Method for transporting mesenchymal stem cells with a transport solution and method for administering stem cells to a wound

Abstract

To provide a method of transporting a stem cell population.SOLUTION: The present invention relates to a method of transporting a stem cell population, the method comprising transporting the stem cell population brought into contact with a liquid carrier. In the preferred embodiment, the liquid carrier is Hypothermosol and comprises Trolox e.g., Hypothermosol-FRS. In addition, the present invention concerns a method of treating a subject having a disease such as a wound, the method comprising topically administering a defined mesenchymal stem cell population to the subject, the mesenchymal stem cell population being administered within about 96 hours from the time point the mesenchymal stem cell population has been harvested. Also concerned is a unit dosage comprising the defined mesenchymal stem cell population derived from the amniotic membrane of the umbilical cord or the like, at least 90% of the cells expressing each of CD73, CD90 and CD105.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 655,198, filed April 9, 2018, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to a method for transporting a stem cell population, comprising transporting the stem cell population in contact with a liquid carrier. Additionally, the present invention relates to a method for treating a subject with a disease, comprising locally administering a defined mesenchymal stem cell population to the subject, wherein the mesenchymal stem cell population is administered within about 96 hours from the time the mesenchymal stem cell population is collected. The present invention also relates to a unit dose containing about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells of the defined mesenchymal stem cell population. [Background technology]

[0003] Background of the Invention Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord and their wound healing properties were first reported in U.S. Patent Application Publication No. 2006 / 0078993 (leading to issued U.S. Patent Nos. 9,085,755, 9,737,568, and 9,844,571) and corresponding International Patent Application WO2006 / 019357. Since then, umbilical cord tissue has attracted attention as a source of multipotent cells; umbilical cords, and specifically stem cells isolated from the amniotic membrane of the umbilical cord (also referred to as "cord lining stem cells"), are widely available and are therefore considered an excellent alternative source of cells for regenerative medicine. See Jeschke et al. Umbilical Cord Lining Membrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities and Differences; The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 21-27.

[0004] Subsequent studies compared the phenotype, proliferation rate, migration, immunogenicity, and immunomodulatory potential of human mesenchymal stem cells (MSCs) derived from the amniotic membrane of the umbilical cord (umbilical cord lining (CL-MSCs)), umbilical cord blood (CB-MSCs), placenta (P-MSCs), and Wharton's gelatin (WJ-MSCs) (Stubbendorf et al., Immunological Properties of Extraembryonic Human Mesenchymal Stromal Cells Derived from Gestational Tissue, STEM CELLS AND DEVELOPMENT Volume 22, Number 19, 2013, 2619-2629). Stubbendorf et al. concluded that MSC populations derived from extraembryonic pregnancy tissues exhibit diverse abilities to evade immune responses and exert immunomodulatory effects. The authors also found that CL-MSCs show the most promising potential for cell-based therapy because they exhibit low immunogenicity and enhanced proliferative and migratory capacity. Therefore, future research should focus on the best disease models in which CL-MSCs can be administered.

[0005] Although amniotic mesenchymal stem cells can be readily obtained using the protocols described in U.S. Patent Application Publication No. 2006 / 0078993 and International Patent Application Publication No. WO2006 / 019357, it would be advantageous for clinical trials using these umbilical cord-lining MSCs to have methods at hand that allow for the isolation of a population of these umbilical cord-lining MSCs that is highly homogeneous and therefore can be used in clinical trials.

[0006] Such a highly homogenous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord was first reported in co-pending U.S. patent application Ser. No. 15 / 725,913, filed October 5, 2018, which claims priority to U.S. provisional patent application Ser. No. 62 / 404,582, filed October 5, 2017 (Patent Document 6), the contents of both of which are incorporated herein by reference in their entireties, and in co-pending PCT application PCT / SG2017 / 050500, filed October 5, 2018, which also claims priority to U.S. provisional patent application Ser. No. 62 / 404,582, filed October 5, 2017 (Patent Document 6), and meets the criteria for mesenchymal stem cells to be used in cell therapy (see the experimental section of U.S. patent application Ser. No. 15 / 725,913, Dominici et al., "Minimal criteria for defining multipotent See also "Mesenchymal stromal cells. The International Society for Cellular Therapy position statement," Cytotherapy (2006) Vol. 8, No. 4, 315-317, or Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," Stem Cell Research & Therapy 2013, 4:66.

[0007] However, stem cells such as above-mentioned mesenchymal stem cells are not typically applied / administered to patients at the place where they are generated.In many cases, a considerable time elapses between the collection of cells and their further use.Therefore, there is a need to provide a specific carrier that can keep cells viable and healthy for the period typically used for transporting or storing cells.

[0008] It is therefore an object of the present invention to provide a method for transporting / storing stem cells that meets this need, particularly a population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent Application 2006 / 0078993 [Patent Document 2] U.S. Patent No. 9,085,755 [Patent Document 3] U.S. Patent No. 9,737,568 [Patent Document 4] U.S. Patent No. 9,844,571 [Patent Document 5] WO2006 / 019357 [Patent Document 6] U.S. Provisional Patent Application No. 62 / 404,582 [Patent Document 7] U.S. Patent Application No. 15 / 725,913 [Patent Document 8] PCT / SG2017 / 050500 [Non-patent literature]

[0010] [Non-Patent Document 1] Jeschke et al. Umbilical Cord Lining Membrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities and Differences; The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 21-27 [Non-patent document 2] Stubbendorf et al, Immunological Properties of Extraembryonic Human Mesenchymal Stromal Cells Derived from Gestational Tissue, STEM CELLS AND DEVELOPMENT Volume 22, Number 19, 2013, 2619-2629 [Non-patent document 3] Dominici et al, “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement”, Cytotherapy (2006) Vol. 8, No. 4, 315-317 [Non-patent document 4] Sensebe et al,. “Production of mesenchymal stromal / stem cells according to good manufacturing practices: a, review”, Stem Cell Research & Therapy 2013, 4:66 Summary of the Invention

[0011] This object is achieved by a method and unit dose having the features of the independent claims.

[0012] In a first aspect, the present invention provides a method of transporting a stem cell population, the method comprising: i) Trolox, ii) Na + , iii) K + , iv) Cl - , v) H2PO4 - , vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione transporting said stem cell population in contact with a liquid carrier comprising:

[0013] In a second aspect, the present invention provides a method of treating a subject having a disease, the method comprising locally administering to the subject a mesenchymal stem cell population described herein, wherein the mesenchymal stem cell population is administered within about 96 hours from the time the mesenchymal stem cell population is harvested.

[0014] In a third aspect, the present invention provides a unit dose comprising about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells of a mesenchymal stem cell population described herein.

[0015] In a fourth aspect, the present invention provides the use of a liquid carrier for transporting a stem cell population, the liquid carrier comprising: i) Trolox, ii) Na+, iii) K+, iv) Cl-, v) H2PO4-, vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione Includes. [The present invention 1001] i) Trolox, ii) Na + , iii) K + , iv) Cl - , v) H2PO4 - , vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione 10. A method of transporting a stem cell population, comprising transporting the stem cell population in contact with a liquid carrier comprising: [The present invention 1002] The method of claim 1001, wherein the transport occurs over a period of about 7 days or less. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein the transport occurs over a period of about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or less than about 1 day. [The present invention 1004] Any of the aforementioned methods of the present invention, wherein the delivery occurs over a period of about 48 hours or about 24 hours or less. [The present invention 1005] Any of the methods of the present invention, wherein the transportation is carried out at a temperature of about -5°C to about 15°C. [The present invention 1006] Any of the methods of the present invention, wherein the transportation is carried out at a temperature of about 2°C to about 8°C. [The present invention 1007] Any of the aforementioned methods of the invention, wherein the transport is carried out at a temperature greater than about -5°C, greater than about -10°C, greater than about -15°C, or greater than about -20°C. [The present invention 1008] Any of the methods of the invention, wherein the stem cell population is delivered at a concentration of about 70 million cells per ml of carrier, about 60 million cells per ml of carrier, about 50 million cells per ml of carrier, about 40 million cells per ml of carrier, about 30 million cells per ml of carrier, about 20 million cells per ml of carrier, about 10 million cells per ml of carrier, about 5 million cells per ml of carrier, about 4 million cells per ml of carrier, about 3 million cells per ml of carrier, about 2 million cells per ml of carrier, about 1 million cells per ml of carrier, about 500,000 cells per ml of carrier, about 100,000 cells per ml of carrier, or less than 100,000 cells per ml of carrier. [The present invention 1009] 1008. The method of claim 1008, wherein the stem cell population is delivered at a concentration of about 10 million cells per ml of carrier to about 1 million cells per ml of carrier. [The present invention 1010] Any of the aforementioned methods of the present invention, wherein the stem cell population is an embryonic stem cell population, an adult stem cell population, a mesenchymal stem cell population, or an induced pluripotent stem cell population. [The present invention 1011] Any of the aforementioned methods of the present invention, wherein the stem cell population is a mesenchymal stem cell population, preferably a human mesenchymal stem cell population. [The present invention 1012] Any of the aforementioned methods of the present invention, wherein the mesenchymal stem cell population is an isolated umbilical cord amniotic membrane mesenchymal stem cell population, preferably an isolated umbilical cord amniotic membrane human mesenchymal stem cell population. [The present invention 1013] 1013. The method of claim 1011 or 1012, wherein at least about 90% or more of the cells of the isolated mesenchymal stem cell population express each of the markers CD73, CD90, and CD105. [The present invention 1014] The method of the present invention 1013, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105. [The present invention 1015] Any of the methods of 1011 to 1014, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells lack expression of the markers CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related). [The present invention 1016] The method of the present invention 1015, wherein at least 97% or more, about 98% or more, or about 99% or more of the cells of the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105, and lack expression of each of CD34, CD45, and HLA-DR. [The present invention 1017] Any of the methods of the invention described above, wherein the stem cell population is contacted with a carrier prior to transport. [The present invention 1018] Any of the aforementioned methods of the invention, wherein the stem cell population is contacted with a carrier after its collection. [The present invention 1019] The method of the present invention, wherein the stem cell population is contacted with the carrier about 0 minutes, about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or longer after collection of the stem cell population. [The present invention 1020] The method of any one of claims 1018 to 1019, wherein recovering comprises separating the stem cell population from the culture medium. [The present invention 1021] 1021. The method of claim 1020, wherein the separation is carried out by centrifuging the stem cells in the culture medium and decanting the culture medium. [The present invention 1022] Any of the methods of the invention described above, wherein the contacting is carried out by suspending the stem cell population in the carrier at a density of about 70 million cells / ml, about 60 million cells / ml, about 50 million cells / ml, about 40 million cells / ml, about 30 million cells / ml, about 20 million cells / ml, about 10 million cells / ml, about 5 million cells / ml, about 4 million cells / ml, about 3 million cells / ml, about 2 million cells / ml, about 1 million cells / ml, about 500,000 cells / ml, about 100,000 cells / ml, or less than 100,000 cells. [The present invention 1023] 1023. The method of claim 1022, wherein the contacting is effected by suspending the population of stem cells in the carrier at a density of about 10 million cells / ml. [The present invention 1024] The method of claim 1022 or 1023, wherein the stem cells contacted with the carrier are dispensed into vials with a volume of carrier of about 50 ml, about 20 ml, about 10 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml, about 1 ml, about 0.5 ml, about 0.25 ml, or less than 0.25 ml. [The present invention 1025] The method of any one of claims 1022 to 1023, wherein the stem cells contacted with the carrier are dispensed into vials in a volume of about 1 ml. [The present invention 1026] Any of the aforementioned methods of the present invention, wherein the carrier is a transport medium or excipient. [The present invention 1027] Any of the preceding methods of the present invention which do not include a thawing or freezing step. [The present invention 1028] Any of the preceding methods of the invention, wherein the carrier does not comprise a dipolar aprotic solvent, particularly DMSO. [The present invention 1029] Any of the methods of the present invention, wherein at most about 50%, about 40%, about 30%, about 20%, about 10%, or less than about 10% of the stem cells in the population die during transport compared to the amount of viable stem cells before transport. [The present invention 1030] Any of the methods of the invention, wherein after transport, the stem cell population secretes approximately the same amount of TGFbeta-1 as before transport. [The present invention 1031] Any of the aforementioned methods of the present invention, wherein PDGF-BB and / or IL-10 are essentially undetectable before and / or after transport. [The present invention 1032] Any of the aforementioned methods of the present invention, wherein after transportation, the mesenchymal stem cell population secretes approximately the same amount of VEGF, PDGF-AA, Ang-1, and / or HGF as before transportation. [The present invention 1033] A unit dose comprising about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells of a mesenchymal stem cell population defined in any of 1012 to 1016 of the present invention. [The present invention 1034] A unit dose of the present invention 1033 comprising about 10 million, about 9 million, about 8 million, about 7 million, about 6 million, about 5 million, about 4 million, about 3 million, about 2 million, about 1 million, about 500,000, about 250,000, or about 100,000 cells. [This invention 1035] A unit dose of the present invention 1033 or 1034 containing approximately 10 million cells. [The present invention 1036] A unit dose of any one of 1033 to 1035 of the present invention contained in a 1 ml vial. [This invention 1037] The unit dose is contained in a liquid carrier, the liquid carrier comprising: i) Trolox, ii) Na+, iii) K+, iv) Cl-, v) H2PO4 - , vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione A unit dose of any one of 1033 to 1035 of the present invention, comprising: [The present invention 1038] The unit dose of any one of 1033 to 1037 of the present invention, wherein the cells are viable cells. [This invention 1039] The method of any one of claims 1001 to 1032, wherein the carrier is HypoThermosol. [The present invention 1040] 1. Use of a liquid carrier for transporting a stem cell population, the liquid carrier comprising: i) Trolox, ii) Na+, iii) K+, iv) Cl-, v) H2PO4 - , vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione The use. [The present invention 1041] 1. Use of a mesenchymal stem cell population, which is an isolated mesenchymal stem cell population of the amniotic membrane of an umbilical cord as defined in any one of claims 1012 to 1016, for the manufacture of a pharmaceutical composition for treating a subject having a disease, The mesenchymal stem cell population is locally administered within about 96 hours from the time the mesenchymal stem cell population is collected. The use. [The present invention 1042] 1041. The use of the present invention, wherein the mesenchymal stem cell population is applied in a unit dose of about 10 million cells. [This invention 1043] The use of invention 1041 or 1042, wherein the mesenchymal stem cell population is applied within about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 6 hours, or less from the time the mesenchymal stem cell population is collected. [This invention 1044] The use of any of claims 1041 to 1043, wherein the disease is a skin disease or a wound. [This invention 1045] The use of the present invention 1044, wherein the wound results from a burn, bite, trauma, surgery, or disease. [The present invention 1046] The use of the present invention 1045, wherein the wound is caused by diabetes, and the wound is preferably a diabetic wound. [This invention 1047] The use of the present invention 1046, wherein the wound is a diabetic foot ulcer. [This invention 1048] 10. The use of any of claims 1041-1047, wherein a unit dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells is administered once or twice a week. [This invention 1049] Use of the present invention 1048, wherein a unit dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells is administered once or twice a week for a period of 3, 4, or 5, or 6, or 7, or 8, or 10 or more weeks. [The present invention 1050] 1049. The use of any of claims 1041 to 1049, wherein the mesenchymal stem cell population is applied topically and covered with a film or bandage. [Brief explanation of the drawings]

[0016] The present invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and drawings.

[0017] [Figure 1-1] FIG. 1 shows Lonza's technical information sheet for Dulbecco's Modified Eagle's Medium, including the catalog number of the DMEM used to make the example medium of the present invention (PTT-6) in the experimental section. [Figure 1-2] See description of Figure 1-1. [Figure 2] 1 shows Lonza's technical information sheet for Ham's F12 medium. [Figure 3] 1 shows Lonza's technical information sheet for DMEM:F12 (1:1) medium, including the catalog number of the DMEM:F12 (1:1) medium used to make the example medium of the present invention (PTT-6) in the experimental section. [Figure 4-1] FIG. 4 shows Life Technologies Corporation's technical information sheet for M171 medium, including the catalog number of the M171 medium used to make the example medium of the present invention (PTT-6) in the experimental section. [Figure 4-2] See description of Figure 4-1. [Figure 5] A list of the ingredients used in the experimental section to make medium PTT-6 is provided, including their commercial suppliers and catalog numbers. [Figure 6a]Figure 6 shows the results of flow cytometry experiments in which mesenchymal stem cells isolated from umbilical cord tissue were analyzed for expression of the mesenchymal stem cell markers CD73, CD90, and CD105. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the tissue in three different culture media, and then subcultured in each medium. In these experiments, three culture media were used: a) 90% (v / v) DMEM supplemented with 10% (v / v) FBS; b) PTT-4 culture medium, described in U.S. Patent Application Publication No. 2006 / 0078993 and corresponding International Patent Application No. WO2006 / 019357 (see paragraph

[0183] of WO2006 / 019357), consisting of 90% (v / v) CMRL1066 and 10% (v / v) FBS; and c) PTT-6 culture medium of the present invention, the composition of which is described herein. In this flow cytometry analysis, two different samples of umbilical cord lining mesenchymal stem cell (CLMC) populations were analyzed for each of the three culture media used. The results are shown in Figures 6a-6c. More specifically, Figure 6a shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and culture in DMEM / 10% FBS, Figure 6b shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and culture in PTT-4, and Figure 6c shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and culture in PTT-6. [Figure 6b] See legend to Figure 6a. [Figure 6c] See legend to Figure 6a. [Figure 7a]7 shows the results of a flow cytometry experiment in which mesenchymal stem cells isolated from umbilical cord were analyzed for their expression of stem cell markers CD73, CD90, and CD105, CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related), which are used to define the suitability of multipotent human mesenchymal stem cells for cell therapy, and compared with the expression of these markers by bone marrow mesenchymal stem cells. For this experiment, amniotic mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in the culture medium PTT-6 of the present invention, while bone marrow mesenchymal stem cells were isolated from human bone marrow using standard protocols. Figure 7a shows the percentage of isolated mesenchymal umbilical cord lining stem cells that express stem cell markers CD73, CD90, and CD105 and lack expression of CD34, CD45, and HLA-DR after isolation and culture from umbilical cord tissue in PTT-6 medium, and Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90, and CD105 and lack expression of CD34, CD45, and HLA-DR. [Figure 7b] See legend to Figure 7a. [Figure 8] The experimental setup for comparing different carriers is shown. First, mesenchymal stem cell populations described herein were expanded in cell culture flasks. The amount of viable mesenchymal stem cells was counted, and then 2 million cells / vial were stored in either PlasmaLyte-A or HypoThermosol™-FRS for various periods. After storage, cells were counted in ≤50 μl samples daily on days 1–5 (total liquid volume removed: 250 μl), and viability was assessed by staining the cells with trypan blue. Additionally, ≤80 μl samples were taken and analyzed on days 1, 3, and 5. Additionally, supernatants were obtained and frozen. PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF, and TGFβ1 were then measured using a FLEXMAP 3D system. [Figure 9]The viability data are summarized. As can be seen from the graph on the left, after 7 days of storage in HypoThermosol™, 73% of the total cell number at the start of storage (approximately 95%) were still viable. In contrast, after 7 days of storage in PlasmaLyte-A, only 42% of the total cell number at the start of storage (approximately 94%) were still viable. All counts were based on duplicate readings within 10% of each other (according to SOP CR D2.600.1). During counting, cells stored in HypoThermosol™ were significantly smaller, with smooth and well-defined contours. In contrast, cells in Plasmalyte-A appeared at a range of sizes. HypoThermosol™ significantly supports membrane integrity and presumably viability over a 6-day period. Similar results are also shown in the graph on the right. [Figure 10] 1 shows the results obtained when measuring the cell diameter of cells. The mesenchymal stem cell populations described herein when maintained in HypoThermosol™ have a narrower diameter range compared to cells maintained in PlasmaLyte A. Comparisons were made after 3 days of storage. [Figure 11]

