscope slides or microchips) or solution-based techniques using spectral analysis (eg, mass spectrometry) The relative performance of genes. An exemplary use of the array in the diagnostic method of the present invention is described in more detail below. Hybridization with the array can be performed' wherein the column can be produced according to any suitable method known in the art. For example, a method for producing a large array of oligonucleotides using photo-guided synthesis techniques is described in U.S. Patent No. 152,716. The heterogeneous array of monomers can be converted to a heterogeneous array of polymers by simultaneous coupling at a number of reaction sites in U.S. Patent No. 5,445,934 and U.S. Patent No. 5,445,934. Alternatively, the microarray can be produced by depositing a pre-synthesized oligonucleotide on a solid substrate, e.g., as described in PCT Publication No. WO 95/35505. Methods for collecting data for hybridization of a sample to an array are also well known in the art. For example, a polynucleotide of a cell sample can be generated using a detectable fluorescent label and the hybridization of the polynucleotide in the sample can be detected by scanning the microarray for the presence of the detectable label. Methods and apparatus for detecting fluorescently-marked objects on a device are known in the art. Generally, such detection devices include a microscope and a light source that directs light to the substrate. The photon counter detects the fluorescence from the substrate and the xy translation stage changes the substrate position. Confocal detection devices that can be used in the subject method are described in U.S. Patent No. 5,631,734. Scanning laser microscopy is described in Shalon et al.' Genome Res.  (1996) 6:639. Each of the fluorophores used was scanned using an appropriate excitation line. The digital images produced by the scan are then combined for subsequent analysis. For any particular array element, the ratio of the fluorescent signal from one sample is compared to the fluorescent signal from another sample and the relative signal strength is determined. Methods for analyzing data collected from hybrid arrays are well known in the art. For example, when the hybridization detection includes a fluorescent marker, the data analysis may include the steps of: determining, according to the collected data, the intensity of the light that changes with the position of the substrate, 'removing the outliers', that is, deviating from the predetermined statistical distribution. Data; and calculate the relative binding affinity of the target based on the remaining data. The resulting data can be presented as images, with the intensity in each region following the knot between the target and the probe 152716. Doc -86- 201130977 Change with affinity. Style matching can be done manually or using a computer program. Methods of preparing substrate matrices (eg, arrays), designing oligonucleotides for use with such matrices, probe labels, hybridization conditions, scanning hybrid matrices, and analyzing the resulting patterns (including comparative analysis) are described, for example, in US Patent No. No. 5,800,992. General methods in molecular and cellular biochemistry can also be found in standard textbooks such as: A Laboratory Manual, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (Ausubel et al.) Edited by John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (edited by Wagner et al., Academic Press 1999); Viral Vectors (edited by Kaplift and Loewy, Academic Press 1995); Immunology Methods Manual (I.  Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle and Griffiths, John Wiley & Sons 1998). The reagents, selection vectors and kits for genetic manipulation referred to in the present invention are commercially available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich and ClonTech®. All individuals received a subcutaneous injection of G-CSF (Neupogen) 480 pg for at least 2 days or less, or 240 pg/kg of a single dose of AMD3100 (Pulesaf, Mozobil®, before administration of cells to mobilize stem cells). Genzyme Corporation, Cambridge, MA) and will be at 152716. Doc -87 - 201130977 After the administration of the drug, it was subjected to separation. Peripheral blood samples (Day 1, Day 2, Day 3, Day 4, and Day 5) were taken to monitor white blood cell count (WBC) and on Days 4 and 5 before undergoing the separation procedure. FACS analysis was performed to assess the modulating efficacy of CD34+ cells in the circulation and to determine the separation parameters. If the individual WBC is higher than 60,000/mm3, the delivery of G-CSF is discontinued and the separation is performed as early as possible. Separation will be performed at the New York Blood Center at the Petri Division in Beth Israel Medical Center (see Medical Director)