[0023] Figure 1 shows the TGFβ1 concentration in supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A after 48 hours of storage. As can be seen from the graph on the right, cells secrete approximately the same amount of TGFβ1 when stored in HypoThermosol™ as when stored in PlasmaLyte-A. In general, the amount of secreted TGFβ1 decreased over time (graph on the right). [Figure 12] Control experiments are shown in which PDGF-BB concentrations were measured in 48-hour supernatants from mesenchymal stem cell populations described herein that were stored in HypoThermosol™ or PlasmaLyte-A. Because PDGF-BB is not normally secreted by the mesenchymal stem cell populations described herein, PDGF-BB was not detectable in any of the samples. [Figure 13]A control experiment is shown in which IL-10 concentrations were measured in 48-hour supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A. Because IL-10 is not normally secreted by the mesenchymal stem cell populations described herein, IL-10 was not detectable in any of the samples. [Figure 14] This graph shows the VEGF concentration in supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A at 48 hours. As can be seen from the graph on the right, on day 0, the cells secrete approximately the same amount of VEGF when stored in HypoThermosol™ or PlasmaLyte-A. On days 1 and 5, the cells secreted more VEGF when stored in PlasmaLyte-A. Notably, when stored for 3 days, the cells secreted more VEGF when stored in HypoThermosol™ than when stored in PlasmaLyte-A. Thus, by the third day of storage, HypoThermosol™ outperforms PlasmaLyte-A. The more VEGF detected, the healthier the culture. Thus, cells were healthier in HypoThermosol™ than in PlasmaLyte-A by secreting more VEGF after 3 days of storage in HypoThermosol™ than when stored in PlasmaLyte-A. From day 5 onwards, storage in PlasmaLyte appears to be more advantageous, as at that point cells stored in PlasmaLyte-A secreted more VEGF. In general, the amount of VEGF secreted decreased over time (graph on the right). [Figure 15]This graph shows the PDGF-AA concentration in the supernatant from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A at 48 hours. As can be seen from the graph on the right, on day 0, cells secrete approximately the same amount of PDGF-AA when stored in HypoThermosol™ as when stored in PlasmaLyte-A. On days 1 and 5, cells secreted more PDGF-AA when stored in PlasmaLyte-A. Notably, when stored for 3 days, cells secreted more PDGF-AA when stored in HypoThermosol™ than when stored in PlasmaLyte-A. Thus, after 3 days of storage, cells stored in HypoThermosol™ are healthier than cells stored in PlasmaLyte-A. After 5 days of storage, PlasmaLyte appears to be a more advantageous carrier, as at that point cells stored in PlasmaLyte-A secreted more PDGF-AA. In general, the amount of PDGF-AA secreted decreased over time (graph on the right). [Figure 16]This figure shows the Ang-1 concentration in supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A at 48 hours. As can be seen from the graph on the right, cells secrete approximately the same amount of Ang-1 when stored in HypoThermosol™ or PlasmaLyte-A on days 0 and 3. On day 5, cells secreted more Ang-1 when stored in PlasmaLyte-A. Notably, after 1 day of storage, cells secreted significantly more Ang-1 when stored in HypoThermosol™ than when stored in PlasmaLyte-A. Thus, cells stored in HypoThermosol™ appear to be healthier than cells stored in PlasmaLyte-A for at least 48 hours up to 3 days of storage. From day 5 onwards, PlasmaLyte appears to be a more advantageous carrier, as at this point cells stored in PlasmaLyte-A secreted more Ang-1. In general, the amount of Ang-1 secreted decreased over time (graph on the right). [Figure 17]This graph shows the HGF concentration in the supernatant from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A after 48 hours of storage. As can be seen from the graph on the right, on day 0, cells secrete approximately the same amount of HGF when stored in HypoThermosol™ compared to when stored in PlasmaLyte-A. On days 3 and 5, cells secreted more HGF when stored in PlasmaLyte-A. Notably, after 1 day of storage, cells secreted significantly more HGF when stored in HypoThermosol™ than when stored in PlasmaLyte-A. Thus, cells stored in HypoThermosol™ appear to be healthier than cells stored in PlasmaLyte-A for at least 1 day (48 hours) up to 3 days of storage. After day 3, PlasmaLyte-A appears to be a more advantageous carrier, as cells stored in PlasmaLyte-A secreted more HGF at days 3 and 5. In general, the amount of secreted HGF decreased over time (graph on the right). [Figure 18-1]Figure 18 shows photographs from a preclinical study using the mesenchymal stem cell population of the present invention in pigs. Pigs were made diabetic with 120 mg / kg streptozotocin and allowed to recover for 45 days before six 5 cm x 5 cm full-thickness wounds were created on their backs. Pigs (n = 2) were treated twice weekly for four weeks with 10 cells per cm² of the human mesenchymal stem cell population described herein. Two control pigs were treated with PBS. Wounds were photographed on post-operative day 0 (PO day 0) and every seven days until post-operative day 35. Wounds were analyzed for surface area size using ImageJ. By day 35, the addition of the mesenchymal stem cell population described herein resulted in closure of 10 of 12 diabetic wounds (83%), compared to only 3 of 12 PBS-treated control wounds (25%). The rate of wound healing was 0.8 cm2 / day using the mesenchymal stem cell population described herein compared to 0.6 cm2 / day in control animals, a 33% improvement. [Figure 18-2] See description of Figure 18-1. [Figure 18-3] See description of Figure 18-1. [Figure 18-4] See description of Figure 18-1. [Figure 18-5] See description of Figure 18-1. [Figure 19-1] Figure 19 is a data sheet for Trolox available from Tocris. [Figure 19-2] See description of Figure 19-1. [Figure 20-1] FIG. 20 shows the data sheet for NaCl available from Sigma Aldrich. [Figure 20-2] See description of Figure 20-1. [Figure 21] A data sheet for KH2PO4 available from Sigma Aldrich is shown. [Figure 22-1] FIG. 22 shows the data sheet for HEPES from Sigma Aldrich. [Figure 22-2] See description of Figure 22-1. [Figure 23-1]Figure 23 shows the product sheet for sodium lactobionate from COMBI-BLOCKS. [Figure 23-2] See description of Figure 23-1. [Figure 23-3] See description of Figure 23-1. [Figure 24-1] FIG. 24 shows the product sheet for sucrose from Sigma Aldrich. [Figure 24-2] See description of Figure 24-1. [Figure 25-1] Figure 25 shows the product sheet for mannitol from avantor. [Figure 25-2] See description of Figure 25-1. [Figure 26] 1 shows a product sheet for glucose from Sigma Aldrich. [Figure 27-1] FIG. 27 shows the product sheet for Dextran-40 from Sigma Aldrich. [Figure 27-2] See description of Figure 27-1. [Figure 28] 1 shows the product sheet for adenosine from Sigma Aldrich. [Figure 29] 1 shows a product sheet for glutathione from Sigma Aldrich. [Figure 30] 1 shows a product sheet for HypoThermosol™-FRS (HTS-FRS) from STEMCELL Technologies. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention As explained above, in a first aspect, the present invention is directed to a method of transporting / storing a stem cell population, the method comprising: i) Trolox, ii) Na + , iii) K + , iv) Cl - , v) H2PO4 - , vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione and transporting / storing said stem cell population in contact with a liquid carrier comprising:

[0019] It has been surprisingly discovered in the present application that the use of the liquid carriers described herein, and in particular liquid carriers such as HypoThermosol™, results in superior survival of stem cells compared to other pharmaceutically approved carriers, such as PlasmaLyte™. For example, after storing the mesenchymal stem cell population described herein in HypoThermosol™ for 7 days, approximately 70% of the cells remained viable. Conversely, after storing in PlasmaLyte™ for 7 days, only approximately 40% of the cells remained viable (as measured by hemocytometer, see Examples). Thus, the use of the liquid carriers described herein allows for the transport / storage of stem cells for a period of time without substantial loss of cell viability. In particular, as detailed in the Experimental Section, storage in HypoThermosol™ for a shorter period of 3 days or less is believed to be particularly beneficial, since stem cells generally secreted more factors than after storage in PlasmaLyte-A.

[0020] As used herein, the term "transport" or "transporting" refers to any transport. Such transport can be performed by any vehicle, such as a car, train, or airplane, or by a person carrying / transporting a container containing stem cells in contact with a liquid carrier from one location to another. In one embodiment, transport is performed from the location where the stem cell population of interest is generated to the location where the stem cells are administered (e.g., from the GMP facility where the stem cell population of interest is generated to the location where the stem cell population is administered, such as a clinic or medical center). However, the term "transporting" is also intended to refer to the storage of cells for a period of time at the same location. For example, stem cells may be stored at a location after collection until they are applied to a subject. The container in which stem cells can be stored or transported may be any container suitable for the method of the present invention.

[0021] The transport / storage can be carried out for any period of time. For example, the transport / storage can be carried out for about 7 days or less. It is also contemplated that the transport / storage can be carried out for about 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or less. Thus, the transport / storage can be carried out for about 48 hours or about 24 hours or less.

[0022] It is also contemplated that transport / storage may occur at any temperature suitable for the methods of the present invention. For example, transport / storage may occur at a temperature of about -5°C to about 15°C. Accordingly, it is also contemplated that transport / storage may occur at a temperature of about 2°C to about 8°C. Transport may also be performed at temperatures greater than about -5°C, greater than about -10°C, greater than about -15°C, or greater than about -20°C. It is further contemplated that transport / storage may occur at temperatures less than 20°C, less than 18°C, less than 15°C, less than 12°C, or less than 10°C.

[0023] The method of the present invention also contemplates that the stem cell population is stored or transported at any suitable concentration. For example, the stem cell population can be transported / stored at a concentration of about 70 million cells per ml of carrier, about 60 million cells per ml of carrier, about 50 million cells per ml of carrier, about 40 million cells per ml of carrier, about 30 million cells per ml of carrier, about 20 million cells per ml of carrier, about 10 million cells per ml of carrier, about 5 million cells per ml of carrier, about 4 million cells per ml of carrier, about 3 million cells per ml of carrier, about 2 million cells per ml of carrier, about 1 million cells per ml of carrier, about 500,000 cells per ml of carrier, about 100,000 cells per ml of carrier, or less than 100,000 cells per ml of carrier. Thus, stem cell populations can be transported / stored at concentrations of about 10 million cells per ml of carrier to about 1 million cells per ml of carrier.

[0024] The method of the present invention relates to the transportation / storage of stem cells. In principle, any stem cells can be used in the method of the present invention. One characteristic property of stem cells is their ability to self-renew. "Self-renewal" refers to the ability to undergo multiple cell cycles of cell division while maintaining an undifferentiated state. Methods for testing whether a cell has the ability to self-renew are known to those skilled in the art. For example, self-renewal can be tested by subculturing cells for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more passages. Subculturing involves splitting cells before replating them as a single-cell suspension. Another characteristic of stem cells is their multipotency or pluripotency, as described elsewhere herein. In principle, multipotency or pluripotency can be tested by differentiating the stem cells into different lineages.

[0025] In particular, the stem cell population used in the methods of the present invention may be an embryonic stem cell population, an adult stem cell population, a mesenchymal stem cell population, or an induced pluripotent stem cell population.

[0026] As used herein, an "embryonic stem cell population" refers to a "pluripotent stem cell population." Pluripotent cells, as referred to herein, refer to cell types that have the ability to self-renew and the potential to differentiate into different cell types. Pluripotent stem cells can differentiate into almost any cell, i.e., cells derived from any of the three primary germ layers: ectoderm, endoderm, and mesoderm. The term "pluripotent stem cells" also encompasses stem cells derived from the inner cell mass of an early embryo, known as a blastocyst. Notably, recent advances in embryonic stem cell research have made it possible to generate new embryonic stem cell lines without destroying the embryo, for example, by using blastomere biopsy-based techniques that do not interfere with the developmental potential of the embryo (Klimanskaya (2006) "Embryonic stem cells from blastomeres maintaining embryo viability." Semin Reprod Med. 2013 Jan;31(1):49-55). Furthermore, numerous established embryonic stem cell lines are available in the art. Therefore, it is possible to manipulate embryonic stem cells without the need for embryo destruction. Pluripotent stem cells may be embryonic stem cells that have not been obtained by destruction of a human embryo. Thus, pluripotent stem cells are embryonic stem cells that have been obtained from an embryo without destroying the embryo.

[0027] As used herein, "adult stem cell population" refers to a multipotent stem cell population. Multipotent stem cell populations can give rise to a limited number of cell types, and therefore they are limited to the fate of somatic cells. For example, neural stem cells can give rise to both neural cells and glial cells. Adult stem cells have the ability to self-renew and can be obtained from any suitable source. For example, adult stem cells can be obtained from bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelium of the skin and digestive system, cornea, retina, liver, or pancreas.

[0028] The stem cell population used in the method of the present invention can also be mesenchymal stem cell population.In this regard, it is noted that the culture medium (for example, PTT-6) described herein makes it possible to isolate mesenchymal stem cell population (also referred to herein as " mesenchymal stem cell ") from amniotic membrane under the condition that allows the cell proliferation of mesenchymal stem / progenitor cell without causing mesenchymal stem / progenitor cell differentiation.Therefore, after isolating mesenchymal stem cell from amniotic membrane as described herein, the isolated mesenchymal stem / progenitor cell population has the ability to differentiate into multiple cell types, as described in, for example, US Patent Application 2006 / 0078993, US Patent No. 9,085,755, International Patent Application WO2006 / 019357, US Patent No. 8,287,854 or WO2007 / 046775. For example, as described in U.S. Patent Application Publication No. 2006 / 0078993, mesenchymal stem cells from the amniotic membrane of the umbilical cord have a spindle shape, express the genes POU5f1, Bmi-1, and leukemia inhibitory factor (LIF), and secrete activin A and follistatin. The mesenchymal stem cells isolated in the present invention can be differentiated into any type of mesenchymal cell, including, but not limited to, adipocytes, dermal fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiac myocytes, smooth muscle cells, skeletal muscle cells, mucin-producing cells, insulin-producing cells (e.g., beta islet cells), and other endocrine-derived cells, or neuroectodermal cells. Stem cells isolated according to the methods described herein can be differentiated in vitro for subsequent use of the differentiated cells for medical purposes. An example of such an approach is the differentiation of mesenchymal stem cells into insulin-producing beta islet cells, which can then be administered, for example, by transplantation, to patients suffering from insulin deficiency, such as diabetes (see also WO2007 / 046775 in this regard). Alternatively, the mesenchymal stem cells described herein can be used in an undifferentiated state for cell-based therapy, for example, for wound healing purposes, such as treating burns or chronic diabetic wounds.In these therapeutic applications, the mesenchymal stem cells of the present invention may help promote wound healing by interacting with surrounding diseased tissue, or may also differentiate into respective skin cells (see, for example, WO2007 / 046775 again).

[0029] In this regard, it is noted that the mesenchymal stem cell populations described herein can be isolated and cultured from (i.e., derived from) any umbilical cord tissue, as long as the umbilical cord tissue contains the amniotic membrane (also referred to as the "umbilical cord lining"). Thus, mesenchymal stem cell populations can be isolated from (a small piece of) the entire umbilical cord, as described in the Experimental Section of this application. Therefore, the umbilical cord tissue may contain any other tissue / component of the umbilical cord in addition to the amniotic membrane. For example, as shown in Figure 16 of U.S. Patent Application Publication No. 2006 / 0078993 or International Patent Application WO2006 / 019357, the amniotic membrane of the umbilical cord is the outermost part of the umbilical cord that covers it. Additionally, the umbilical cord contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-depleted blood away from the fetus). For protection and mechanical support, these three blood vessels are embedded in Wharton's gelatinous substance, primarily mucopolysaccharides. Thus, umbilical cord tissue, as used herein, can also include this one vein, two arteries, and Wharton's gel. The use of such an entire (intact) portion of the umbilical cord has the advantage that it is not necessary to separate the amniotic membrane from the other components of the umbilical cord. This reduces the isolation steps, thereby making the methods described herein simpler, faster, less error-prone, and more economical—all important aspects of GMP production required for therapeutic applications of mesenchymal stem cells. Thus, mesenchymal stem cell isolation can begin from tissue explants, and the isolated mesenchymal stem cells can then be subsequently subcultured (cultured) if larger quantities of mesenchymal stem cells are desired, for example, for use in clinical trials. Alternatively, it is also possible to isolate mesenchymal umbilical cord lining stem cells from the amniotic membrane by first separating the amniotic membrane from the other components of the umbilical cord and culturing the amniotic membrane in a culture medium, such as PTT-6. This culture can also be performed on tissue explants, optionally followed by subculture of the isolated mesenchymal stem cells.In this context, the term "tissue explant" or "tissue explant method" is used in its ordinary sense in the art to refer to a method in which a collected tissue or a tissue fragment thereof is placed in a cell culture dish containing a culture medium (growth medium), and over time, stem cells migrate from the tissue onto the surface of the dish. These primary stem cells can then be further expanded and transferred to a new dish by micropropagation (subculture), as also described herein. In this context, it is noted that in the first step of isolating amniotic mesenchymal stem cells from the umbilical cord, in terms of generating cells for therapeutic purposes, a master cell bank of the isolated mesenchymal stem cells can be obtained, and subsequent subculture can result in a working cell bank. In certain embodiments, the stem cell population is therefore a mesenchymal stem cell population.

[0030] A mesenchymal stem cell population can be isolated from the amniotic membrane of the umbilical cord by a method comprising culturing in a culture medium containing DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum). Using such a medium, a mesenchymal stem cell population can be isolated from the amniotic membrane of the umbilical cord, with more than 90% or even 99% or more of the cells being positive for the three mesenchymal stem cell markers CD73, CD90, and CD105, and at the same time, these stem cells lack expression of CD34, CD45, and HLA-DR (see Experimental Section), meaning that 99% or even more of the cells in this population express the stem cell markers CD73, CD90, and CD105, but do not express the markers CD34, CD45, and HLA-DR.Such a highly homogeneous and well-defined cell population was first reported in co-pending U.S. patent application Ser. No. 15 / 725,913, filed October 5, 2018, which claims priority to U.S. provisional patent application Ser. No. 62 / 404,582, filed October 5, 2017 (the contents of both of which are incorporated herein by reference in their entireties), and in co-pending PCT application PCT / SG2017 / 050500, filed October 5, 2018, which also claims priority to U.S. provisional patent application Ser. No. 62 / 404,582, filed October 5, 2017; and this stem cell population has been described, for example, in Dominici et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement," Cytotherapy (2006) Vol. 8, No. 4, 315-317; Sensebe et al., "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement," Cytotherapy (2006) Vol. 8, No. 4, 315-317; They are ideal candidates for clinical trials and cell-based therapies because they fully meet the generally accepted standards for human mesenchymal stem cells to be used in cell therapy, as defined by Vonk et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," Stem Cell Research & Therapy 2013, 4:66; Vonk et al., Stem Cell Research & Therapy (2015) 6:94; or Kundrotas Acta Medica Lituanica. 2012, Vol. 19, No. 2, pp. 75-79. Furthermore, by using bioreactors such as the Quantum Cell Expansion System, it is possible to obtain large numbers of mesenchymal stem cells, such as 300-700 million mesenchymal stem cells per run (see also the Experimental Section).Therefore, the present invention allows for the cost-effective transport / storage of stem cells in the amount required for therapeutic applications, such as wound healing. In addition, all components used to prepare the culture medium of the present invention are commercially available with GMP quality. Thus, the present invention opens a route for the transport / storage of GMP-produced and highly homogeneous mesenchymal stem cell populations derived from the amniotic membrane of umbilical cord.

[0031] Therefore, in some embodiments, the mesenchymal stem cell population is an isolated mesenchymal stem cell population from the amniotic membrane of the umbilical cord.It is further assumed that at least about 90% or more of the cells in the isolated stem cell population express each of the markers CD73, CD90 and CD105.For example, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells in the isolated mesenchymal stem cell population express each of CD73, CD90 and CD105. Additionally, or alternatively, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cell population lacks expression of the following markers: CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related).

[0032] The marker CD73 is known to those skilled in the art. In this regard, CD73 refers to cluster of differentiation 73, also known as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase. The sequence of the human CD73 protein may have the sequence of SEQ ID NO. 1. The marker CD90 is known to those skilled in the art. In this regard, CD90 refers to cluster of differentiation 90, also known as thymocyte differentiation antigen 1 (Thy-1). The sequence of the human CD90 protein may have the sequence of SEQ ID NO: 2. The marker CD105 is known to those skilled in the art. CD105 is also known as endoglin (ENG). The sequence of the human CD105 protein may have the sequence of SEQ ID NO: 3.

[0033] When the mesenchymal stem cell population of the present invention (specifically, a population of mesenchymal stem cells, at least about 98% or 99% of which express the markers CD73, CD90, and CD105, and lack expression of the markers CD34, CD45, and HLA-DR) is used for clinical trials or as an approved therapy, a cell population from a working cell bank is typically used for this purpose. As explained, the mesenchymal stem cell population may lack expression of the following markers: CD34, CD45, and HLA-DR. In this regard, it is noted that the markers CD34, CD45, and HLA-DR are known to those skilled in the art. The human CD34 protein may have the sequence of SEQ ID NO. 4. The human CD45 protein may have the sequence of SEQ ID NO: 5. The human HLA-DR protein may have the sequence of SEQ ID NO: 6.

[0034] Both the stem cell population at the isolation stage (which may constitute a master cell bank) and the stem cell population at the subculture stage (which may constitute a working cell bank) may be stored, for example, in a cryopreserved form.

[0035] As described above, the present method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord has the advantage that all of the components used in the culture medium of the present invention are available in GMP quality, thus providing the possibility for mesenchymal stem cells to be isolated under GMP conditions for subsequent therapeutic administration.

[0036] Therefore, stem cell population can also be induced pluripotent stem cell population.As used herein, " induced pluripotent stem cell " refers to the adult somatic cell that is genetically reprogrammed into embryonic stem cell-like state by forcing the expression of the genes and factors that are important for maintaining the definitive characteristics of embryonic stem cell.Therefore, induced pluripotent stem cell can be induced / produced from non-pluripotent cell.

[0037] Induced pluripotent stem cells (iPSCs) represent a significant advance in stem cell research because they allow for the generation of pluripotent stem cells without the use of embryos. Mouse iPSCs were first reported in 2006 (Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." Cell 126 (4): 663-76), and human iPSCs (hiPSCs) were first reported in 2007 (Takahashi et al. (2007) "Induction of pluripotent stem cells from adult human fibroblasts by defined factors." Cell; 131(5):861-72). Mouse iPSCs exhibit key characteristics of pluripotent stem cells, including the expression of stem cell markers, the ability to form tumors containing cells derived from all three germ layers, and the ability to contribute to many different tissues when injected into mouse embryos at very early developmental stages. Human iPSCs also express stem cell markers and can generate cells characteristic of all three germ layers. These markers include Oct3 / 4, Sox2, Nanog, alkaline phosphatase (ALP), and stem cell-specific antigens 3 and 4 (SSEA3 / 4). The chromatin methylation patterns of iPSCs are also similar to those of embryonic stem cells (Tanabe, Takahashi, Yamanaka (2014) "Induction of pluripotency by defined factors." Proc. Jpn. Acad., 2014, Ser. B 90).

[0038] In addition, iPSCs can self-renew in vitro and differentiate into all three germ layers. The pluripotency or potential to differentiate into different cell types of iPSCs can be tested, for example, by in vitro differentiation into neural or glial cells or by blastocyst injection to generate germline chimeric animals.

[0039] Methods for producing human induced pluripotent stem cells are known to those skilled in the art and are described, for example, in WO2009115295, WO2009144008, or EP2218778. Therefore, those skilled in the art can obtain iPSCs by any method. In principle, induced pluripotent cells can be obtained from any adult somatic cells (of the subject). Exemplary somatic cells include peripheral blood mononuclear cells (PBMCs) from blood or fibroblasts obtained from skin tissue biopsies.

[0040] The method of the present invention comprises contacting the stem cell population as described herein with a liquid carrier.In the method of the present invention, it is assumed that the stem cell population as described herein is contacted with a carrier before transport / storage.In addition, or alternatively, the stem cell population is contacted with a carrier after its collection.How collection can be carried out is described in detail elsewhere in this specification and in the experimental section.For example, the stem cell population can be contacted with a carrier about 0 minutes, about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 45 minutes, about 60 minutes or longer after its collection.

[0041] Recovery can include separating stem cell population from culture medium, for example, from PTT-6.Suitable techniques for such separation are known to those skilled in the art.For example, separation can be carried out by centrifuging stem cells in culture medium and decanting the culture medium.

[0042] The stem cell population is i) Trolox, ii) Na + , iii) K + , iv) Cl - , v) H2PO4 - , vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione contacting the composition with a liquid carrier comprising:

[0043] "Trolox" refers to 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, CAS number 53188-07-1. It is a water-soluble analog of vitamin E and has been suggested to reduce oxidative stress or damage. Figure 19 shows a data sheet for Trolox, available from Tocris. It is also commercially available from Sigma Aldrich (product number: 238813).

[0044] Na + and Cl - are all well-known ions. Those skilled in the art know how to obtain them. For example, these ions may be added to the carrier as NaCl salts. GMP quality NaCl is available from Sigma Aldrich. Figure 20 shows the data sheet for NaCl available from Sigma Aldrich.

[0045] K + and H2PO4 - (Dihydrogen phosphate) is also well known to those skilled in the art. It can be used, for example, as KH2PO4, available from Sigma Aldrich. Figure 21 shows the data sheet for KH2PO4, available from Sigma Aldrich.

[0046] HEPES, also known as 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS number 7365-45-9), is commonly used as a zwitterionic organic chemical buffer. Those skilled in the art also know where to obtain HEPES, which is commercially available. For example, it can be obtained from Sigma Aldrich; the corresponding data sheet is shown in Figure 22.

[0047] Lactobionate is the carboxylate anion of lactobionic acid. Lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid) is a sugar acid. Lactobionate can be used in different ways. When used as potassium lactobionate, it can provide osmotic support and prevent cell swelling, and when combined with sodium, it can have a preservative function. Alternatively, mineral salts of lactobionic acid can be used for mineral supplementation. For pharmaceutical applications, the antibiotic erythromycin is often used, inter alia, as erythromycin lactobionate. Those skilled in the art also know where to obtain lactobionates, such as sodium lactobionate (Cas No.: 27297-39-8), for example, from COMBI-BLOCKS, see the product sheet in Figure 23.

[0048] Sucrose, also known as D-Glc-(1→2)-β-D-Fru, α-D-glucopyranosyl β-D-fructofuranoside, β-D-fructofuranosyl-α-D-glucopyranoside, D(+)-sucrose, or sugar (CAS number 57-50-1), is commercially available, as are other substances, and those skilled in the art will know where to purchase it as well. The corresponding product sheet for sucrose from Sigma Aldrich is shown in Figure 24.

[0049] Mannitol is a type of sugar alcohol (CAS Registry Number: 69-65-8). Those skilled in the art know how to obtain mannitol. For example, it can be obtained from Avantor. The respective product sheets are shown in Figure 25.

[0050] Glucose (CAS number: 50-99-7) is also well known to those skilled in the art and is commercially available. The respective product sheet from Sigma Aldrich is shown in Figure 26.

[0051] Dextran is a branched glucan composed of linear α(1→6)-linked glucose units and an α(1→3)-linked initial branch. Dextran sizes range from 10,000 to 150,000 Kd. Dextrans are used in many applications, including as bulking agents, stabilizers, matrix components, binding platforms, lubricants, and physical structural components. Dextran-40 (CAS No. 9004-54-0), used in the carriers described herein, is typically used in the development of new and improved preservation solutions for organ transplantation. Dextran-40 can be used to determine cell firmness and transcellular flow parameters. Dextran-40 can also be used as a colloid plasma expander. Dextran-40 is commercially available, particularly from Sigma-Aldrich (product sheet shown in Figure 27).

[0052] Adenosine (CAS number 58-61-7) is a purine nucleoside composed of a molecule of adenine linked to a ribose sugar molecule (ribofuranose) moiety via a β-N9-glycosidic bond. Adenosine is commercially available from, among others, Sigma-Aldrich (the corresponding product sheet is shown in Figure 28).

[0053] Glutathione is also known as (2S)-2-amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}butanoic acid. This component is commercially available and can be obtained from, among others, Sigma Aldrich (corresponding product sheet shown in Figure 29).

[0054] In principle, any liquid carrier containing the substances listed in i) to xiii) above can be used in the method of the present invention. The carrier is a liquid carrier. Therefore, to form a solution / suspension, the substances listed in i) to xiii) can be dissolved in a liquid. The liquid can be any suitable liquid. For example, the liquid can be a culture medium, water, a buffer solution, or the like.

[0055] The carrier may additionally contain additional pH buffers, energy substrates, free radical scavengers, and osmotic / colloid stabilizing agents—all of which are known to those skilled in the art. Furthermore, the liquid carrier may be serum-free and / or protein-free. The liquid carrier may be free of dipolar aprotic solvents, such as DMSO. In particular, the liquid carrier may be a carrier described in WO 2010 / 064054. The carrier may be HypoThermosol™ or HypoThermosol™-FRS (HTS-FRS). HypoThermosol™-FRS (HTS-FRS) can be purchased from STEMCELL Technologies (according to the respective product sheets shown in FIG. 30).

[0056] It is further envisioned that the carrier is a transport / storage medium or excipient. The transport / storage medium may be a natural medium, which consists solely of a naturally occurring biological fluid additionally containing the substances listed in i) to xiii) as described herein. The medium may also contain the substances listed in i) to xiii) as described herein, as well as the addition of (additional) nutrients (both organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, and / or cofactors. In certain embodiments, the medium is serum-free and / or protein-free.

[0057] The carrier may also be an excipient. An "excipient" is a substance that is formulated with an active ingredient of a pharmaceutical. In this method, the active ingredient is a stem cell population.

[0058] The carrier may further comprise a biocompatible scaffold or microcarrier. The scaffold or microcarrier may be, for example, a biodegradable polymeric material, most preferably poly(D,L lactic-co-glycolic acid) (PLGA). Alternatively, the scaffold or microcarrier may be a smooth, macroporous, or microporous structure containing materials including poly-L-lactide (PLLA), collagen, fibronectin, glycosaminoglycans (GAGs), fibrin, starch, cellulose arabinogalactan (larch gum), alginate, agar, carrageenan, chitin, hyaluronic acid, dextran, gellan gum, pullulan, hydroxyapatite, polyhydroxyalkanoic acid (PHA), hydrogels, or other self-assembling materials such as peptide-based nanostructured fibrous scaffolds.

[0059] In principle, any amount of stem cells can be contacted with any amount of liquid carrier.In this regard, contact can be carried out by suspending stem cell population at a density of about 70 million / ml, about 60 million / ml, about 50 million / ml, about 40 million / ml, about 30 million / ml, about 20 million / ml, about 10 million / ml, about 5 million / ml, about 4 million / ml, about 3 million / ml, about 2 million / ml, about 1 million / ml, about 1 million / ml, about 500,000 / ml, about 100,000 / ml or less than 100,000 cells in 1 ml of carrier.In some embodiments, contact can be carried out by suspending stem cell population at a density of about 10 million / ml of carrier.

[0060] After contacting the stem cell population with the carrier, the stem cells contacted with the carrier can be dispensed into a vial with a volume of about 50 ml, about 20 ml, about 10 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml, about 1 ml, about 0.5 ml, about 0.25 ml, or less than 0.25 ml of the carrier. For example, the stem cells contacted with the carrier can be dispensed into a vial with a volume of about 1 ml.

[0061] It is further envisioned that the methods of the present invention do not include a thawing or freezing step, which may involve transporting / storing the stem cell population after collection without the need to freeze and thaw the stem cell population.

[0062] The carrier used in the method for transporting / storing stem cell population described herein is particularly suitable for this purpose.One advantage of this carrier is that substantially all stem cells transported / stored therein remain viable.A " viable cell " is a cell that can survive.Those skilled in the art know how to detect viable cells.One such method is to stain cells with dye trypan blue.Vivable cells are not stained positively by trypan blue.

[0063] In this regard, in the methods of the present invention, at most about 50%, about 40%, about 30%, about 20%, about 10%, or less than about 10% of the stem cells in the population may die during transport / storage compared to the number / quantity of viable stem cells before transport / storage.

[0064] The method of the present invention also contemplates that the stem cell population may have any cell diameter after transportation / storage. Those skilled in the art know how to measure cell diameter. For example, cell size / diameter can be determined by taking a microscopic image and measuring the cell diameter using secondary software. The majority of stem cells in the stem cell population may therefore have a cell diameter of about 9 μm to about 20 μm after transportation / storage. It is also contemplated that the majority of stem cells in the stem cell population may have a cell diameter of about 12 μm to about 16 μm after transportation.

[0065] Stem cells transported / stored in the carriers described herein secrete the same proteins / factors as live stem cells. For example, the method of the present invention contemplates that after transport / storage, the (mesenchymal) stem cell population can secrete approximately the same amount of TGF beta 1 as before transport / storage. TGF beta 1 (transforming growth factor beta, TGF-β1) is known to those skilled in the art and may include the sequence shown in SEQ ID NO. 7. Additionally, or alternatively, after transport / storage, the (mesenchymal) stem cell population can secrete approximately the same amount of VEGF (vascular endothelial growth factor), PDGF-AA (platelet-derived growth factor subunit AA), Ang-1 (angiogenin-1), and / or HGF (hepatocyte growth factor) as before transport / storage. VEGF, PDGF-AA, Ang-1, and / or HGF are all known to those skilled in the art for their involvement in wound healing. In particular, VEGF may have the sequence shown in SEQ ID NO. 8, PDGF-AA may have the sequence shown in SEQ ID NO. 9, Ang-1 may have the sequence shown in SEQ ID NO. 10, while HGF may have the sequence shown in SEQ ID NO. 11. Additionally or alternatively, PDGF-BB and / or IL-10 are essentially undetectable before and / or after transport. PDGF-BB (platelet-derived growth factor subunit BB) and / or IL-10 (interleukin-10) are also known to those skilled in the art. PDGF-BB may have the sequence shown in SEQ ID NO. 12, while IL-10 may have the sequence shown in SEQ ID NO: 13. Secretion of these factors can be determined by any suitable method, for example, by measuring the amount of protein (e.g., PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF, or TGFβ1) secreted by stem cells into the carrier. The amount of protein can be measured by commercially available antibody / immunoassays in an automated format, for example using systems such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).In this regard, it is noted that the involvement of the proteins angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF in the wound healing process is known to those skilled in the art. For the involvement of angiopoietin 1 in wound healing, see, for example, Li et al. Stem Cell Research & Therapy 2013, 4:113 "Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing." For the involvement of hepatocyte growth factor (HGF) in wound healing, specifically in the healing of chronic / non-healing wounds, see, for example, Yoshida et al., "Neutralization of Hepatocyte Growth Factor Leads to Retarded Cutaneous Wound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation," J. Invest. Dermatol. 120:335-343, 2003; Li, Jin-Feng et al., "HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through β1-Integrin / ILK Pathway," BioMed Research International 2013 (2013):470-418; or Conway et al., "Hepatocyte growth factor regulation: An integral part of why wounds become chronic," Wound Rep Reg (2007) 15 683-692.For the involvement of vascular endothelial growth factor (VEGF) in wound healing, particularly in the healing of chronic / non-healing wounds, see, for example, Froget et al., Eur. Cytokine Netw., Vol. 14, March 2003, 60-64, or Bao et al., "The Role of Vascular Endothelial Growth Factor in Wound Healing," J Surg Res. 2009 May 15; 153(2): 347-358.

[0066] For the involvement of transforming growth factor beta (including TGF-β1, TGF-β2, and TGF-β3) in wound healing, particularly in the healing of chronic / non-healing wounds, see, for example, Ramirez et al., "The Role of TGFβ Signaling in Wound Epithelialization," Advances In Wound Care, Volume 3, Number 7, 2013, 482-491, or Pakyari et al., "Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing," Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.

[0067] Turning now to the culture medium used in the present invention, the culture medium for the isolation or culture of mesenchymal umbilical cord lining stem cells may contain a final concentration of about 55-65% (v / v) DMEM, a final concentration of about 5-15% (v / v) F12, a final concentration of about 15-30% (v / v) M171, and a final concentration of about 1-8% (v / v) FBS. As used herein, the "% (v / v)" refers to the volume of an individual component relative to the final volume of the culture medium. This means that if DMEM is present in the culture medium at a final concentration of, for example, about 55-65% (v / v), one liter of culture medium will contain about 550-650 ml DMEM.