Vijay Shah's accompanying letter). MSC is mixed and broadcasted. All individual isolates were processed as described below to isolate MSCs for cell therapy according to standard techniques. » The various cell populations in the mobilized PB will use Fic〇I1_ Paque, followed by 50 The ml conical tube was separated by gradient centrifugation. Two layers of PB, such as PB, were formed on each of the 10 ml Fico-Parker liquids that had been divided into 4 ml of 50 ml. This is achieved by slowly and gently flowing pb down the tube side to the flow. The layered tube was centrifuged at 400 x g for 30 minutes at room temperature. Once pB was centrifuged, the intermediate white blood cell layer was placed in a T25 culture flask. Each τ25 flask will use 7 ml MSC complete medium. The contamination and cell morphology of the cells were examined by light microscopy twice a week and the medium was changed. Wash unadhered cells when changing the medium. After the cells are confluent, they will be passaged into T75 flasks. For cell therapy for patients, early passage (up to 5 times) will be used. Quality control tests were performed on the final cell product prior to delivery of the cells to the patient' including phenotypic characterization, karyotyping, mycoplasma and bacterial testing. The cell dose will be "〇〇, 〇〇〇 cells. 152716.doc -88- 201130977 f潆身培培# of M5T. Remove the medium from the T-75 flask. Wash each flask with 1〇 as lx sterile PBS 2 Minutes. Remove PBS and add 2.2 mi chymotrypsin (TryPsin)-EDTA to each flask. Rotate the flask to allow trypsin to completely coat the bottom of each flask. Return the flask to the incubator for i 〇 minutes. Check for proper trypsinization by examining cell roundness and cell mobility. Adenosine-EDTA is not activated by adding 5 ml of MSC medium to each flask. Use a pipette to gently relax the bottle of shampoo Bottom medium to maximize cell capture. Transfer the cell/culture mixture to the centrifuge tube using the same pipette. Combine cells and media from all flasks into one 50 ml centrifuge tube. At room temperature at 400 The cells were centrifuged for 7 minutes at g (1400 rpm). The supernatant was carefully removed and the pellet was resuspended in 10 ml sterile saline (standard 0.9 〇 / saline obtained from the pharmacy). 400 g (14〇 The cells were centrifuged for 7 minutes at rpm. This procedure was repeated twice. Carefully remove the supernatant and dilute it 10 times by suspending it in 1.5 ml of saline and obtaining this suspension (1〇4 in 1〇〇^ medium) A small aliquot of the sample is used to count the cells. The hemacytometer well requires 10 μΐ of the fluid sample. The window is invited to # bath therapy; sputum combination: MSC is then mixed with the fibrin gel as described below and 4 Weekly intervals were applied to the wound 3 times. Preparation and application of diluted fibrin gel/cell mixture. Description of the fly The diluted fibrin gel was prepared using 1 ml TISSEEL VH kit (Baxter Healthcare, inc.). The system utilizes two liquid phases that can be extruded via a dual chamber applicator or via an applicator with an inert gas carrier. In this diluted fibrin gel, the coated protein (sealer 152716.doc -89 - 201130977) The final concentration of protein and thrombin was 25 units/ml, respectively. Prepared by adding 0.221 g of CaClz powder to 50 ml of sterile physiological saline and filtering the mixture through a 0.22 μπι top filter in a tissue culture hood. 30 A sterile mixture of mM calcium (CaCh) in physiological saline (0.99% NaCl). To prepare the clotting factor solution, dissolve 1 ml of the bottled clotting factor in a heated mixer for 20 minutes. Once the mixture is dissolved, use a syringe from the bottle. 0.5 ml was withdrawn and mixed with 4.5 ml of sterile CaCb mixture. This mixture was kept warm in the heating mixer. This diluted clotting factor solution was used within 4 hours of the initial dissolution step. To prepare the encapsulated protein, 1 ml of sterile physiological saline was added to the coated protein vial using an i W syringe and dissolved in a heated mixer for 20 minutes. Once the physiological saline was dissolved, 0.5 ml was taken from the bottle with a syringe and mixed with 4.5 ml of sterile physiological saline. This mixture was kept warm in the heating mixer. This diluted coating protein was used within 4 hours of the initial dissolution step. The gel is formed as follows: first depending on the size of the wound, the total amount of application of the gel is determined. Each of the two liquids will be one-half of this total. The same amount of encapsulated protein as necessary to coat the final volume of the protein/cell mixture was aspirated in a 1 ml pipette. The cells and the coated protein were mixed by gently pipetting up and down (4 times) in the centrifuge tube containing the cells. The coated protein/cell mixture is pumped up into a 1 ml syringe that has been removed. The volume of the coagulation factor solution of the same volume as the coated protein/cell mixture is pumped up another 1 ml to inject nt; any bubbles are knocked out again. The needle was removed and the two syringes were snapped into the red applicator. The white./transparent applicator fitting is then placed over the syringe opening.嗔 fog 152716.doc 201130977, the sterile gas hose is connected to the applicator joint and the gas supply flow is maintained during application to prevent condensation in the applicator joint. If the gel is to be extruded rather than ejected, continue to steadily and slowly squeeze until all fluid is used up to avoid condensation in the applicator fitting. CFU-C test (community forming unit culture) Adjust the number of femoral bone marrow cells in MEM (flow) to 2 5 x i 〇 6 cells/ml. 0.2 ml of this suspension with 〇·5 mi horse serum, 〇.丨mi thioglycerol (20 mM 'diluted 4 times with MEM), 1. 