[0068] In another embodiment, the culture medium may contain DMEM at a final concentration of about 57.5-62.5% (v / v), F12 at a final concentration of about 7.5-12.5% ​​(v / v), M171 at a final concentration of about 17.5-25.0% (v / v), and FBS at a final concentration of about 1.75-3.5% (v / v). In a further embodiment, the culture medium may contain DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0069] In addition to the above components, the culture medium may contain supplements beneficial to the culture of mesenchymal umbilical cord lining stem cells. The culture medium of the present invention may contain, for example, epidermal growth factor (EGF). When present, EGF may be present in the culture medium at a final concentration of about 1 ng / ml to about 20 ng / ml. In some of these embodiments, the culture medium may contain EGF at a final concentration of about 10 ng / ml.

[0070] The culture medium may also contain insulin. If present, insulin may be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some of these embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml.

[0071] The culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may contain all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may contain adenine at a final concentration of about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0072] In one embodiment, to obtain the highly purified mesenchymal stem cell population described and used herein, mesenchymal stem cells are cultured in PTT6 medium. In this regard, the PTT6 medium described herein is used to obtain a final culture volume of 500 ml: i. 250 ml of DMEM ii. M171 118 ml iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v / v) v. EGF at a final concentration of 10 ng / ml vi. Insulin at a final concentration of 5 μg / ml vii. Insulin 0.175 ml (final concentration 5 μg / ml) It is noted that the above can be obtained by mixing

[0073] "DMEM" refers to Dulbecco's Modified Eagle's Medium (BME), a modification of Basal Medium Eagle's Medium, developed in 1969 (see Figure 1, which shows the data sheet for DMEM available from Lonza). The original DMEM formulation contained 1000 mg / L glucose and was first reported for the culture of embryonic mouse cells. DMEM has since become a standard medium for cell culture and is commercially available from a variety of sources, including ThermoFisher Scientific (catalog number 11965-084), Sigma Aldrich (catalog number D5546), or Lonza, to name just a few. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is Sigma Aldrich catalog number D5546 DMEM medium, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0074] "F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed to allow the cultivation of a wide variety of mammalian cells and hybridoma cells when used with serum in combination with hormones and transferrin (see Figure 2, which shows the data sheet for Ham's F12 medium from Lonza). Any commercially available Ham's F12 medium (e.g., from ThermoFisher Scientific (catalog number 11765-054), Sigma Aldrich (catalog number N4888), or Lonza, to name just a few suppliers) can be used in the present invention. In a preferred embodiment, Ham's F12 medium from Lonza is used.

[0075] "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham's F12 medium (see Figure 3, which shows the data sheet for DMEM:F12 (1:1) medium from Lonza). DMEM / F12 (1:1) medium is a basal medium widely used to support the growth of many different mammalian cells and is commercially available from various suppliers, such as ThermoFisher Scientific (catalog number 11330057), Sigma Aldrich (catalog number D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM / F12 (1:1) medium (DMEM:F12 with L-glutamine, 15 mM HEPES, and 3.151 g / L glucose) available from Lonza under catalog number 12-719F.

[0076] "M171" refers to medium 171, which was developed as a basal medium for the culture and growth of normal human mammary epithelial cells (see Figure 4, which shows the data sheet for M171 medium from Life Technologies Corporation). This basal medium is widely used and commercially available from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in the present invention. In a preferred embodiment, the M171 medium used herein is M171 medium available from Life Technologies Corporation under catalog number M171500.

[0077] "FBS" refers to fetal bovine serum (also called fetal bovine serum), i.e., the blood fraction remaining after natural blood clotting and subsequent removal of any remaining red blood cells by centrifugation. Fetal bovine serum is the most widely used serum supplement for in vitro cell culture of eukaryotic cells because it has very low levels of antibodies, contains many growth factors, and allows for versatility in many different cell culture applications. FBS is preferably obtained from a member of the International Serum Industry Association (ISIA), whose focus is the safety and safe use of serum and animal-derived products through proper origin tracking, authenticity of labeling, and appropriate standardization and oversight. ISIA member suppliers of FBS include Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, to name a few. In a currently preferred embodiment, FBS is obtained from GE Healthcare under catalog number A15-151.

[0078] As described above, the method for preparing a culture medium for isolating a mesenchymal stem cell population used in the present invention comprises the steps of: i. 250 ml of DMEM ii. M171 118 ml iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v / v) The method includes the step of mixing the above components.

[0079] As explained above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Therefore, using this method of making culture medium, the final concentrations (v / v) in a total volume of 500 ml are as follows: -DMEM:250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v). - M171: 118 ml, equivalent to 118 / 500 = 23.6% (v / v). - F12: 59 ml, equivalent to 59 / 500 = 11.8% (v / v).

[0080] An embodiment of the present method for producing a culture medium includes: v. 1 ml of EGF stock solution (5 μg / ml) to achieve a final EGF concentration of 10 ng / ml, and vi. 0.175 ml of insulin stock solution (14.28 mg / ml) to achieve a final insulin concentration of 5 μg / ml The method further comprises the step of adding

[0081] In these embodiments, it is noted herein that the above volumes of these components i-vi result in a culture medium with a final volume of 499.675 ml. If no additional components are added to the culture medium, the remaining 0.325 ml (total volume of 500 ml) can be any of components i-iv, meaning, for example, DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of the EGF or insulin stock solution can be adjusted so that the total volume of the culture medium is 500 ml. In addition, it is also noted that components i-iv do not necessarily have to be added in the order listed, and that these components can be mixed in any order to arrive at the culture medium of the present invention. This means, for example, that M171 and DMEM / F12 can be mixed together and then combined with DMEM and FBS to achieve the final concentrations described herein, i.e., a final DMEM concentration of about 55-65% (v / v), a final F12 concentration of about 5-15% (v / v), a final M171 concentration of about 15-30% (v / v), and a final FBS concentration of about 1-8% (v / v).

[0082] In other embodiments, the method further comprises adding one or more of the supplements adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) to the DMEM in a volume of 0.325 ml, thereby bringing the total volume to 500 ml. In this embodiment, the final concentrations of these supplements in the DMEM may be as follows: about 0.05 to 0.1 μg / ml adenine, for example about 0.025 μg / ml adenine; hydrocortisone at approximately 1–10 μg / ml; About 0.5-5 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0083] Consistent with the above disclosure, the cell culture medium used herein is obtainable or obtained by the methods of making medium described herein.

[0084] Additionally, described herein is a method for isolating mesenchymal stem cells from the amniotic membrane of an umbilical cord, the method comprising culturing the amniotic tissue in a culture medium prepared by the method.

[0085] Therefore, the present invention also provides a method for producing a method for manufacturing a semiconductor device comprising: - DMEM at a final concentration of approximately 55-65% (v / v), - F12 at a final concentration of approximately 5-15% (v / v), - M171 at a final concentration of approximately 15-30% (v / v), and -Final concentration of approximately 1-8% (v / v) FBS The present invention relates to (the use of) a cell culture medium containing

[0086] In certain embodiments of the culture medium described herein, the medium contains DMEM at a final concentration of about 57.5-62.5% (v / v), F12 at a final concentration of about 7.5-12.5% ​​(v / v), M171 at a final concentration of about 17.5-25.0% (v / v), and FBS at a final concentration of about 1.75-3.5% (v / v). In other embodiments, the culture medium may contain DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0087] Additionally, the culture medium may further comprise epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. In certain embodiments, the culture medium comprises EGF at a final concentration of about 10 ng / ml. The culture medium described herein may further comprise insulin at a final concentration of about 1 μg / ml to 10 μg / ml. In such embodiments, the culture medium may comprise insulin at a final concentration of about 5 μg / ml.

[0088] The cell culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In certain embodiments, the culture medium includes all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). When present, the culture medium may include adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine or about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml hydrocortisone or about 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0089] In an embodiment of the cell culture medium, 500 ml of the cell culture medium of the present invention comprises: i. 250 ml of DMEM ii. M171 118 ml iii. 118 ml of DMEM / F12 iv. Fetal bovine serum (FBS) 12.5 ml (final concentration 2.5%) In a further embodiment, the cell culture medium comprises: v. EGF at a final concentration of 10 ng / ml, and vi. Insulin at a final concentration of 5 μg / ml Both insulin and EGF can be added to the culture medium using an optimal storage solution so that the total volume of the culture medium does not exceed 500 ml.

[0090] In a specific example, components i to vi of the culture medium used in the present invention are the components shown in Figure 5, which means that they are obtained from the respective manufacturers using the catalog numbers shown in Figure 5. The medium obtained by mixing components i to vi as shown in Figure 5 is also referred to herein as "PTT-6." In this regard, it is again noted that components i to vi and any other components, such as antibiotics, from any other commercial supplier can be used in making the medium of the present invention.

[0091] Additionally, the cell culture medium of the present invention may contain adenine at a final concentration of about 0.01 to about 0.1 μg / ml or about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 0.1 to 10 μg / ml, about 0.5 to about 10 μg / ml, or about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.1 to about 5 ng / ml or about 0.5 to about 5 ng / ml.

[0092] To obtain the mesenchymal stem cell populations described herein, umbilical cord tissue can be cultured until an appropriate number of (primary) mesenchymal umbilical cord lining stem cells proliferate from the tissue. In a typical embodiment, umbilical cord tissue is cultured until cell proliferation of amniotic mesenchymal stem cells reaches approximately 70 to 80% confluence. It is noted that the terms "confluence" or "confluence" are used herein in their usual sense in the art of cell culture and refer to an estimate / measure of the number of adherent cells in a culture dish or flask, referring to the percentage of the surface covered by cells. For example, 50 percent confluence means that approximately half of the surface is covered, with room for cells to grow. 100 percent confluence means that the surface is completely covered by cells, with no room left for cells to grow as a monolayer.

[0093] Once an appropriate number of primary cells (mesenchymal umbilical cord lining stem cells) have been obtained from the umbilical cord lining tissue by tissue explantation, the mesenchymal stem cells are removed from the culture vessel used for culturing. This allows for the creation of a master cell bank containing the (primary) isolated amniotic mesenchymal stem cells. Typically, because mesenchymal stem cells are adherent cells, the removal is performed using standard enzymatic treatment. For example, the enzymatic treatment may include trypsinization, as described in International Patent Application No. 2006 / 0078993, International Patent Application No. WO2006 / 019357, or International Patent Application No. WO2007 / 046775, which means that the proliferating cells can be recovered by trypsinization (0.125% trypsin / 0.05% EDTA) for further expansion. When the recovered mesenchymal stem cells are used, for example, to create a master cell bank, the cells can also be cryopreserved and stored for further use, as described herein below.

[0094] Once collected, the mesenchymal stem cells can be transferred to a culture vessel for subculture. Subculture can also be initiated from frozen primary cells, i.e., from a master cell bank. For subculture, any suitable amount of cells can be seeded into a culture vessel, such as a cell culture plate. For this purpose, mesenchymal stem cells can be seeded at a concentration of, for example, about 0.5 × 10 6 cells / ml ~ approx. 5.0×10 6 The cells can be suspended in an appropriate medium for subculturing (most conveniently medium PTT-6) at a concentration of about 1.0 x 10 cells / ml for subculturing. In one embodiment, the cells can be suspended in an appropriate medium for subculturing (most conveniently medium PTT-6) at a concentration of about 1.0 x 10 cells / ml for subculturing. 6The cells are suspended at a concentration of 10 ... An example of a commercially available hollow fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT, Inc.), which has been used, for example, to expand bone marrow mesenchymal stem cells for clinical trials (see Hanley et al., Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048-1058). Another example of a commercially available bioreactor that can be used to subculture the mesenchymal stem cell populations of the present invention is the Xuri Cell Expansion System, available from GE Healthcare. Culturing mesenchymal stem cells in an automated system such as the Quantum® Cell Expansion System is particularly effective when working cell banks for therapeutic applications are to be generated under GMP conditions and large numbers of cells are required.

[0095] The subculture of mesenchymal umbilical cord lining stem cells described herein is carried out in a culture medium described herein, such as PTT-6 medium. Thus, a culture medium such as PTT-6 can be used for both isolating mesenchymal stem cells from the amniotic membrane and subsequently culturing the isolated primary cells by subculture. Similarly, for subculture, mesenchymal stem cells can be cultured until an appropriate number of cells have proliferated. In an exemplary embodiment, mesenchymal stem cells are subcultured until they reach about 70 to about 80% confluency.

[0096] Isolation / culturing of a population of mesenchymal umbilical cord lining stem cells can be performed under standard conditions for culturing mammalian cells. Typically, the method of isolating a population of mesenchymal umbilical cord lining stem cells of the present invention is typically performed under conditions (temperature, atmosphere) normally used for culturing cells of the species from which the cells originate. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are typically cultured at 37°C in an air atmosphere containing 5% CO2. In this regard, it is noted that mesenchymal cells can be derived from any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey, or human, and in one embodiment, mesenchymal stem cells of human origin are preferred.

[0097] Once a desired / appropriate number of mesenchymal umbilical cord lining stem cells have been obtained from the subculture, they can be recovered by removing the mesenchymal stem cells from the culture vessel used for subculture. Recovery of mesenchymal stem cells is typically accomplished by enzymatic treatment, again involving trypsinization of the cells. The isolated mesenchymal stem cells are then collected and used directly or stored for further use. Typically, preservation is accomplished by cryopreservation. The term "cryopreservation" is used herein in its ordinary sense to refer to a process in which mesenchymal stem cells are preserved by cooling to subzero temperatures, such as (typically) -80°C or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be performed as known to those skilled in the art and may include the use of cryoprotectants, such as dimethyl sulfoxide (DMSO) or glycerol, which retard the formation of ice crystals in the umbilical cord cells.

[0098] The isolated population of mesenchymal umbilical cord lining stem cells obtained by the isolation method described herein is highly distinct and highly homogeneous. In typical embodiments of the method, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD105. In addition, in these embodiments, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45, and HLA-DR. In certain embodiments, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cell population express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.

[0099] Thus, consistent with the above disclosure, a mesenchymal stem cell population isolated from the amniotic membrane of an umbilical cord, wherein at least about 90% or more of the cells in the stem cell population express each of the following markers: CD73, CD90, and CD105. In a preferred embodiment, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the cells in the isolated mesenchymal stem cell population are CD73+, CD90+, and CD105+, meaning that this percentage of the isolated cell population expresses each of CD73, CD90, and CD105 (see the Experimental Section of this application), and may be used herein. Additionally, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: In certain embodiments, about 97% or more, about 98% or more, or about 99% or more of the cells of the isolated mesenchymal stem cell population express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR. Such a highly homogenous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord was first reported in U.S. Provisional Patent Application No. 62 / 404,582, filed October 5, 2016, and co-pending U.S. Patent Application No. 15 / 725,913, filed October 5, 2017, and co-pending PCT application PCT / SG2017 / 050500, also filed October 5, 2017, and meets the criteria for mesenchymal stem cells to be used in cell therapy (see Experimental Section, and also, e.g., Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," supra).In this regard, it is noted that this mesenchymal stem cell population can be obtained by the isolation method of the present invention, but can also be obtained by a different method, such as cell sorting, if desired.

[0100] The method for preparing a culture medium for isolating mesenchymal stem cells described herein comprises the steps of: i. 250 ml of DMEM ii. M171 118 ml iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v / v) The method includes the step of mixing the above components.

[0101] As explained above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium.

[0102] Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Using this method of making cultures, the final concentrations (v / v) in a total volume of 500 ml are therefore: DMEM:250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v). M171: 118 ml, equivalent to 118 / 500 = 23.6% (v / v). F12: 59 ml, equivalent to 59 / 500 = 11.8% (v / v).

[0103] The present invention also relates to a method of treating a subject having a disease, the method comprising the step of locally administering to the subject a mesenchymal stem cell population described herein, wherein the mesenchymal stem cell population is administered within about 96 hours from the time the mesenchymal stem cell population is harvested.

[0104] Similarly, the present invention also relates to the mesenchymal stem cell population described herein for use in a method of treating a disease in a subject, wherein the mesenchymal stem cell population is administered locally within about 96 hours from the time the mesenchymal stem cell population is harvested.

[0105] The subject to be treated may be any suitable subject. The subject may be a vertebrate, more preferably a mammal. Mammals include, but are not limited to, livestock, sport animals, pets, primates, dogs, horses, mice, and rats. Mammals may also be humans, dogs, cats, cows, pigs, mice, rats, etc. Thus, in one embodiment, the subject is a vertebrate. The subject may also be a human subject. Thus, the subject may be a subject in need of treatment. As such, the subject may suffer from a disease described elsewhere herein. In some embodiments, the subject suffers from type I or type II diabetes with chronic foot ulcers. Preferably, the subject is negative for HLA antibodies against mesenchymal stem cell populations.

[0106] The mesenchymal stem cell population can be applied in any dosage. The dosage can be therapeutically effective. As will be clear to those skilled in the art, the "therapeutically effective amount / dosage" can vary depending on factors including, but not limited to, the activity of the cells used, the stability of the cells in the patient's body, the severity of the condition to be alleviated, the age and sensitivity of the patient to be treated, adverse events, and the like. As various factors change over time, the amount of administration can be adjusted.

[0107] The dosage of mesenchymal stem cell that is applied can also be unit dosage.For example, mesenchymal stem cell population can be applied at unit dosage of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells or less than 250,000 cells.In certain embodiments, mesenchymal stem cell population is applied at unit dosage of about 10 million cells.

[0108] Mesenchymal stem cells can be applied to the same subject several times. For example, the stem cells can be applied once, twice, three times, or more times per week. In principle, any unit dose of mesenchymal stem cells can be applied as many times as appropriate to cure or alleviate a disease. For example, the mesenchymal stem cell population can be applied once, twice, three times, or more times per week. The mesenchymal stem cell population can also be applied for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or more.

[0109] Thus, a unit dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells is administered once or twice per week. A unit dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells can also be administered once or twice per week for a period of 3, 4, or 5, or 6, or 7, or 8, or 10 or more weeks.

[0110] Mesenchymal stem cell population is approximately 1000 cells / cm 2 ~About 5 million cells / cm 2 It is also contemplated by the methods of treatment of the present invention that the administration of a dose of 2 The term refers to the area of ​​the wound / skin to which the stem cells are applied. The mesenchymal stem cell population is approximately 100,000 cells / cm 2 , 300,000 cells / cm 2 , or 500,000 cells / cm 2 It is also contemplated that the mesenchymal stem cell population may be administered at a dosage of about 100,000 cells / cm.2 , about 300,000 cells / cm 2 , or approximately 500,000 cells / cm 2 can be applied twice a week for about eight weeks.

[0111] The mesenchymal stem cell population is administered within about 96 hours from the time the mesenchymal stem cell population is collected. How collection can be performed is described elsewhere herein. The mesenchymal stem cell population can also be applied within about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 6 hours, or less from the time the mesenchymal stem cell population is collected. Between the time of collection and the time of application, the mesenchymal stem cell population can be transported or stored using the transport / storage method of the present invention. Accordingly, aspects described in the transport / storage method of the present application are equally relevant, mutatis mutandis, to the treatment method of the present invention.

[0112] The treatment method of the present invention is useful for alleviating the disease suffered by the subject. In principle, any disease that can be treated by the mesenchymal stem cell group described herein is meant here. In particular, the disease can be a skin disease or a wound. The wound can be caused by any cause, for example, a burn, a bite, trauma, surgery, or disease. The wound can also be caused by diabetes. Therefore, the wound can also be a diabetic wound. The wound can also be a diabetic foot ulcer. It is worth noting that the mesenchymal stem cell group can be placed directly on the wound, for example, a burn or a diabetic wound (see International Patent Application WO2007 / 046775).

[0113] As described herein, between the collection of the mesenchymal stem cell population described herein and its application to a subject, the cells can be transported / stored in a carrier as defined herein.Therefore, the method of treating a subject of the present invention can also include a step of separating the mesenchymal stem cell population from the carrier before administering the mesenchymal stem cell population to the subject.Those skilled in the art know how to separate cells from the carrier.For example, separating the mesenchymal stem cell population from the carrier can include centrifugation.In addition, or instead, separating the mesenchymal stem cell population from the carrier can include removing the cell population from the vial with a syringe.

[0114] After separating the stem cells from the carrier, or after collecting the mesenchymal stem cells, or after obtaining the mesenchymal stem cell population described herein by any other method, these cells are topically applied to a subject. In principle, any method of topical administration is intended herein. The mesenchymal stem cell population can be administered by syringe. However, before applying the mesenchymal stem cells to a subject, the mesenchymal stem cells can also be contacted in a cream, ointment, gel, suspension, or any other suitable substance. After applying the mesenchymal stem cells to a subject, the mesenchymal stem cell population can be held in place by a dressing, such as Tegaderm® dressing, and a crepe bandage to cover the Tegaderm® dressing. For more uniform cell distribution, the application site can be gently massaged.

[0115] The present invention also relates to unit doses comprising about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells of the mesenchymal stem cell populations described herein.

[0116] It is also contemplated that the unit dose may comprise about 10 million, about 9 million, about 8 million, about 7 million, about 6 million, about 5 million, about 4 million, about 3 million, about 2 million, about 1 million, about 500,000, about 250,000, or about 100,000 cells. Preferably, the unit dose comprises about 10 million cells. It is further contemplated that the unit dose may comprise about 1,000 cells to about 5 million cells. The unit dose may be applied at a dose of about 100,000 cells, 300,000 cells, or 500,000 cells. As described herein, the unit dose may be applied topically. For example, the unit dose may be administered over a 1 cm area. 2 It can be applied topically to the skin.

[0117] The unit dose can be applied once, twice, three times, or more times per week. For example, the unit dose can be applied for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more weeks. A unit dose containing about 100,000 cells, about 300,000 cells, or about 500,000 cells is preferably applied in a volume of 1 cm. 2 It can be applied twice a week for eight weeks.

[0118] The unit dose may be contained in any suitable container. For example, the unit dose may be contained in a 1 ml vial. In such a case, for example, 0.1 ml of the vial is administered to a subject, preferably 1 cm 2 The unit dose may alternatively be contained in a syringe.

[0119] In the unit dosage of the present invention, cells can be in contact with the liquid carrier as defined herein.In this case, mesenchymal stem cells are separated from the carrier before administration.For example, before administration to subject, cells can be separated by centrifugation.Carrier can be any carrier as described herein, such as HypoThermosol™ or Hypothermosol™-FRS.

[0120] The methods of treatment and unit dosages of the present invention may involve the use of viable cells. How viability can be tested is described elsewhere herein.

[0121] The present invention is further illustrated by the following non-limiting experimental examples.

[0122] The sequences used herein are shown in Table 1 below.

[0123] Table 1: Sequences used herein TIFF0007804359000001.tif230170TIFF0007804359000002.tif233170TIFF0007804359000003.tif237170TIFF0007804359000004.tif47170 [Example]

[0124] Experimental Example 1. Cryopreservation of umbilical cord tissue prior to mesenchymal stem cell isolation Umbilical cord tissue (umbilical cords were donated with the mother's informed consent) was processed as follows for the subsequent isolation of mesenchymal stem cells from the amniotic membrane of the umbilical cord.

[0125] 1.1 Washing of umbilical cord tissue samples: a. Remove the scalpel from its protective cover. b. Hold the umbilical cord firmly with forceps and cut it into 10 cm pieces with a scalpel. Place the unused cord back into the original tissue cup. c. Transfer a 10 cm piece of umbilical cord to a new 150 mm culture dish. A 150 mm culture dish can also be used in place of the cup. d. Use the cover of a 150 mm culture dish as a resting place for forceps and a scalpel. e. Withdraw 25 ml of Plasmalyte A (Baxter, catalog # 2B2543Q) into a 30 ml syringe. Hold the syringe at a 45° angle with one hand and dispense the Plasmalyte A directly onto the umbilical cord tissue. f. While holding the culture dish at a slight angle, remove the Plasmalyte A with a 30 ml syringe and blunt needle. g. Collect the used Plasmalyte A in a 300 ml transfer bag that serves as a waste container and dispose of it in a biohazard trash can. h. Repeat the washing procedure as needed, using a new culture dish for each wash. Ensure all blood clots on the surface are removed. Additional Plasmalyte A can be used if tissue cleaning is required. i. Place the tissue in a new labeled tissue culture dish and continue cutting the tissue. To prevent the tissue from drying out during cutting, add 20 ml of Plasmalyte A to the dish. j. Cut the umbilical cord into equal, approximately 1-cm sections for a total of 10 sections. k. Each 1 cm section is further cut into smaller pieces measuring approximately 0.3 cm x 0.3 cm to 0.5 cm x 0.5 cm per section. l. Remove all Plasmalyte A from the dish. m. Withdraw 25 ml of Plasmalyte A from the original Plasmalyte A bag with a 30 ml syringe and dispense directly onto the umbilical cord tissue piece. n. Hold the culture dish at an angle to collect all of the Plasmalyte A used to wash the tissue to one side and remove it with a syringe and blunt needle. o. Repeat the wash once more. No blood clots should remain.

[0126] NOTE: If the umbilical cord is not frozen immediately, the umbilical cord tissue is maintained in Plasmalyte A until immediately prior to freezing.

[0127] 1.2 Cryopreservation of umbilical cord tissue: a. Prepare the cryopreservation solution: i. Prepare 50 ml of freezing solution consisting of 60% Plasmalyte A, 30% 5% human serum albumin, and 10% dimethyl sulfoxide (DMSO). ii. Label a 150 ml transfer bag "Tissue Freezing Solution" and attach the plasma transfer set to the port using aseptic technique. iii. Withdraw 30 ml of Plasmalyte A from the original Plasmalyte A bag with a 30 ml syringe and place into a transfer bag labeled "Tissue Freezing Solution" along with the date and time the solution was made. iv. With a 20 ml syringe, withdraw 15 ml of 5% human serum albumin and transfer it into a labeled transfer bag. v. Add 5 ml of DMSO to the transfer bag. vi. Mix thoroughly and record the mixture of the freezing solution. b. Remove Plasmalyte A from the tissue before adding the freezing solution. c. Using a 60 ml syringe, draw up the entire 50 ml of freezing solution into the syringe and add approximately 30 ml of freezing solution to the 150 mm cell culture dish containing the umbilical cord tissue. Attach a blunt needle onto the syringe to keep it sterile. d. Swirl the culture dish containing the tissue and freezing solution every minute for 10 minutes. e. Using forceps, select eight randomly selected sections and place them into each of four 4 ml cryovials. Select four randomly selected sections and place them into one 1.8 ml cryovial. These sections should be free of blood clots. f. Fill each cryovial containing umbilical cord tissue with the remaining freezing solution up to the 3.6 ml fill line for 4 ml tubes and up to the 1.8 ml line for 1.8 ml Nunc vials. g. Label one bottle of Bactec Lytic / 10 - Anaerobic / F and one bottle of Bactec Pluc Aerobic / F with your organization's ID. h. Remove 20 ml of frozen solution from the culture dish using a syringe and blunt needle, wipe the Bactec vial with an alcohol swab, then replace the blunt needle with an 18g needle and inoculate 10 ml each into an aerobic and anaerobic Bactec bottle. i. Start the controlled speed freezer. j. After the controlled rate freezer is completed, the units are placed in a liquid nitrogen freezer with continuous temperature monitoring until further use.

[0128] 2. Isolation of mesenchymal umbilical cord lining stem cells from umbilical cord tissue 2.1. Preparation of medium for processing MSCs from umbilical cord tissue: a. To make 500 ml of PTT6 (culture / growth medium), add the following in the order listed: i. 250 ml of DMEM ii. M171 118 ml iii. 118 ml of DMEM F12 iv. FBS 12.5 ml (final concentration 2.5%) v. EGF 1 ml (final concentration 10 ng / ml) vi. 0.175 ml of insulin (final concentration 5 μg / ml).

[0129] The above volumes of components i-vi result in a final culture volume of 499.675 ml. If no additional components are added to the culture, the remaining 0.325 ml (total volume of 500 ml) can be any of components i-iv, meaning, for example, DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of a stock solution of EGF or insulin can be adjusted to bring the total volume of the culture to 500 ml. Alternatively, a stock solution of antibiotics such as penicillin-streptomycin-amphotericin can be added to bring the final volume to 500 ml. One or more of the supplements adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) can be added to the culture in a volume of 0.325 ml to bring the total volume of the culture to 500 ml.

[0130] vii. Label the bottle "PTT6" with the date the medium was prepared, the operator's initials, and the phrase "Expire" followed by the expiration date, which is the earliest expiration date of any of the components or one month after the date of preparation, whichever comes first.

[0131] b. To make rinse medium (Hank's Buffered Salt Solution (HBSS) without calcium and magnesium and containing 5% FBS), add 2.5 ml of FBS to 47.5 ml of HBSS in a 50 ml centrifuge tube. Label the tube "Rinse Medium" with your initials and the date the medium is made. c. Test all media for sterility using Bactec Lytic / 10 - Anaerobic / F (Becton Dickinson & Company) and Bactec Plus + Aerobic / F (Becton Dickinson & Company). Add 20 ml of prepared media to each bottle.

[0132] 2.2 Thawing of umbilical cord tissue for MSC recovery: Thawing begins when the operator is ready to process the specimens in the clean room. Do not thaw more than one vial at a time unless the vials are from the same donor. b. Wipe down the water bath with disinfectant and then 70% isopropanol, then fill it with 1 L of sterile water. Heat the water bath to 36-38°C. c. Under a biosafety cabinet in a clean room, prepare 10 ml of rinse medium consisting of 70%–90% PlasmaLyte A. Sterile filter this solution with a 0.2-μm syringe filter attached to a 10 ml syringe and keep the solution refrigerated until use. d. Label the 50 ml conical tube with the treatment label. e. Ensure the water bath temperature is 36-38°C. f. Remove tissue vials from liquid nitrogen storage and rapidly thaw them in a 37°C water bath filled with 1 L of sterile water. The vial holder in the Mr. Frosty Nalgene Cryo 1°C freezing container holds the vial in place and allows it to float, serving as a floating rack for thawing samples. g. Remove the vials from the water bath and spray them with 70% isopropanol solution. A good time to remove the vials from the water bath is when you see small pieces of ice floating in the vial - indicating that the internal temperature of the vial is below 37°C. h. Place the vial in the pass-through and notify the cleanroom processing technician.

[0133] 2.3 Preparation for tissue processing: Umbilical cord tissue processing must be performed in an Environmentally Monitored (EM) clean room. Complete cleaning of the room and hood at the end of each shift. b. Prepare / clean biosafety cabinet. c. Conduct bioparticle counts while working in the biosafety cabinet. d. Assemble all necessary items into the biosafety cabinet, checking each for damaged packaging and expiration dates. When handling syringes, serological pipettes, sterile forceps, scalpels, tissue plates, and needles, never touch any surfaces that will come into contact with the sterile product. Only the syringe barrel, tubing, plunger tip, and / or the exterior of the needle cap or casing may be safely handled. Discard the item if it touches any surface or if the surface comes into contact with a non-sterile surface. e. Record the lot numbers and expiration dates (if applicable) of all reagents and supplies used. f. Receive thawed vials by cleaning them with lint-free wipes moistened with 70% alcohol and then moving them into the biosafety cabinet. g. Using the aspirating needle attached to the syringe, withdraw as much liquid as possible from the vial, being careful not to aspirate any tissue. h. Using sterile forceps, transfer the tissue to a sterile 100 mm Petri dish. i. Add a 5 ml aliquot of rinse medium to the tissue fragments. j. Swirl the contents for 15-30 seconds, then remove the rinse medium with a pipette or syringe with an aspirator. Repeat this rinse process twice. k. Add 2 mL of rinsing medium to the tissue to prevent it from drying out.

[0134] 2.4. Initiating MSC expansion from tissue: Label the bottom of a 6-well plate "Expansion 1" along with the MSC lot number or umbilical cord tissue ID and the date expansion will begin. If using a 60 mm tissue culture dish, draw a grid on the bottom of the dish to divide the plate into four sections. b. Using sterile disposable forceps, place one piece of tissue measuring 3x3 mm to 5x5 mm into each well. If using a 60 mm tissue culture dish, place the tissue in the center of each section and keep the tissues separated (more than 1 cm from each other). c. Fill each well with 3 ml of PTT6. d. Using an aspirating needle attached to a 30 ml syringe, withdraw enough medium to just barely cover the tissue. Do not tilt the plate. Do not touch the bottom of the well with the aspirating needle. e. Cell growth is monitored daily (24 ± 6 hours) using an inverted optical microscope. A real-time cell culture imaging system may be used instead of an optical microscope. f. Change the medium daily, always equilibrate the medium to room temperature before use. i. Aspirate the medium. ii. Add 3 ml of PTT6. iii. Apply aspirate until the tissue is just barely submerged in the medium. g. Once cell growth is observed from the tissue, transfer the tissue to a new 6-well plate using the same procedure as in 4.a-4.e above, except label the plate "Growth 2." Maintain cell growth in the "Growth 1" plate by adding 2 ml of PTT6 to each well. Monitor daily for confluency. Replace the medium every 2-3 days (be sure to equilibrate the medium to room temperature before use). h. Once cell growth is observed in the "Growth 2" plate, repeat steps 4.a-4.e except label the plate as "Growth 3." Maintain cell growth in the "Growth 2" plate by adding 2 ml of PTT6 to each well. Observe daily for confluency. Replace medium every 2-3 days (be sure to equilibrate medium to room temperature before use). i. Once growth is observed in the "Growth 3" plate, discard the tissue. If the tissue is very small and does not interfere with cell growth, discard the tissue during subculture. j. Once the cells reach 40-50% confluence, observe the cells daily to prevent over-expansion. k. Subculture cells when they reach 70-80% confluence. Do not allow cells to grow beyond 80% confluence.

[0135] When tissue explants are approximately 1-3 mm in size and tissue explant / cell culture is performed in a 175 mm square culture dish, the average number of mesenchymal stem cells recovered from the explants is typically about 4,000-6,000 cells / explant. Thus, if mesenchymal stem cells are simultaneously grown from 48 explants, approximately 300,000 cells can be obtained at harvest. These 300,000 mesenchymal stem cells harvested from the explants are then cultured in a 175 cm square culture dish, as described in Example 2.5 below. 2 By seeding a cell culture flask with 300,000 such cells, they can be used for subculture (which may be referred to as passage 1). The mesenchymal stem cells obtained from this passage 1 can then be used to culture again at 175 cm as described in Example 2.5 below. 2 The cells can be expanded by seeding into flasks (passage 2). Cells obtained from both passage 1 and passage 2 can be "banked" by cryopreservation, with the mesenchymal stem cells obtained after passage 2 being considered to represent a master cell bank, for further expansion of mesenchymal stem cells, for example in bioreactors, as described in Example 2.7 below.

[0136] 2.5. Subculture of MSCs in cell culture dishes a. Perform bioparticle counts while working in the biosafety cabinet. Equilibrate all media to room temperature before use. b. When cell growth reaches approximately 70-80% confluency, subculture the cells. i. Remove the PTT6 from the Petri dish. ii. Rinse with calcium- and magnesium-free HBSS. iii. Add 0.2 ml of 1x TrypLE-EDTA and swirl for 1-2 minutes. iv. Tilt the dish 30-45° to allow the cells to move downward by gravity flow. Gently tap the side of the plate to facilitate detachment. v. Add 1 ml of PTT6. Gently pipette up and down, then transfer the cells to a 15 ml centrifuge tube. Use a clean pipette tip for each well. Pool the cells from all 6 wells into a single 15 ml tube. vi. Centrifuge at 1200 rpm for 10 minutes. vii. Remove the supernatant and resuspend the cells in 5 ml of PTT6. c. Subculture the MSCs. i. 50 μl of cell suspension is aliquoted and assayed for TNC and viability by trypan blue exclusion assay. ii. Count the cells using a hemocytometer. Expect to count 20-100 cells / section. If the number is greater than 100, dilute the original sample 1:5 and repeat the trypan blue method using a hemocytometer. iii. Count viable cells / ml and total viable cells: 1. Viable cells / ml = number of viable cells x dilution factor x 10 4 2. Total viable cells = number of viable cells x dilution factor x total volume x 10 4 iv. Count % Viability: 1. % Viability = Number of viable cells x 100 / (Number of viable cells + Number of dead cells) v. Cell suspension 1.0 × 10 6 Dilute to cells / ml: 1. "X" volume = total viable cells / 10 6 cells / ml 2. For example, if the total viable cell count is 1.0 x 10 7 If it is an individual; 3. "X" = 10 7 / 10 6 cells / ml, ie 10 ml, so add 5 ml to the cell suspension (which is 5 ml) to bring the total cell volume to 10 ml. vi. The cell suspension is 10 6 If the number of cells / ml is less than 150 mm, use 175 cm 2 2 x 10 cells in a flask 6Determine the volume required to sow the seeds. 1. Cells 2×10 6 Volume per cell = 2 x 10 cells 6 cells ÷ viable cells / ml 2. For example, 8 x 10 viable cells / ml 5 If cells / ml, 2 x 10 cells 6 pieces ÷ 8×10 5 cells / ml, ie 2.5 ml is required. vii. Set aside 0.5 ml for MSC marker analysis. viii. 2×10 cells 6 6 30 ml of PTT in each 150 mm Petri dish or 175 cm 2 Seed the flasks. ix. Observe cells every 3 days for adhesion, colony formation, and confluence. Once cells reach 40-50% confluence, observe cells daily to every 2 days to prevent overgrowth. Do not allow cells to grow beyond 80% confluence. A real-time cell culture monitoring system can be used instead of light microscopy. x. Replace the medium every 2-3 days.