〇mi曱 based cellulose in mem), 〇·6 ml MEM ( Flow) and Oi mi additional medium or standardized stimulated mouse serum (200-fold dilution of serum taken 3 hours after intraperitoneal administration of 2.5 mg/kg lipopolysaccharide (LPS)) or 5 ng/ml rhG-CSF . The fully mixed semi-solid suspension was drawn into a 4 CIn Petri dish and incubated for 6 days at 37 ° C '5% C02 and 95% relative humidity. After adding 0.5 ml of a purple solution of p-i〇d〇nitrotetrazolium (0.5 mg/ml PBS), the culture dish was incubated for another 24 hours. The community was counted using a community counter and calibrated to 1 to 6 bone marrow cells. Example 1 Isolation of MSCs from Peripheral Blood and Amplification The present inventors utilized MSCs derived from mobilized peripheral blood, wherein granulocyte community stimulating factor (G-CSF) was administered to individuals to mobilize MSCs from the bone marrow to the peripheral circulation. . The mobilized peripheral blood is enriched in stem cells by Fico separation and collection of leukocytes containing monocytes. The cells from the white blood cell layer were then seeded in a cell culture flask containing MSC growth medium. Unadhered cells were washed off during the first two weeks of regular (every 3 days) medium change. Adhesive 152716.doc •91· 201130977 The attached cells were then expanded for up to 5 passages in MSC growth medium. Proof that MSC is mobilized from G-CSF to peripheral blood. The inventors have demonstrated that as a response to two consecutive subcutaneous injections of G-CSF (480 pg per day), MSCs are mobilized to the peripheral blood of healthy individuals. The 'inventors' demonstrated that the MSC phenotype (ie, CD45-, CD34-, CD105+, CD90+) is enriched in the pre-mobilization and mobilized peripheral blood samples analyzed by multiparameter flow cytometry analysis. , CD29+) a large number of cells. Figure 6 shows a significant increase in the number of MSCs in the peripheral blood of healthy human donors (η = 5) after G-CSF mobilization. The one-tailed paired τ test has shown that 'MSCs with CD45-, CD34-, CD90+, CD105+, CD29+ respond adequately to G-CSF mobilization and the number of MSCs in the sample after mobilization is significantly increased compared to the sample before mobilization (ρ= 〇.03). Use of MSC samples obtained from mobilized peripheral blood Chronic ischemic, diabetic, and venous stasis wounds affect millions of Americans and cause a large number of morbidities, including loss of function, chronic pain syndrome, and systemic infections Even the risk of fatal infections increases. Even when the latent condition has improved, the resistance to skin wound healing can be related to the following: cells in the wound (specifically, stem cells in the "tissue") aging' disease-based pair exists in successful wound healing Abnormal reactions of normal cell-cell and cell-matrix signaling; or necrotic debris and/or scars that disrupt coarse and microscopic tissue, preventing the action of body fluids or cells on the healing process. In particular, systemic cell migration of bone marrow-derived stem cells can play an important role in wound healing and can be important for grafting such endogenous barriers. Human autologous bone marrow-derived (non-peripheral blood-derived) mesenchymal stem cells (MSC) isolated from ex vivo culture and applied to wounds can be affected by isolation from patients with chronic wounds 152716.doc •92· 201130977 Significant functional tissue recovery and minimal scar formation were observed in all tissue layers of the limb. Thus, one aspect of the invention pertains to the use of PB-derived MSCs obtained from an individual according to methods disclosed herein, which may be from individuals having circulating MSCs that have been more readily available from bone marrow mobilization (where such individuals have Induction of granulocyte community stimulating factor) was obtained and obtained from peripheral blood. Thus, this approach is significantly superior to previous studies directly isolated from patient bone marrow cells (a procedure that can be laborious, painful, and expensive, and in some cases not practical). The inventors have demonstrated that the procedure for isolating and capturing Msc from blood makes the procedure for obtaining MSC for wound healing simpler and actually allows for the collection and storage of stem cells prior to injury for subsequent use. Thus, the inventors have demonstrated that direct administration of cultured autologous circulating Msc to chronic non-healing skin wounds can achieve significant improvement or complete wound healing and functional recovery with minimal scar formation. G_CSF induces MSC transfer from the bone marrow to the peripheral circulation, followed by performing a ligation procedure to isolate the MSC. MSCs were grown in culture, passaged up to 5 times, and then topically applied (in the form of a fibrin mixture) to the wound at 3 time points with an interval of 4 weeks at each time point.