[0137] 2.6 Cryopreservation of MSC cells a. Conduct bioparticle counts while working in the biosafety cabinet. b. When cells reach 70-80% confluence, plate each 150 mm Petri dish or 175 cm 2 Detach the cells with 2 ml of 1×TrypLE-EDTA per flask. i. Remove the PTT6 from the Petri dish. ii. Wash with 5 ml of calcium- and magnesium-free HBSS or PBS. iii. Add 2 ml of 1x TrypLE-EDTA and swirl for 1-2 minutes. iv. Tilt the dish 30-45° to allow the cells to move downwards by gravity flow. Gently tap the side of the Petri dish to help promote detachment. v. Add 10 ml of PTT6 to inactivate TrypLE. Mix thoroughly to dissociate cell clumps. vi. Using a Pasteur pipette, transfer the cells to a 15 ml centrifuge tube. vii. Centrifuge at 1200 rpm for 10 minutes. viii. Aspirate the medium and resuspend in 10 ml of PTT6. ix. Take a 50 μl aliquot and determine total viable cell count and % viability as above. Cell counts should be performed within 15 minutes as cells may begin to clump. c. Prepare cells for cryopreservation. i. Prepare cell suspension medium and cryopreservation medium and freeze cells.

[0138] 2.7. Subculture (expansion) of MSCs in Quantum bioreactors (Terumo BTC, Inc.) MSCs can also be expanded using the Quantum bioreactor. The starting cell number for expansion in the Quantum bioreactor should be 20-30 million cells per run. Typical yields per run are 300-700 million MSCs at harvest. The bioreactor is operated according to the manufacturer's protocol. The mesenchymal stem cells so obtained are typically cryopreserved (see below) and become a working cell bank.

[0139] Materials / Reagents: 1. Quantum Augmentation Set 2. Quantum Waste Bag 3. Quantum Media Bags 4. Quantum Inlet Bag 5. PTT6 6. PBS 7. Fibronectin 8. TrypLE 9. 3 ml syringe 10. Glucose test strips 11. Lactic Acid Test Strips 12. 60 ml cell culture plate or equivalent 13. Medical Grade 5% CO2 Gas Mixture 14. 50 ml Combi Tip

[0140] Device: 1. Biosafety Cabinet 2. Glucose meter (Bayer Healthcare / Ascensia Contour blood glucose meter) 3. Lactate Plus (Nova Biomedical) 4. Peristaltic pump with head 5. Centrifuge, Eppendorf 5810 6. Sterile tubing connectors 7. M4 repeating pipettor 8. RF Sealer

[0141] procedure: 1. Quantum Bioreactor Preparation a) Quantum Bioreactor Preparation b) Bioreactor Coating: 1) Prepare the fibronectin solution in a biosafety cabinet. 1) Allow the lyophilized fibronectin to acclimate to room temperature (≥15 minutes at room temperature). 2) Add 5 ml of sterile distilled water; do not swirl or stir. 3) Allow the fibronectin to come into solution for 30 minutes. 4) Using a 10 ml syringe fitted with an 18 g needle, transfer the fibronectin solution into the cell inlet bag containing 95 ml of PBS. 2) Connect the bag to the "reagent" line. 3) Check for air bubbles (air bubbles can be removed by using "IC Air Removal" or "EC Air Removal" and by using "Clean" as the inlet source). 4) Open or set up the program for coating the bioreactor (Figure 1, steps 3-5). 5) Run the program. 6) While the program is running to coat the bioreactor, prepare a media bag with 4 L of PTT6 media. 7) Connect the media bag to the IC media line using a sterile tubing connector. 8) Once the bioreactor coating step is complete, use an RF sealer to remove the cell inlet bag used for the fibronectin solution. c) Washing to remove excess fibronectin d) Conditioning the bioreactor with culture medium 2. Cultivating cells in the Quantum bioreactor a) Cell loading and adhesion using a homogenous suspension: b) Cell feeding and cultivation 1) Select the medium flow rate to nutrient the cells. 2) Daily sampling for lactate and glucose. 3) Adjust the medium flow rate as lactate levels increase. The actual maximum tolerable lactate concentration will be determined by the flask culture from which the cells were derived. Ensure there is enough PTT6 medium in the medium bag. Replace the PTT6 medium bag with a fresh PTT6 medium bag, if necessary. 4) Once the flow rate reaches the desired value, measure the lactate level every 8-12 hours. If the lactate level does not decrease or continues to increase, harvest the cells. 3. Harvesting Cells from the Quantum Bioreactor a) Once the lactate concentration has not decreased, cells are harvested after a final sampling for lactate and glucose. b) Cell harvesting: 1) Using a sterile tubing connector, connect the cell inlet bag filled with 100 ml of TrypLE to the "reagent" line. 2) Make sure there is enough PBS in the PBS bag. If not, use a sterile tubing connector to connect a new bag containing at least 1.7 liters of PBS to the "Wash" line. 3) Implement a take-back program. 4. Cell Cryopreservation 1) Once the cells have been harvested, transfer the cells to a 50 ml centrifuge tube and pellet the cells. 2) Resuspend in 25 ml of cold cell suspension solution. Count cells using a Sysmex or Biorad cell counter. Attach the cell count report to each Quantum processing batch record. 3) Increase the cell concentration to 2 x 10 7 Adjust to pieces / ml. 4) Add an equal volume of cryopreservation solution and mix thoroughly (do not shake or vortex). 5) Using a repeating pipettor, add 1 ml of the cell suspension in cryopreservative to each 1.8 ml vial. Freeze and store in a controlled rate freezer using the CRF program as described in SOP D6.100 CB Cryopreservation. 6) Store the vials in the designated liquid nitrogen storage space. 7) Attach the CRF execution report to each MSC P3-Quantum processing batch record form.

[0142] 3. Analysis of stem cell marker expression in mesenchymal umbilical cord lining stem cell populations isolated from umbilical cord tissue using different culture media Flow cytometry experiments were performed to analyze mesenchymal stem cells isolated from umbilical cords for expression of mesenchymal stem cell markers CD73, CD90, and CD105.

[0143] For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the tissue in three different culture media, as described in Example 2, and then subcultured in each medium.

[0144] In these experiments, three culture media were used: a) 90% (v / v) DMEM supplemented with 10% (v / v) FBS; b) PTT-4 culture medium, described in U.S. Patent Application Publication No. US2008 / 0248005 and corresponding International Patent Application No. WO2007 / 046775 (see paragraph

[0183] of WO2007 / 046775), consisting of 90% (v / v) CMRL1066 and 10% (v / v) FBS; and c) PTT-6 culture medium of the present invention, the composition of which is described herein. In this flow cytometry analysis, two different samples of umbilical cord lining mesenchymal stem cell (CLMC) populations were analyzed for each of the three culture media used.

[0145] The following protocol was used for flow cytometry analysis.

[0146] material and method TIFF0007804359000005.tif195152

[0147] procedure a) Isolation and culture of cells from umbilical cord lining membrane 1. As described in Example 2, explant tissue samples were incubated in cell culture plates, immersed in the respective media, and then maintained in a CO2 incubator at 37°C. 2. The medium was changed every 3 days. 3. Cell growth from tissue culture explants was monitored under a light microscope. 4. At approximately 70% confluence, cells were detached from the dish by trypsinization (0.0125% trypsin / 0.05% EDTA) and used for flow cytometry experiments. b) Trypsinization of experimental cells 1. Remove the medium from the cell culture plate. 2. Remove traces of FBS by gently rinsing with sterile 1x PBS, as FBS will interfere with the enzymatic action of trypsin. 3. Add 1X trypsin to the cell culture plate and incubate at 37°C for 3-5 minutes. 4. Observe the cells under a microscope to ensure they are removed. Neutralize the trypsin by adding complete medium containing FBS (DMEM with 10% FBS). 5. Using a pipette, break up cell clumps by pipetting the cells against the wall of the plate in the medium. Collect the cell suspension and transfer to a 50 ml centrifuge tube. 6. Add sterile 1x PBS to the plate to rinse it and collect the cell suspension in the same centrifuge tube. 7. Centrifuge this at 1800 rpm for 10 minutes. 8. Discard the supernatant and resuspend the cell pellet in PBA medium. c) Cell counting 1. Ensure that the hemocytometer and its cover slip are clean and dry, preferably by washing them with 70% ethanol, allowing them to dry, and then wiping them with a Kimwipe (lint-free paper). 2. Aliquot a small amount of cells in suspension into a microcentrifuge tube and remove from the BSC hood. 3. Stain the cells in suspension with an equal volume of trypan blue, for example, add 500 μl of trypan blue to 500 μl of suspension (dilution factor = 2X, resulting in a 0.2% trypan blue solution). 4. Do not expose cells to trypan blue for more than 30 minutes, as trypan blue is toxic and can lead to an increase in non-viable cells, resulting in false cell counts. 5. Add 20 μl of the cell suspension mixture to each chamber of the hemocytometer and view under a light microscope. a. Count the number of viable cells (bright cells; nonviable cells readily take up trypan blue and are therefore dark) in each section of the hemocytometer for a total of eight sections in the upper and lower chambers. The total cell count is calculated as (average cell count / section) x 10 4 Given as cells / ml. d) Cell staining i. Preparation before staining cells Aliquot the cell suspensions, each containing 50,000 cells, in duplicate into three tubes (CD73, CD90, CD105) and two tubes (negative control). ii. Primary antibody (Ab) staining Add 1 μl of primary antibody [0.5 mg / ml Ab] to 100 μl of cell suspension and incubate at 4°C for 45 minutes. Adjust to 1 ml with PBA. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. Add 1 ml of PBA and resuspend the pellet. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. Resuspend in 100 ul of PBA. iii. Secondary Ab staining In the dark Add 1 ul of secondary antibody [0.5 mg / ml ab] to 100 ul of cell suspension and incubate at 4°C for 30 minutes. Adjust to 1 ml with PBA. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. Add 1 ml of PBA and resuspend the pellet. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. For flow cytometry analysis, resuspend in 200-300 ul of PBA. Transfer cells to FACS tubes for reading on a BD FACS CANDO flow cytometer.

[0148] The results of flow cytometry analysis are shown in Figures 6a-6c. Figure 6a shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and culture in DMEM / 10% FBS. Figure 6b shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and culture in PTT-4. Figure 6c shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and culture in PTT6. As can be seen from Figure 6a, the population isolated using DMEM / 10% FBS as the medium culture had approximately 75% CD73+ cells, 78% CD90+ cells, and 80% CD105+ cells (average values ​​for two experiments), whereas after isolating and culturing umbilical cord tissue using PTT-4 medium (see Figure 6b), the number of mesenchymal stem cells that were CD73-positive, CD90-positive, and CD105-positive was approximately 87% (CD73+ cells), 93% (CD90+ cells), and 86% (CD105+ cells), average values ​​for two experiments. The purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention was at least 99.0% for all three markers (CD73, CD90, CD105), which means that the purity of this cell population was significantly higher than that obtained by culturing using PTT-4 medium or DMEM / 10% FBS. In addition, and even more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is essentially a 100% pure and distinct stem cell population. This makes the stem cell population of the present invention an ideal candidate for stem cell-based therapy. Therefore, this population of mesenchymal umbilical cord lining stem cells may become the gold standard for such stem cell-based therapeutic approaches.

[0149] The findings shown in Figure 6 are further supported by the results of flow cytometry analysis shown in Figures 7a and 7b. Figure 7a shows the percentage of isolated mesenchymal umbilical cord lining stem cells (mesenchymal stem cells of the amniotic membrane of the umbilical cord) that express stem cell markers CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR, after isolation from umbilical cord tissue and culture in PTT-6 medium. As shown in Figure 7a, the mesenchymal stem cell population contained 97.5% viable cells, 100% of which expressed each of CD73, CD90, and CD105 (see columns "CD73+CD90+" and "CD73+CD105+"), whereas 99.2% of the stem cell population did not express CD45, and 100% of the stem cell population did not express CD34 and HLA-DR (see columns "CD34-CD45-" and "CD34-HLA-DR-"). Thus, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is essentially a 100% pure and well-defined stem cell population that meets the criteria for realizing the use of mesenchymal stem cells in cell therapy (95% or more of the stem cell population express CD73, CD90, and CD105, while 98% or more of the stem cell population lacks expression of CD34, CD45, and HLA-DR; see Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," supra). It is noted herein that the mesenchymal stem cells of the amniotic membrane of the present invention adhere to plastic under standard culture conditions and differentiate in vitro into osteoblasts, adipocytes, and chondroblasts (see U.S. Patent No. 9,085,755, U.S. Patent No. 8,287,854, or WO2007 / 046775), thus meeting the generally accepted criteria for the use of mesenchymal stem cells in cell therapy.

[0150] Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90, and CD105, and lack the expression of CD34, CD45, and HLA-DR. As shown in Figure 7b, the bone marrow mesenchymal stem cell population contains 94.3% viable cells, of which 100% express CD73, CD90, and CD105 (see the "CD73+CD90+" and "CD73+CD105+" columns), while only 62.8% of the bone marrow stem cell population lacks the expression of CD45, and 99.9% of the stem cell population lacks the expression of CD34 and HLA-DR (see the "CD34-CD45-" and "CD34-HLA-DR-" columns). Thus, bone marrow mesenchymal stem cells, which are considered to be the gold standard of mesenchymal stem cells, are much less homogeneous / purified with respect to stem cell markers than the mesenchymal stem cell population (amniotic membrane of umbilical cord) of the present application. This finding also indicates that the stem cell population of the present invention may be an ideal candidate for stem cell-based therapy and may represent the gold standard for stem cell-based therapeutic approaches.

[0151] Experiments demonstrating that the mesenchymal stem cell populations of the present invention can be transported / stored in HypoThermosol™: To analyze the health and viability of mesenchymal stem cells described herein in different storage or transport carriers, two different carriers were compared to each other. Namely, the carrier HypoThermosol™-FRS was compared to the carrier PlasmaLyte-A. Both are commercially available. The product sheet for HypoThermosol™-FRS is shown in FIG. 30, and its composition is described elsewhere herein. Each 100 mL of PlasmaLyte contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H 11 NaO7); 368 mg sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg potassium chloride, USP (KCl); and 30 mg magnesium chloride, USP (MgCl2·6H2O). PlasmaLyte does not contain antimicrobial agents. The pH of PlasmaLyte is adjusted to 7.4 (6.5-8.0) with sodium hydroxide.

[0152] The experimental setup for comparison is shown in Figure 8. Initially, mesenchymal stem cell populations described herein were expanded in cell culture flasks. The number of viable mesenchymal stem cells was counted, and then 2 million cells / vial were stored for various periods in either PlasmaLyte-A or HypoThermosol™-FRS. After storage, cells were counted daily in ≤50 μl samples (total liquid volume removed: 250 μl) on days 1–5, and viability was assessed by staining the cells with trypan blue. Additionally, ≤80 μl samples were taken and analyzed on days 1, 3, and 5. Additionally, supernatants were obtained and frozen. PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-1, HGF, and TGFβ1 were then measured using a FLEXMAP 3D system.

[0153] Figure 9 summarizes the viability data. As can be seen from the graph on the left, after 7 days of storage in HypoThermosol™, 73% of the total cells (approximately 95%) at the start of storage were still viable. In contrast, after 7 days of storage in PlasmaLyte-A, only 42% of the total cells (approximately 94%) at the start of storage were still viable. All counts were based on duplicate readings within 10% of each other (according to SOP CR D2.600.1). During counting, cells stored in HypoThermosol™ were significantly smaller, with smooth and well-defined contours. In contrast, cells in Plasmalyte-A appeared at a range of sizes. HypoThermosol™ significantly supported membrane integrity and presumably viability over a 1-week period (6 days). Similar results are also shown in the graph on the right.

[0154] Figure 10 shows the results obtained when measuring the cell diameter of the cells. The mesenchymal stem cell populations described herein when maintained in HypoThermosol™ have a narrower diameter range compared to cells maintained in PlasmaLyte A. The comparison was made after 3 days of storage.

[0155] 11 shows the TGFβ1 concentration in supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A after 48 hours of storage. As can be seen from the graph on the right, cells secrete approximately the same amount of TGFβ1 when stored in HypoThermosol™ as when stored in PlasmaLyte-A. In general, the amount of TGFβ1 secreted decreased over time (graph on the right).

[0156] Figures 12 and 13 show control experiments in which PDGF-BB and IL-10 concentrations were measured in 48-hour supernatants from mesenchymal stem cell populations described herein that were stored in HypoThermosol™ or PlasmaLyte-A. Because neither PDGF-BB nor IL-10 are normally secreted by the mesenchymal stem cell populations described herein, neither PDGF-BB nor IL-10 was detectable in any of the samples.

[0157] Figure 14 shows VEGF concentrations in supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A at 48 hours. As can be seen from the graph on the right, on day 0, cells secrete approximately the same amount of VEGF when stored in HypoThermosol™ or PlasmaLyte-A. On days 1 and 5, cells secreted more VEGF when stored in PlasmaLyte-A. Notably, when stored for 3 days, cells secreted more VEGF when stored in HypoThermosol™ than when stored in PlasmaLyte-A. Thus, after 3 days of storage, HypoThermosol™ outperforms PlasmaLyte-A. The more VEGF detected, the healthier the culture. Thus, cells are healthier in HypoThermosol™ than in PlasmaLyte-A by secreting more VEGF after 3 days of storage in HypoThermosol™ than when stored in PlasmaLyte-A. From 5 days of storage onwards, PlasmaLyte appears to be a more advantageous carrier, as at 5 days, cells stored in PlasmaLyte-A secreted more VEGF. In general, the amount of VEGF secreted decreased over time (graph on the right).

[0158] Figure 15 shows the PDGF-AA concentration in supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A at 48 hours. As can be seen from the graph on the right, on day 0, cells secrete approximately the same amount of PDGF-AA when stored in HypoThermosol™ compared to when stored in PlasmaLyte-A. On days 1 and 5, cells secreted more PDGF-AA when stored in PlasmaLyte-A. Notably, when stored for 3 days, cells secreted more PDGF-AA when stored in HypoThermosol™ than when stored in PlasmaLyte-A. Thus, after 3 days of storage, cells stored in HypoThermosol™ are healthier than cells stored in PlasmaLyte-A. After 5 days of storage, PlasmaLyte appears to be a more advantageous carrier, as cells stored in PlasmaLyte-A secreted more PDGF-AA at day 5. In general, the amount of PDGF-AA secreted decreased over time (graph on the right).

[0159] Figure 16 shows Ang-1 concentrations in supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A over a 48-hour period. As can be seen from the graph on the right, cells secrete approximately the same amount of Ang-1 when stored in HypoThermosol™ or PlasmaLyte-A on days 0 and 3. On day 5, cells secreted more Ang-1 when stored in PlasmaLyte-A. Notably, after 1 day of storage, cells secreted significantly more Ang-1 when stored in HypoThermosol™ than when stored in PlasmaLyte-A. Thus, cells stored in HypoThermosol™ appear to be healthier than cells stored in PlasmaLyte-A for at least 48 hours, up to 3 days of storage. After 5 days of storage, PlasmaLyte appears to be a more advantageous carrier, as at this point cells stored in PlasmaLyte-A secreted more Ang-1. In general, the amount of Ang-1 secreted decreased over time (graph on the right).