锗广从SC广忑兴4砰龙居若]^^(:for wound repair and healing', not all wounds may have the same degree of healing. Determine the latent disease that may tend to or inhibit wound healing in each patient It will be useful. Therefore, MSCs can be stored for future analysis. It is possible to expect complementary studies including, but not limited to, comparative quantitative analysis (by ELISA alone or via comprehensive proteomic research screening) before and after mobilization. Related circulating MSC group, serum 152716.doc •93· 201130977 cytokines and chemokines; differentiation, activation, proliferation and apoptosis markers of cells and expansion and diffusion factors in the biopsy of the mouth Histochemistry and in situ hybridization analysis. The inventors can use MSC to pre-sample and recycle the patient's circulating cell population, serum and wound sites before and after treatment for use including, but not limited to, the following further Study. Pre-separation and each day of treatment (but before treatment), and serum 1 month after the final treatment. These samples can be used for related individual cycles. ELISA for chemokine/cytokine assessment, or promising for complete proteomic analysis and comparison between patients and patients at different time points. Every day of treatment (but before treatment), 1 month after final treatment Drilling biopsy around the wound and at the center. Each biopsy will be split in half, one for frozen tissue and one for formalin fixation and paraffin embedding. These tissues can be used to target cell populations and parts according to standard protocols. Immunohistochemical staining of diffuse cytokines and chemokines and receptors, proliferation and apoptosis markers on the cell membrane. Before mobilization with G-CSF and at the time of separation (ie after GSF) in peripheral blood Different subpopulations of circulating stem cells are included, including but not limited to: CD34+, c-kit+, thy-11, Sca-1+, lin. ("KTLS cells"), minimal embryonic stem cells ( VSELS) and MSC. Example 2 MSCs obtained using the methods described herein can be used to treat or repair bone damage and for other bridge-shaped indications. Of particular concern are PB-derived MSCs and BM-derived MSCs. Use of stress fractures during stress physical training (stress 152716.doc -94·201130977 fracture), or repair of bone injuries (using conventional treatments (eg, monthly stimulation, hormones and fixation) without healing). There are about 8 million fractures and nearly 1% of the fractures are damaged every year. In general, the stress fracture rate in men in the basic training period is up to 5%, and the ratio in women is as high as 21%. In the Army, the male fracture number is 2.6% and the female (four) discount number is 8.1%. The special bone site at the risk of fracture is the ulna (step grab training); the femoral neck (stress fracture); the upper and lower limbs and A blast injury of the mandible, craniofacial bone, and clavicle. These fractures result in a 10,000-dollar medical cost and loss of duty time per year for all branches of the military. Thus, autologous MSCs obtained from an individual using methods as disclosed herein can promote healing of non-healing fractures. Fracture nonunion treatment using percutaneous placement and culture of expanded autologous mesenchymal stem cells Non-healing of long bone fractures is a serious health problem that significantly increases the cost of average fracture treatment. Treatment can take several forms and usually requires surgery, including bone grafting and/or initial open reduction, internal fixation (〇RIF) procedures for revision. More conservative options include the use of bone morphogenetic proteins and bone stimulators in conjunction with surgical procedures. These procedures have a relatively high failure rate, indicating a clear need for newer and more effective methods to promote the treatment of non-healing fractures. In this study, the inventors demonstrated that PB-derived MSCs and BM-derived MSCs obtained by the methods disclosed herein are safe and effective for repairing bone damage 'and demonstrate that radiographically-directed injections are administered to PB-derived Mg匸 and BM-derived MSCs have the initial effect of promoting bone regeneration in fracture nonunion. These PB-derived MSCs and BM-derived MSCs increased the tissue environment and recovery function of 152716.doc-95·201130977 by increasing bone regeneration. Without wishing to be bound by theory, bone is a specialized tissue that undergoes continuous remodeling involving a balance between new formation and resorption. When the bone is destroyed, the mesenchymal stem cells (MSC) are recruited to the site of injury. MSCs are pluripotent cells found in human adult tissues, including bone marrow (BM), synovial tissue, adipose tissue, and peripheral blood (PB). Because it is derived from the mesoderm, MSCs are able to differentiate into bone, cartilage, muscle and adipose tissue. Normal fracture healing cannot be performed in the absence of endogenous MSCs, as these cells differentiate into the various cells required for bone repair. Although drugs have not been particularly effective in promoting fracture healing, many studies have shown that direct administration of MSC to non-healing fracture sites can effectively heal damaged bone. The MSC lineage has successfully regenerated soft bone and bone in many animal models and humans. Thus, autologous PB-derived MSCs and BM-derived MSCs obtained by the methods disclosed herein can allow cells to differentiate into osteoblasts and to deliver to the fracture site in a manner that is desirable for other mature cells. Thus, PB-derived MSCs