[0160] Figure 17 shows the HGF concentration in supernatants from mesenchymal stem cell populations described herein stored in HypoThermosol™ or PlasmaLyte-A after 48 hours of storage. As can be seen from the graph on the right, on day 0, cells secrete approximately the same amount of HGF when stored in HypoThermosol™ compared to when stored in PlasmaLyte-A. On days 3 and 5, cells secreted more HGF when stored in PlasmaLyte-A. Notably, after 1 day of storage, cells secreted significantly more HGF when stored in HypoThermosol™ than when stored in PlasmaLyte-A. Thus, cells stored in HypoThermosol™ appear to be healthier than cells stored in PlasmaLyte-A for at least 1 day (48 hours) up to 3 days of storage. After day 3, PlasmaLyte-A appears to be a more advantageous carrier, as cells stored in PlasmaLyte-A secreted more HGF at days 3 and 5. In general, the amount of secreted HGF decreased over time (graph on the right).

[0161] Summarizing the above data, it can be concluded that storage of the mesenchymal stem cell population of the present invention in HypoThermosol™ is superior to storage in PlasmaLyte-A, particularly for the first 3 days of storage.

[0162] Experiment demonstrating that the mesenchymal stem cell population of the present invention has wound healing properties by local treatment in pigs: A preclinical study was also conducted using 10-week-old female Yorkshire Landrace pigs (50 kg). Treatments were performed at the SingHealth Experimental Medicine Centre in Singapore. The pigs were made diabetic with 120 mg / kg streptozotocin and allowed to recover for 45 days before six 5 cm x 5 cm full-thickness wounds were made on their backs (see Figure 18). The pigs (n = 2) were placed in a 1 cm 2 10 per 5Two control pigs were treated with the human mesenchymal stem cell population described herein twice weekly for four weeks. Two control pigs were treated with PBS. Wounds were photographed on post-operative day 0 (PO day 0) and every seven days until post-operative day 35. Wounds were analyzed for surface area size using ImageJ. By day 35, the addition of the mesenchymal stem cell population described herein had resulted in closure of 10 of 12 diabetic wounds (83%), compared to only 3 of 12 (25%) PBS-treated control wounds. The rate of wound healing was 0.6 cm in control animals. 2 / day compared to 0.8 cm using the mesenchymal stem cell populations described herein 2 / day, a 33% improvement. The results of this study are summarized in Figure 18.

[0163] Although the pig model is not naturally occurring, its skin structure most closely resembles that of humans. This data suggests that the umbilical cord lining mesenchymal stem cell population of the present invention improves wound healing without the risk of serious adverse side effects. Therefore, these data strongly support the hypothesis that the human umbilical cord lining mesenchymal stem cell population described herein can promote the healing of chronic wounds by suppressing inflammation and promoting angiogenesis. Furthermore, whether xenogeneic mesenchymal stem cells are used in mice or pigs, there is clearly no sign of inflammation, and therefore it is highly unlikely that allogeneic mesenchymal stem cells will cause any serious adverse effects in humans.

[0164] Experiments demonstrating that the mesenchymal stem cells described herein are effective in localized treatment in humans: Experiments demonstrating that the mesenchymal stem cells described herein are effective in localized treatment in humans are described in WO 2007 / 046775. In particular, as described in Examples 23-26 of WO 2007 / 046775, umbilical cord amniotic membrane mesenchymal stem cells (UCMC) were able to alleviate full-thickness burns (Example 23), partial-thickness wounds (Example 24), non-healing radiation wounds (Example 25), and non-healing diabetic wounds and non-healing diabetic foot wounds (Example 26). Notably, according to Example 2 of WO 2007 / 046775, mesenchymal stem cells were resuspended in PTT-4 medium.

[0165] Notably, as shown in Figures 6b and 6c, the stem cell population obtained by culturing using the PTT6 (used herein) medium is significantly more homogeneous than the population of cells obtained by using the PTT4 medium (used in WO 2007 / 046775). Since PTT-4 was used as the medium for mesenchymal stem cells in Examples 23-26 of WO 2007 / 046775, it is clear that the even more homogeneous mesenchymal stem cell population isolated after culturing in PTT-6 (used herein) has similar beneficial effects in wound healing applications such as full-thickness burn wounds, partial-thickness wounds, non-healing radiation wounds, and non-healing diabetic wounds and non-healing diabetic foot wounds.

[0166] Experiments demonstrating that the mesenchymal stem cells described herein are effective in localized treatment in humans: This is a planned dose-escalation study of a mesenchymal stem cell population obtained as described herein, conducted at the University of Colorado Anschutz Medical Campus in Aurora, Colorado. The purpose of this study is to determine a safe dose of the mesenchymal stem cell population (human umbilical cord lining mesenchymal stem cells) described herein. This is a single-center, dose-escalation study, enrolling five subjects at each of three dose levels for a total of 15 subjects. The first group of five patients will receive 100,000 MSCs / cm. 2(skin / wound area) twice a week for 8 weeks. A second group of 5 patients received 300,000 MSCs / cm 2 The third group of five patients received 500,000 MSCs / cm twice a week for eight weeks. 2 Patients will receive 100 mg of the mesenchymal stem cell population derived herein twice weekly for 8 weeks. This schedule will continue until the highest dose is reached, or until at least two subjects at a dose level experience a Grade 2 or higher allergic reaction suspected to be related to the mesenchymal stem cell population derived herein, or until two or more subjects at a dose level experience an unexpected, treatment-related serious adverse event or dose-limiting toxicity within 14 days after the first dose of the mesenchymal stem cell population derived herein. All patients will be evaluated for anti-HLA antibody development and wound closure 30 days after treatment. While HLA antibody development is not currently considered an absolute contraindication to a particular dose, it will be a factor in the overall safety assessment. This is an open-label study; all subjects will receive the study drug, and all study personnel will know the dose each subject receives. The secondary endpoint of this study is a significant improvement in wound condition. The endpoints are based on the rate of wound closure, the percentage of wound area successfully closed, and the percentage of wounds completely closed, as measured using the Silhouette Wound Measurement and Documentation System, a device approved by the FDA for this purpose.

[0167] Subject population Patients with type I or type II diabetes who have chronic foot ulcers that have not healed after at least 30 days of conventional treatment and who are negative for HLA antibodies against the mesenchymal stem cell population described herein are enrolled. Patients continue conventional wound healing treatment for the first two weeks, starting at the time of enrollment, and at that time have already been screened for diabetic foot ulcers that have not healed for 30 days. At this time, photographic recording and measurement of wound parameters are initiated. For the first two weeks, conventional dressing changes are performed twice a week, and then the mesenchymal stem cell population described herein is applied to the wound at the specified concentration twice a week. Wounds treated with the mesenchymal stem cell population described herein are also covered with Tegaderm® and crepe dressing.

[0168] Dose Levels The purpose of this study is to determine a safe dose of the human umbilical cord lining mesenchymal stem cells described herein for further testing. Patients were treated with three doses: 100,000 cells / cm 2 skin / wound area, 300,000 cells / cm 2 , or 500,000 cells / cm 2 Patients are treated twice weekly for 8 weeks with one of the following: Each 100,000 cell dose represents 0.1 ml of the mesenchymal stem cell population described herein from a vial containing 1 million cells / ml in HypoThermosol.

[0169] Dosage regimen This is a safety and tolerability study of increasing doses of the mesenchymal stem cells described herein. The purpose of this study is to determine a safe dose of the human umbilical cord lining mesenchymal stem cells described herein for further testing. Five subjects will be enrolled at each of three dose levels. The first group of five patients will receive 100,000 MSCs / cm. 2 The second group of five patients will receive 300,000 MSCs / cm2 of MSCs per skin / wound site twice a week for eight weeks. 2 The third group of five patients received 500,000 MSCs / cm twice a week for eight weeks.2 Patients will receive 100 mg of the mesenchymal stem cell population twice weekly for 8 weeks. This schedule will continue until the maximum dose is reached, or until at least two subjects at a dose level experience a Grade 2 or greater allergic reaction suspected to be related to the mesenchymal stem cells described herein, or until two or more subjects at a dose level experience an unexpected, treatment-related serious adverse event or dose-limiting toxicity within 30 days after the first dose of the mesenchymal stem cell population described herein. All patients will be evaluated for anti-HLA antibody production and the extent of wound closure 30 days after treatment. At this time, HLA antibody production is not considered an absolute contraindication to a particular dose, but it will be a factor in the overall safety assessment. This is an open-label study; all subjects will receive the study drug, and all study personnel will know the dose each subject will receive.

[0170] Administration route The mesenchymal stem cell populations described herein are applied topically to debrided diabetic foot ulcers and held in place by a Tegaderm® dressing.

[0171] Administration Procedure After appropriate debridement, if necessary, the patient is placed in a prone position with the affected leg bent at a 90° angle. The vial of mesenchymal stem cell population described herein is gently swirled to ensure even distribution of the cells. A sterile syringe is then used to gently inject 1 cm of the vial. 2 The elevated paw is treated by removing 100,000 (0.1 ml) to 500,000 (0.5 ml) cells per wound and placing them in the center of the wound. The wound is then sealed with a Tegaderm® membrane and gently massaged to evenly distribute the cells. The paw is kept elevated for 5 minutes to allow the cells to settle and adhere. A crepe bandage is then placed over the paw, covering the Tegaderm® dressing.

[0172] The present invention is further characterized by the following items. 1. i) Trolox, ii) Na + , iii) K + , iv) Cl - , v) H2PO4 - , vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione 10. A method of transporting a stem cell population, comprising transporting the stem cell population in contact with a liquid carrier comprising: 2. The method of item 1, in which the transportation takes place over a period of about 7 days or less. 3. The method of item 1 or 2, wherein the transportation occurs over about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or less than about 1 day. 4. The method of any one of the preceding items, wherein the transport occurs over a period of about 48 hours or about 24 hours or less. 5. The method of any one of the preceding items, wherein the transport is carried out at a temperature of about -5°C to about 15°C. 6. The method of any one of the preceding items, wherein the transport is carried out at a temperature of about 2°C to about 8°C. 7. The method of any one of the preceding items, wherein the transport is carried out at a temperature greater than about -5°C, greater than about -10°C, greater than about -15°C, or greater than about -20°C. 8. The method of any one of the preceding items, wherein the stem cell population is delivered at a concentration of about 70 million cells per ml of carrier, about 60 million cells per ml of carrier, about 50 million cells per ml of carrier, about 40 million cells per ml of carrier, about 30 million cells per ml of carrier, about 20 million cells per ml of carrier, about 10 million cells per ml of carrier, about 5 million cells per ml of carrier, about 4 million cells per ml of carrier, about 3 million cells per ml of carrier, about 2 million cells per ml of carrier, about 1 million cells per ml of carrier, about 500,000 cells per ml of carrier, about 100,000 cells per ml of carrier, or less than 100,000 cells per ml of carrier. 9. The method of item 8, wherein the stem cell population is delivered at a concentration of about 10 million cells per ml of carrier to about 1 million cells per ml of carrier. 10. The method of any one of the preceding items, wherein the stem cell population is an embryonic stem cell population, an adult stem cell population, a mesenchymal stem cell population, or an induced pluripotent stem cell population. 11. The method of any one of the preceding items, wherein the stem cell population is a mesenchymal stem cell population. 12. The method of any one of the preceding items, wherein the mesenchymal stem cell population is an isolated mesenchymal stem cell population from the amniotic membrane of an umbilical cord. 13. The method of item 11 or 12, wherein at least about 90% or more of the cells of the isolated mesenchymal stem cell population express each of the markers CD73, CD90, and CD105. 14. The method of item 13, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the cells in the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105. 15. The method of any one of paragraphs 11-14, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells lack expression of the markers CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related). 16. The method of any one of the preceding items, wherein the stem cell population is contacted with a carrier prior to transport. 17. The method of any one of the preceding items, wherein the stem cell population is contacted with a carrier after its collection. 18. The method of item 17, wherein the stem cell population is contacted with the carrier about 0 minutes, about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or longer after collection of the stem cell population. 19. The method of items 17 or 18, wherein recovering comprises separating the stem cell population from the culture medium. 20. The method of item 19, wherein the separation is carried out by centrifuging the stem cells in the culture medium and decanting the culture medium. 21. The method of any one of the preceding items, wherein the contacting is carried out by suspending the stem cell population in the carrier at a density of about 70 million cells / ml, about 60 million cells / ml, about 50 million cells / ml, about 40 million cells / ml, about 30 million cells / ml, about 20 million cells / ml, about 10 million cells / ml, about 5 million cells / ml, about 4 million cells / ml, about 3 million cells / ml, about 2 million cells / ml, about 1 million cells / ml, about 500,000 cells / ml, about 100,000 cells / ml, or less than 100,000 cells. 22. The method of item 21, wherein the contacting is carried out by suspending the stem cell population in the carrier at a density of about 10 million cells / ml. 23. The method of item 21 or 22, wherein the stem cells contacted with the carrier are dispensed into vials with a volume of carrier of about 50 ml, about 20 ml, about 10 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml, about 1 ml, about 0.5 ml, about 0.25 ml, or less than 0.25 ml. 24. The method of item 21 or 22, wherein the stem cells contacted with the carrier are dispensed into vials in a volume of about 1 ml. 25. The method of any one of the preceding items, wherein the carrier is a transport medium or excipient. 26. The method of any one of the preceding items, which does not include a thawing or freezing step. 27. The method of any one of the preceding items, wherein the carrier does not include a dipolar aprotic solvent, particularly DMSO. 28. The method of any one of the preceding items, wherein at most about 50%, about 40%, about 30%, about 20%, about 10%, or less than about 10% of the stem cells in the population die during transport compared to the amount of viable stem cells before transport. 29. The method of any one of the preceding items, wherein the majority of stem cells in the stem cell population have a cell diameter of about 9 μm to about 20 μm after transport. 30. The method of any one of the preceding items, wherein the majority of stem cells in the stem cell population have a cell diameter of about 12 μm to about 16 μm after transport. 31. The method of any one of the preceding items, wherein after transport, the stem cell population secretes approximately the same amount of TGF beta-1 as before transport. 32. The method of any one of the preceding items, wherein PDGF-BB and / or IL-10 are essentially undetectable before and / or after transport. 33. The method of any one of the preceding items, wherein after transport, the mesenchymal stem cell population secretes approximately the same amount of VEGF, PDGF-AA, Ang-1, and / or HGF as before transport. 34. A method for treating a subject having a disease, comprising a step of locally administering to the subject a mesenchymal stem cell population defined in any one of items 12 to 15, wherein the mesenchymal stem cell population is administered within about 96 hours from the time the mesenchymal stem cell population is collected. 35. The method of item 34, wherein the mesenchymal stem cell population is applied in a unit dosage of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells. 36. The method of items 34 or 35, wherein the mesenchymal stem cell population is applied at a unit dose of about 10 million cells. 37. The method of any one of items 34 to 36, wherein the mesenchymal stem cell population is applied within about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 6 hours, or less from the time the mesenchymal stem cell population is harvested. 38. The method of any one of items 34 to 37, wherein the disease is a skin disease or a wound. 39. The method of item 38, wherein the wound results from a burn, bite, trauma, surgery, or disease. 40. The method of item 39, wherein the wound is caused by diabetes, and the wound is preferably a diabetic wound. 41. The method of item 40, wherein the wound is a diabetic foot ulcer. 42. The method of any one of paragraphs 34-41, wherein a unit dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells is administered once or twice a week. 43. The method of item 42, wherein a unit dose of about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells is administered once or twice weekly for a period of 3, 4, or 5, or 6, or 7, or 8, or 10 or more weeks. 44. The method of any one of items 34 to 43, wherein the mesenchymal stem cell population is applied topically and covered with a film or bandage. 45. Mesenchymal stem cell populations are approximately 1000 cells / cm 2 ~About 5 million cells / cm 2 45. The method of any one of items 34 to 44, wherein the method is applied at a dosage of 46. ​​Mesenchymal stem cell populations are approximately 100,000 cells / cm 2, about 300,000 cells / cm 2 , or approximately 500,000 cells / cm 2 46. ​​The method of any one of items 34 to 45, wherein the method is applied at a dose of 47. The method of any one of paragraphs 34 to 46, wherein the mesenchymal stem cell population is applied once, twice, or more frequently per week. 48. The method of any one of paragraphs 34 to 47, wherein the mesenchymal stem cell population is applied for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or more. 49. Mesenchymal stem cell populations are approximately 100,000 cells / cm 2 , about 300,000 cells / cm 2 , or approximately 500,000 cells / cm 2 49. The method of any one of items 34 to 48, wherein the dose is twice a week for about 8 weeks. 50. The method of any one of items 34 to 49, wherein the mesenchymal stem cell population is administered to the subject after separating the mesenchymal stem cell population from the carrier. 51. The method of item 50, wherein separating the mesenchymal stem cell population from the carrier comprises centrifugation. 52. The method of items 50 and 51, wherein separating the mesenchymal stem cell population from the carrier comprises removing the cell population from the vial with a syringe. 53. The method of any one of items 34 to 52, comprising administering the mesenchymal stem cell population by syringe. 54. A unit dose comprising about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 500,000 cells, about 250,000 cells, or less than 250,000 cells of the mesenchymal stem cell population defined in any one of paragraphs 12 to 15. 55. A unit dose of item 54 comprising about 10 million, about 9 million, about 8 million, about 7 million, about 6 million, about 5 million, about 4 million, about 3 million, about 2 million, about 1 million, about 500,000, about 250,000, or about 100,000 cells. 56. A unit dose of item 54 or 55 containing approximately 10 million cells. 57. A unit dose of any one of items 54 to 56, comprising from about 1,000 cells to about 5,000,000 cells. 58. The unit dose of any one of items 54 to 57, applied in a dose of about 100,000 cells, 300,000 cells, or 500,000 cells. 59. A unit dose of any one of items 54 to 58, to be applied topically. 60.1 cm 2 A unit dose of any one of items 54 to 59, to be applied topically per 61. A unit dose of any one of items 54 to 60, to be applied once, twice, three times or more weekly. 62. A unit dose of any one of items 54 to 61 applied for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or more. 63. A unit dose of any one of items 54 to 62, wherein the unit dose contains about 100,000 cells, about 300,000 cells, or about 500,000 cells of any one of items, and is applied twice a week for 8 weeks. 64. A unit dose of any one of items 54 to 63 contained in a 1 ml vial. 65. The unit dose of item 64 applies to 0.1 ml of the vial. 66. The unit dosage of any one of items 54 to 65, wherein the cells are in contact with a liquid carrier as defined in item 1. 67. The unit dose of any one of items 54 to 66, wherein the cells are isolated by centrifugation prior to administration to the subject. 68. The unit dose of any one of items 54 to 67, wherein the carrier is HypoThermosol™. 69. The method of any one of items 34 to 53 and the unit dosage of any one of items 54 to 68, wherein the cells are viable cells. 70. The method of any one of items 1 to 33, wherein the carrier is HypoThermosol. 71. Use of a liquid carrier for transporting a stem cell population, the liquid carrier comprising: i) Trolox, ii) Na+, iii) K+, iv) Cl-, v) H2PO4-, vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione The use. 72. Use of item 71 where the transportation takes place over a period of about 7 days or less. 73. Use of item 71 or 72, where the transportation takes place over about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or less than about 1 day. 74. The use of any one of items 71 to 73, wherein the delivery is over a period of about 48 hours or about 24 hours or less. 75. Use of any one of the preceding items 71 to 74, wherein the transportation is carried out at a temperature of about -5°C to about 15°C. 76. Use of any one of the preceding items 71 to 75, wherein the transportation is carried out at a temperature of about 2°C to about 8°C. 77. The use of any one of the preceding items 71 to 76, wherein the transport is carried out at a temperature greater than about -5°C, greater than about -10°C, greater than about -15°C, or greater than about -20°C. 78. The use of any one of items 71 to 77, wherein the stem cell population is delivered at a concentration of about 70 million cells per ml of carrier, about 60 million cells per ml of carrier, about 50 million cells per ml of carrier, about 40 million cells per ml of carrier, about 30 million cells per ml of carrier, about 20 million cells per ml of carrier, about 10 million cells per ml of carrier, about 5 million cells per ml of carrier, about 4 million cells per ml of carrier, about 3 million cells per ml of carrier, about 2 million cells per ml of carrier, about 1 million cells per ml of carrier, about 500,000 cells per ml of carrier, about 100,000 cells per ml of carrier, or less than 100,000 cells per ml of carrier. 79. The use of item 78, wherein the stem cell population is delivered at a concentration of about 10 million cells per ml of carrier to about 1 million cells per ml of carrier. 80. The use of any one of items 71 to 79, wherein the stem cell population is an embryonic stem cell population, an adult stem cell population, a mesenchymal stem cell population, or an induced pluripotent stem cell population. 81. The use of any one of items 71 to 80, wherein the stem cell population is a mesenchymal stem cell population. 82. The use of any one of items 71 to 81, wherein the mesenchymal stem cell population is an isolated mesenchymal stem cell population from the amniotic membrane of an umbilical cord. 83. Use of item 81 or 82, wherein at least about 90% or more of the cells in the isolated mesenchymal stem cell population express each of the markers CD73, CD90, and CD105. 84. Use of item 83, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the cells in the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105. 85. The use of any one of paragraphs 81-84, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells lack expression of the markers CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D related). 86. The use of any one of items 71 to 85, wherein the stem cell population is contacted with a carrier before transport. 87. The use of any one of items 71 to 86, wherein the stem cell population is contacted with the carrier after its collection. 88. The use of item 87, wherein the stem cell population is contacted with the carrier about 0 minutes, about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or longer after collection of the stem cell population. 89. The use of items 87 or 88, wherein recovering comprises separating the stem cell population from the culture medium.