and BM-derived MSCs as disclosed herein are useful as bone regeneration products for the treatment of non-unsaturated fractures. Thus, PB-derived MSCs and BM-derived MSCs obtained from mobilized blood according to the methods disclosed herein can be used to treat a variety of conditions, including fracture non-healing. The inventors have demonstrated a method of isolating viable MSCs from mobilized peripheral blood, and such MSCs are useful in methods of autologous MSC cell therapy for chronic wounds and orthopedic indications. Because MSCs are self-contained, the risk of rejection is limited or risk free. The inventors have demonstrated that administration of G-CSF (480 gg, 2 days, subcutaneous) shows that MSC can be mobilized from human BM to PB. 152716.doc • 96· 201130977 Alternatively, the inventors have demonstrated that MSC can be mobilized into peripheral blood by a single dose of AMD 3100 (240 pg/kg, subcutaneously). The inventors demonstrate that PB-derived MSCs and BM-derived MSCs can be collected by methods as disclosed herein and subsequently expanded and/or cryopreserved as appropriate. Efficacy of autologous BM-MSCs and PB-MSCs in the treatment of fracture nonunion In order to evaluate the efficacy of MSC in the treatment of nonunion fractures, the inventors performed a 12-month randomized, placebo-controlled, double-blind, multicenter phase 1 clinical trial using a total of 100 trials. The patients were divided into 4 groups of 25 patients each. One group was treated with BM-MSC, the second group was treated with PB-MSC and the two controls were treated with autologous platelet lysate (PL, for amplifying MSC) or phosphate buffered saline (PBS). Patients were recruited and treated in 4 orthopaedic professional hospitals. The cells were processed and expanded in a central cell culture facility. The primary endpoint of nonunion fracture healing can be determined by thin-layer CT measurements of the fracture site. If the fracture nonunion is significantly less than the (P<〇.05) control treatment group, the efficacy of the MSC will be assessed. METHODS: #礼衮序汔·· 1. Whole bone marrow aspiration: Consistent with the bone marrow collection procedure, 200 cubic centimeters (CC) of heparinized intravenous venous blood was drawn for the production of autologous PL. The bone marrow aspirate was drawn into two 30 ml syringes containing 30,000 IU of heparin. The nucleated cells were isolated and washed once in PBS, counted, and then resuspended in DMEM + 10% PL and placed in a 4 ° C shipping package along with the collected 200 ml of total intravenous blood for 24 hours. Transport to the cell culture site. 2. MSO's ####说磨和从# .· Use AMD3100 (240 pg/kg, subcutaneous) to mobilize the patient 4 hours before collection 152716.doc -97- 201130977. Separation was performed to collect approximately 300 cc of mobilized peripheral blood. About 2 cc of heparinized intravenous venous blood was drawn for the production of autologous platelet lysate (pL). The separated product was placed in 4 along with the collected 200 ml of total intravenous blood. Transfer to the cell culture site via a 24-hour transport. 3 · Coldly invite MSC# to increase cells. The cells were inoculated in a single-layer flask at 1 X [〇6 cells/cm2 and cultured in a moisture-containing environment at 37 C /5% C〇2. After 3 days, the medium (containing autologous PL) was replaced, and unadhered cells were removed, and the MSC group was formed 6-12 days after inoculation. Each culture was passaged 1:3 after reaching 4〇_5〇% confluence. MSCs were grown to passage 1st through passage 3, followed by suspension in phosphate buffered saline (PB S) and autologous pL concentrates that meet their maximum in vitro culture expansion rates.矣治# 忑 忑 忑 忑. Transplant procedure: The final MSC dose is the maximum dose that can be utilized for up to 1.0 x 107 cells. The MSCs are placed in a sterile syringe, sterile delivery bag, and 4»c cooler for transport to the operating room. The instructions required by the staff will be included with the product. The patient will return to the sterile operating room and the area of the non-healing site will be prepared using betadine and sterile gloves. The sterile trocar is then inserted into the fracture site at several locations on the c-arm. Visipaque, diluted 2 times with PBS, will be used to determine the flow of radiographic contrast agents in the fracture site. The injected sample is injected into the area where the dye flow can be determined. Remove the trocar. The patient is instructed to remain stationary for 3 minutes to allow for cell attachment. RESULTS Endpoint measurement: Fractures were used before, at 1, 3, 6 and 12 months of operation. 152716.doc -98- 201130977 The thin-layer CT scan of the site determined the efficacy of MSC in improving the healing of the fracture site. A scout film is used to determine the area of the fracture site. To limit radiation exposure, the field of view was determined to be 2 cm proximal to the fracture site and 2 cm distal. Example 3 The inventors have previously demonstrated that human umbilical cord blood contains small (size smaller than red blood cells) CXCR4+CD133+CD34+SSEA-4+Oct-4+liiTCD45·cell population 2007:21; 297-303) and these cells are During tissue damage, mobilize to the surrounding blood, such as in the case of myocardial infarction / Co / /.

CarAo/., 2009:53;1-9.) and stroke (see Siroh. 2009:40;1237_). Similar cells in rodent organs have also been reported, and more importantly, cells that have been described to differentiate into all three germ layers in vitro have been described (Zewkwz'a 2006: 20; 857-869). In order to investigate the possibility that human VSELs can be a source of pluripotent stem cells in regenerative medicine, the inventors have developed an efficient strategy for isolating such cells from adult patients. The inventors have demonstrated that VSELs are similar to their murine counterparts and can be injected. The granulocyte community stimulating factor (G-CSF) is then mobilized into the peripheral blood (汾em Ce//i 2008:26; 2083-2092). The present inventors demonstrated this phenomenon by recruiting a group of young healthy donors who were mobilized using G-CSF (480 pg per day, subcutaneous) for 2 consecutive days. On day 3, nucleated cells (TNC) were collected by bloodpheresis. The inventors evaluated the number of VSELs in the peripheral blood (PB) samples before and after G-CSF mobilization and the final number in the fractionated product. At least 1 million TNCs were obtained and analyzed by FACS Diva software. Three different fractions of non-hematopoietic stem cells rich in VSEL (Lin7CD457CD133+, Lin7CD457CD34+, Lin*/CD457 152716.doc ·99·201130977 CXCR4+) and their CD45-positive hematopoietic counterparts were analyzed. The absolute number of cells contained in the 1 pL sample was calculated based on the percentage content of each population of each sample and the TNC count. The inventors determined that after G-CSF mobilization, human peripheral blood contains a population of 0-114, €034, and €0133 antigens such as - 045-monocytes. These lin_CD45XXCR4+CD133+CD34+ cells are highly enriched in mRNA of nuclear intraprimary embryonic transcription factors such as Oct-4, Sox2 and Nanog. More importantly, the inventors have found that 〇ct-4 is expressed in the nucleus of the mobilized VSEL and that these cells also exhibit the cell surface marker SSEA-4, which is commonly used as a marker for undifferentiated universal human embryonic stem cells. Early embryonic glycoester antigens" The inventors observed that the blood-borne VSELs of such adults were slightly larger than their counterparts identified in the bone marrow of adult rats, but were still extremely small. In addition, such human peripheral blood-derived VSELs have large nuclei containing embryonic amorphous euchromatin. The inventors have clearly demonstrated that only very few VSELs are detectable in peripheral blood prior to G-CSF mobilization, whereas after G-CSF-induced mobilization, VSELs in the separation products that account for less than 0.01% of TNC increase significantly. More than 106. The inventors have demonstrated that although VSELs are relatively rare cells, they are mobilized into peripheral blood and G-CSF-induced mobilization is suitable for obtaining human pluripotent stem cells for use in regenerative medicine. REFERENCES. All references, patents, and patent applications, which are hereby incorporated by reference in its entirety herein in its entirety herein in its entirety [Simple illustration] 152716.doc -100- 201130977 Figure 1 A-1D shows in vitro adhesion and expansion of mobilized peripheral blood-derived human MSCs. Figures 1A and 1B show cultures containing 1 〇 ° /. The mobilized peripheral blood-derived human MSCs in FMEM (fetal calf serum) in DMEM and inoculated with 1, 〇〇〇, 细胞 cells/plates (Fig. 1A) and 2,000,000 cells/plate (Fig. 1B). Figures 1C and 1D show mobilized peripheral blood-derived humans incubated in MesenCult-MSC (**Trademark?) and inoculated with 1,000,000 cells/plate (Figure 1C) and 2,000,000 cells/plate (Figure 1D). MSC. Figures 2A-2D show peripheral blood-derived human MSCs as CD45- and CD105+. Figure 2A shows the FAC of the isolated product expressed based on the label of CD45. Figure 2B shows the FAC of adherent cells expressed on CD45-based markers. Adhesive cells are rich in cells that do not express CD45. Figure 2C shows the FAC of the isolated product expressed based on the label of CD 105. Figure 2D shows that FACs based on adherent cells expressed by CD105 markers are adherent to cells that are positive for CD105. Figure 3-8 shows peripheral blood-borne humans 1\^0: €090+ and 00105+. Figure 3A shows FAC of floating cells expressed on CD90-based markers. Most floating cells are negative for CD90 expression. Fig. 3B shows the FAC of adherent cells after one passage (P1) based on the expression of CD45. Adherent cells (1 passage) are rich in cells expressing CD90. Figure 3C shows the FAC of floating cells based on the label of CD105. Most floating cells show negative for CD105. Figure 3D shows the FAC of adherent cells after one passage (P1) based on the label of CD105. Adherent cells (1 passage) are rich in cells expressing CD 105. Figures 4A-4B show peripheral blood-derived human MSCs as CD44+. Figure 4A shows the FAC of floating cells expressed by the CD44-based marker 152716.doc -101 - 201130977. Most floating cells were negative for CD44. Figure 4B shows FAC of adherent cells after 1 passage (P1) based on the CD44 marker. Adherent cells (1 passage) are rich in cells expressing CD44. Figure 5 is a schematic diagram showing the steps for separating MSC populations from the peripheral blood of the mobilization. Figure 6 shows the number of MSCs in peripheral blood before and after G-CSF mobilization. 152716.doc -102-

Claims (1)

201130977 VII. Patent Application Range 1. A method for obtaining peripheral blood-derived human mesenchymal stem cells from an individual, comprising: a. administering to the individual an effective amount of G-CSF or GM-CSF for 4 days or less 4 days; b. obtaining human mesenchymal stem cell population from peripheral blood samples of the individual, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and for CD44+, CD90+, CD105+, CD29+ and The CD73+ cell surface marker is positive; and c. Human mesenchymal stem cells are isolated relative to other adult stem cells in the peripheral blood. 2. A method of increasing a human mesenchymal stem cell population in a peripheral blood of an individual, comprising: a. administering to the peripheral blood of the individual an effective amount of G-CSF or GM-CSF for 4 days or less; b A human mesenchymal stem cell population is obtained from a peripheral blood sample of the individual, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and for CD44+, CD90+, CD105+, CD29+, and CD73+ cell surface markers Positive; and c. Isolation of human mesenchymal stem cells relative to other adult stem cells in the peripheral blood. 3. A method of obtaining human mesenchymal stem cells from an individual, comprising: a. contacting a peripheral blood sample from the individual with an effective amount of G-CSF or GM-CSF to increase human mesenchymal stem cells in the peripheral blood sample 152716.doc 201130977 number; b. obtaining human mesenchymal stem cell population from the peripheral blood sample, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers' and for CD44+, CD90+, CD105+, CD29+ and The CD73+ cell surface marker is positive; and c. Human mesenchymal stem cells are isolated relative to other adult stem cells in the peripheral blood. 4. The method of any one of claims 1 or 2, wherein the effective amount of the G-CSF or GM-CSF is administered to the individual for a period of 3 days or less. The method of any one of claims 1 or 2, wherein the effective amount of the G-CSF or GM-CSF is administered to the individual for 2 days or less. 6. The method of any one of claims 1 or 2, wherein the effective amount of G-CSF or GM-CSF is administered to the individual for a period of one day. 7. The method of any one of clauses 1 to 6, wherein the individual is a healthy individual. 8. The method of any one of clauses 1 to 7, wherein the human mesenchymal stem cell population is obtained from the peripheral blood sample using a size-based separation method. 9. The method of any one of clauses 1 to 8, wherein the human mesenchymal stem cell population is subjected to a negative selection method that excludes non-mesenchymal stem cells, and the self-contained interstitial is obtained from the peripheral blood sample The stem cell population and the sample of the non-mesenchymal stem cell population were isolated and collected for the human mesenchymal stem cell population. The method of any one of claims 1 to 9, wherein the human mesenchymal stem cell population is obtained from the peripheral blood using a combination of any of the following methods: positive selection based on cell surface markers, cell surface based labeling Negative 152716.doc 201130977 selection method, size-based positive selection method, size-based negative selection method. π. A method for obtaining human mesenchymal stem cells from an individual, comprising: a. contacting a peripheral blood sample with an effective amount of 〇_(:817 or GM-CSF in vitro to increase human interstitial in the peripheral blood sample) The number of stem cells; b. The human mesenchymal stem cell population is isolated from the peripheral gold sample, wherein the human mesenchymal stem cells are negative for CD34-, CD45-cell surface markers, and for CD44+, CD90+, CD105+, CD29+, and CD73+ The cell surface marker is positive. I2. The method of claim 3*U, wherein the peripheral blood sample is a cultured peripheral blood sample. 13. The method of claim 1, 2, μη, wherein the peripheral blood sample is via Blood apheresis is obtained from the individual. 14. The method of claim 13, wherein the hematocrit of the separated product is in the range of 3%. K. The method of claim (4), wherein the peripheral blood sample is Fresh peripheral blood samples. 16. As in the above-mentioned request, the root, soil, and θ ^ Τ1 items are selected, and the human mesenchymal stem cell population obtained from the peripheral blood sample is cultured and expanded. In the above request The one-and-a-side, the earth/the sapphire side, wherein the human mesenchymal stem cell population obtained from the peripheral blood sample is cryopreserved. Class 18. The method of the __ blocking item in the above request, wherein The individual is a human. 19. A pure lineage of cell lines comprising a substantially pure human mesenchymal stem cell population isolated using the method of 152, 716. doc 201130977, as claimed in any of the above claims. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. A container comprising a suitable medium and a pure cell line of the human mesenchymal stem cell population of claim 18. The container of claim 19, wherein the suitable medium and human mesenchymal stem cell population The pure cell line is cryopreserved. A cryopreserved human mesenchymal stem cell population obtained by any of the methods of claims 1 to 17. A human mesenchymal stem cell population or a differentiated progeny thereof is used for treatment. Use of the disease or condition of an individual, wherein the human mesenchymal stem cell population is isolated using the method of any one of the above claims, and wherein the individual is administered to a subject in need thereof A human mesenchymal stem cell population or a differentiated progeny thereof for use in autologous regenerative therapy. A method of treating a disease or condition in an individual with autologous mesenchymal stem cells (MSC), comprising: a. utilizing a Msc population of peripheral blood samples, Wherein the peripheral blood sample is obtained from an individual who has received the administration of the mobilizer for 4 days or less, and wherein the MSC population is enriched with the Msc population relative to other stem cells in the peripheral blood; and b. is administered to the individual Msc group' to treat the disease or condition. The method of claim 23, wherein the method is rich by #正c. The method of claim 23, wherein the msc is enriched by a panning method. The method of claim 23, wherein the sell group has been cryopreserved. The method of claim 23, wherein the enriched population comprises non-sputum cells. The method of claim 23 wherein the enriched MSC group comprises at least 1% 152716.doc 201130977 MSC. 30. The method of claim 23, wherein the enriched Msc group is a substantially pure msc group. The method of claim 23 or 26, wherein the MSC population has been expanded in vitro prior to administration to the individual. 32. The method of claim 23, wherein the individual is a human individual. 3 3. A method for obtaining peripheral blood-derived human stem cells from an individual, comprising: a. administering to the individual an effective amount of a mobilizer for a period of 4 days or less; b. obtaining a peripheral blood sample from the individual Obtaining a population of human stem cells; and c. isolating human stem cells relative to other adult stem cells in the periphery. 34. The method of claim 33 wherein the stem cells are mesenchymal stem cells. 35. The method of claim 33, wherein the stem cells are "very small embryonic stem cells (VSELs)". 3. The method of claim 33, wherein the mobilizing agent is G-CSF or GM-CSF » 3. The method of claim 33, wherein the mobilizing agent is selected from the group consisting of interleukin _17, AMD3 100, ring A group consisting of Cypress (CyCi〇ph〇Sphamide; Cy), European Taxol (Docetaxel) and (DXT). 152716.doc