[0173] It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0174] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0175] The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, or limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are to be read inclusively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description, rather than as terms of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof, recognizing that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed in terms of preferred embodiments and optional features, it should be understood that modifications and variations of the invention embodied therein as disclosed herein may be subject to those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of the invention. This includes generic descriptions of the invention using conditional or negative limitations that exclude any subject matter from the genus, regardless of whether the excluded material is specifically recited herein. Additionally, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of that Markush group. Further aspects of the invention will become apparent from the appended claims.

[0176] As used herein, the term "about" is understood to mean that there may be a variation in each value or range (pH, concentration, ratio, molarity, number of amino acids, time, etc.) of up to 5%, 10%, 15%, or even 20%, inclusive, of the given value. For example, if a formulation contains about 5 mg / ml of a compound, this is understood to mean that the formulation may have 4-6 mg / ml, preferably 4.25-5.75 mg / ml, more preferably 4.5-5.5 mg / ml, and even more preferably 4.75-5.25 mg / ml, and most preferably 5 mg / ml. As used herein, an interval defined as "between X and Y" is considered equivalent to an interval defined as "between X and Y." Any interval explicitly includes the upper and lower limits. This means, for example, that the interval "5 mg / ml to 10 mg / ml" or "between 5 mg / ml and 10 mg / ml" includes concentrations of 5, 6, 7, 8, 9, and 10 mg / ml, as well as any given intermediate value.

[0177] Sequence information SEQUENCE LISTING <110> CELLRESEARCH CORPORATION PTE. LTD. <120> A METHOD OF TRANSPORTING MESENCHYMAL STEM CELLS BY MEANS OF A TRANSPORTING SOLUTION AND A METHOD OF ADMINISTERING STEM CELLS TO WOUNDS <150> US 62 / 655,198 <151> 2018-04-09 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 574 <212> PRT <213> human <400> 1 Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu 1 5 10 15 Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu 20 25 30 His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser 35 40 45 Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu 50 55 60 Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu 65 70 75 80 Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr 85 90 95 Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala 100 105 110 Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile 115 120 125 Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro Ile Leu Ser Ala Asn Ile 130 135 140 Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro 145 150 155 160 Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr 165 170 175 Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val 180 185 190 Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu Val Asp Lys Leu Lys 195 200 205 Thr Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu 210 215 220 Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val 225 230 235 240 Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys 245 250 255 Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile Val Thr Ser Asp Asp Gly 260 265 270 Arg Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly 275 280 285 Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His 290 295 300 Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile 305 310 315 320 Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr 325 330 335 Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser 340 345 350 Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met 355 360 365 Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val 370 375 380 Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu 385 390 395 400 Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro 405 410 415 Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys 420 425 430 Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu 435 440 445 Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys 450 455 460 Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg 465 470 475 480 Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile 485 490 495 Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys 500 505 510 Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val 515 520 525 Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly 530 535 540 Arg Ile Lys Phe Ser Thr Gly Ser His Cys His Gly Ser Phe Ser Leu 545 550 555 560 Ile Phe Leu Ser Leu Trp Ala Val Ile Phe Val Leu Tyr Gln 565 570 <210> 2 <211> 161 <212> PRT <213> human <400> 2 Met Asn Leu Ala Ile Ser Ile Ala Leu Leu Leu Thr Val Leu Gln Val 1 5 10 15 Ser Arg Gly Gln Lys Val Thr Ser Leu Thr Ala Cys Leu Val Asp Gln 20 25 30 Ser Leu Arg Leu Asp Cys Arg His Glu Asn Thr Ser Ser Ser Pro Ile 35 40 45 Gln Tyr Glu Phe Ser Leu Thr Arg Glu Thr Lys Lys His Val Leu Phe 50 55 60 Gly Thr Val Gly Val Pro Glu His Thr Tyr Arg Ser Arg Thr Asn Phe 65 70 75 80 Thr Ser Lys Tyr Asn Met Lys Val Leu Tyr Leu Ser Ala Phe Thr Ser 85 90 95 Lys Asp Glu Gly Thr Tyr Thr Cys Ala Leu His His Ser Gly His Ser 100 105 110 Pro Pro Ile Ser Ser Gln Asn Val Thr Val Leu Arg Asp Lys Leu Val 115 120 125 Lys Cys Glu Gly Ile Ser Leu Leu Ala Gln Asn Thr Ser Trp Leu Leu 130 135 140 Leu Leu Leu Leu Ser Leu Ser Leu Leu Gln Ala Thr Asp Phe Met Ser 145 150 155 160 Lion <210> 3 <211> 658 <212> PRT <213> Humana <400> 3 Met Asp Arg Gly Thr Leu Pro Leu Ala Val Ala Leu Leu Leu Ala Ser 1 5 10 15 Cys Ser Leu Ser Pro Thr Ser Leu Ala Glu Thr Val His Cys Asp Leu 20 25 30 Gln Pro Val Gly Pro Glu Arg Gly Glu Val Thr Tyr Thr Thr Ser Gln 35 40 45 Val Ser Lys Gly Cys Val Ala Gln Ala Pro Asn Ala Ile Leu Glu Val 50 55 60 His Val Leu Phe Leu Glu Phe Pro Thr Gly Pro Ser Gln Leu Glu Leu 65 70 75 80 Thr Leu Gln Ala Ser Lys Gln Asn Gly Thr Trp Pro Arg Glu Val Leu 85 90 95 Leu Val Leu Ser Val Asn Ser Ser Val Phe Leu His Leu Gln Ala Leu 100 105 110 Gly Ile Pro Leu His Leu Ala Tyr Asn Ser Ser Leu Val Thr Phe Gln 115 120 125 Glu Pro Pro Gly Val Asn Thr Thr Glu Leu Pro Ser Phe Pro Lys Thr 130 135 140 Gln Ile Leu Glu Trp Ala Ala Glu Arg Gly Pro Ile Thr Ser Ala Ala 145 150 155 160 Glu Leu Asn Asp Pro Gln Ser Ile Leu Leu Arg Leu Gly Gln Ala Gln 165 170 175 Gly Ser Leu Ser Phe Cys Met Leu Glu Ala Ser Gln Asp Met Gly Arg 180 185 190 Thr Leu Glu Trp Arg Pro Arg Thr Pro Ala Leu Val Arg Gly Cys His 195 200 205 Leu Glu Gly Val Ala Gly His Lys Glu Ala His Ile Leu Arg Val Leu 210 215 220 Pro Gly His Ser Ala Gly Pro Arg Thr Val Thr Val Lys Val Glu Leu 225 230 235 240 Ser Cys Ala Pro Gly Asp Leu Asp Ala Val Leu Ile Leu Gln Gly Pro 245 250 255 Pro Tyr Val Ser Trp Leu Ile Asp Ala Asn His Asn Met Gln Ile Trp 260 265 270 Thr Thr Gly Glu Tyr Ser Phe Lys Ile Phe Pro Glu Lys Asn Ile Arg 275 280 285 Gly Phe Lys Leu Pro Asp Thr Pro Gln Gly Leu Leu Gly Glu Ala Arg 290 295 300 Met Leu Asn Ala Ser Ile Val Ala Ser Phe Val Glu Leu Pro Leu Ala 305 310 315 320 Ser Ile Val Ser Leu His Ala Ser Ser Cys Gly Gly Arg Leu Gln Thr 325 330 335 Ser Pro Ala Pro Ile Gln Thr Thr Pro Pro Lys Asp Thr Cys Ser Pro 340 345 350 Glu Leu Leu Met Ser Leu Ile Gln Thr Lys Cys Ala Asp Asp Ala Met 355 360 365 Thr Leu Val Leu Lys Lys Glu Leu Val Ala His Leu Lys Cys Thr Ile 370 375 380 Thr Gly Leu Thr Phe Trp Asp Pro Ser Cys Glu Ala Glu Asp Arg Gly 385 390 395 400 Asp Lys Phe Val Leu Arg Ser Ala Tyr Ser Ser Cys Gly Met Gln Val 405 410 415 Ser Ala Ser Met Ile Ser Asn Glu Ala Val Val Asn Ile Leu Ser Ser 420 425 430 Ser Ser Pro Gln Arg Lys Lys Val His Cys Leu Asn Met Asp Ser Leu 435 440 445 Ser Phe Gln Leu Gly Leu Tyr Leu Ser Pro His Phe Leu Gln Ala Ser 450 455 460 Asn Thr Ile Glu Pro Gly Gln Gln Ser Phe Val Gln Val Arg Val Ser 465 470 475 480 Pro Ser Val Ser Glu Phe Leu Leu Gln Leu Asp Ser Cys His Leu Asp 485 490 495 Leu Gly Pro Glu Gly Gly Thr Val Glu Leu Ile Gln Gly Arg Ala Ala 500 505 510 Lys Gly Asn Cys Val Ser Leu Leu Ser Pro Ser Pro Glu Gly Asp Pro 515 520 525 Arg Phe Ser Phe Leu Leu His Phe Tyr Thr Val Pro Ile Pro Lys Thr 530 535 540 Gly Thr Leu Ser Cys Thr Val Ala Leu Arg Pro Lys Thr Gly Ser Gln 545 550 555 560 Asp Gln Glu Val His Arg Thr Val Phe Met Arg Leu Asn Ile Ile Ser 565 570 575 Pro Asp Leu Ser Gly Cys Thr Ser Lys Gly Leu Val Leu Pro Ala Val 580 585 590 Leu Gly Ile Thr Phe Gly Ala Phe Leu Ile Gly Ala Leu Leu Thr Ala 595 600 605 Ala Leu Trp Tyr Ile Tyr Ser His Thr Arg Ser Pro Ser Lys Arg Glu 610 615 620 Pro Val Val Ala Val Ala Ala Pro Ala Ser Ser Glu Ser Ser Ser Thr 625 630 635 640 Asn His Ser Ile Gly Ser Thr Gln Ser Thr Pro Cys Ser Thr Ser Ser 645 650 655 Met Ala <210> 4 <211> 385 <212> PRT <213> human <400> 4 Met Leu Val Arg Arg Gly Ala Arg Ala Gly Pro Arg Met Pro Arg Gly 1 5 10 15 Trp Thr Ala Leu Cys Leu Leu Ser Leu Leu Pro Ser Gly Phe Met Ser 20 25 30 Leu Asp Asn Asn Gly Thr Ala Thr Pro Glu Leu Pro Thr Gln Gly Thr 35 40 45 Phe Ser Asn Val Ser Thr Asn Val Ser Tyr Gln Glu Thr Thr Thr Pro 50 55 60 Ser Thr Leu Gly Ser Thr Ser Leu His Pro Val Ser Gln His Gly Asn 65 70 75 80 Glu Ala Thr Thr Asn Ile Thr Glu Thr Thr Val Lys Phe Thr Ser Thr 85 90 95 Ser Val Ile Thr Ser Val Tyr Gly Asn Thr Asn Ser Ser Val Gln Ser 100 105 110 Gln Thr Ser Val Ile Ser Thr Val Phe Thr Thr Pro Ala Asn Val Ser 115 120 125 Thr Pro Glu Thr Thr Leu Lys Pro Ser Leu Ser Pro Gly Asn Val Ser 130 135 140 Asp Leu Ser Thr Thr Ser Thr Ser Leu Ala Thr Ser Pro Thr Lys Pro 145 150 155 160 Tyr Thr Ser Ser Ser Pro Ile Leu Ser Asp Ile Lys Ala Glu Ile Lys 165 170 175 Cys Ser Gly Ile Arg Glu Val Lys Leu Thr Gln Gly Ile Cys Leu Glu 180 185 190 Gln Asn Lys Thr Ser Ser Cys Ala Glu Phe Lys Lys Asp Arg Gly Glu 195 200 205 Gly Leu Ala Arg Val Leu Cys Gly Glu Glu Gln Ala Asp Ala Asp Ala 210 215 220 Gly Ala Gln Val Cys Ser Leu Leu Leu Ala Gln Ser Glu Val Arg Pro 225 230 235 240 Gln Cys Leu Leu Leu Val Leu Ala Asn Arg Thr Glu Ile Ser Ser Lys 245 250 255 Leu Gln Leu Met Lys Lys His Gln Ser Asp Leu Lys Lys Leu Gly Ile 260 265 270 Leu Asp Phe Thr Glu Gln Asp Val Ala Ser His Gln Ser Tyr Ser Gln 275 280 285 Lys Thr Leu Ile Ala Leu Val Thr Ser Gly Ala Leu Leu Ala Val Leu 290 295 300 Gly Ile Thr Gly Tyr Phe Leu Met Asn Arg Arg Ser Trp Ser Pro Thr 305 310 315 320 Gly Glu Arg Leu Gly Glu Asp Pro Tyr Tyr Thr Glu Asn Gly Gly Gly 325 330 335 Gln Gly Tyr Ser Ser Gly Pro Gly Thr Ser Pro Glu Ala Gln Gly Lys 340 345 350 Ala Ser Val Asn Arg Gly Ala Gln Glu Asn Gly Thr Gly Gln Ala Thr 355 360 365 Ser Arg Asn Gly His Ser Ala Arg Gln His Val Val Ala Asp Thr Glu 370 375 380 Leu 385 <210> 5 <211> 1304 <212> PRT <213> human <400> 5 Met Tyr Leu Trp Leu Lys Leu Leu Ala Phe Gly Phe Ala Phe Leu Asp 1 5 10 15 Thr Glu Val Phe Val Thr Gly Gln Ser Pro Thr Pro Ser Pro Thr Gly 20 25 30 Leu Thr Thr Ala Lys Met Pro Ser Val Pro Leu Ser Ser Asp Pro Leu 35 40 45 Pro Thr His Thr Thr Ala Phe Ser Pro Ala Ser Thr Phe Glu Arg Glu 50 55 60 Asn Asp Phe Ser Glu Thr Thr Thr Ser Leu Ser Pro Asp Asn Thr Ser 65 70 75 80 Thr Gln Val Ser Pro Asp Ser Leu Asp Asn Ala Ser Ala Phe Asn Thr 85 90 95 Thr Gly Val Ser Ser Val Gln Thr Pro His Leu Pro Thr His Ala Asp 100 105 110 Ser Gln Thr Pro Ser Ala Gly Thr Asp Thr Gln Thr Phe Ser Gly Ser 115 120 125 Ala Ala Asn Ala Lys Leu Asn Pro Thr Pro Gly Ser Asn Ala Ile Ser 130 135 140 Asp Val Pro Gly Glu Arg Ser Thr Ala Ser Thr Phe Pro Thr Asp Pro 145 150 155 160 Val Ser Pro Leu Thr Thr Thr Leu Ser Leu Ala His His Ser Ser Ala 165 170 175 Ala Leu Pro Ala Arg Thr Ser Asn Thr Thr Ile Thr Ala Asn Thr Ser 180 185 190 Asp Ala Tyr Leu Asn Ala Ser Glu Thr Thr Thr Leu Ser Pro Ser Gly 195 200 205 Ser Ala Val Ile Ser Thr Thr Thr Ile Ala Thr Thr Pro Ser Lys Pro 210 215 220 Thr Cys Asp Glu Lys Tyr Ala Asn Ile Thr Val Asp Tyr Leu Tyr Asn 225 230 235 240 Lys Glu Thr Lys Leu Phe Thr Ala Lys Leu Asn Val Asn Glu Asn Val 245 250 255 Glu Cys Gly Asn Asn Thr Cys Thr Asn Asn Glu Val His Asn Leu Thr 260 265 270 Glu Cys Lys Asn Ala Ser Val Ser Ile Ser His Asn Ser Cys Thr Ala 275 280 285 Pro Asp Lys Thr Leu Ile Leu Asp Val Pro Pro Gly Val Glu Lys Phe 290 295 300 Gln Leu His Asp Cys Thr Gln Val Glu Lys Ala Asp Thr Thr Ile Cys 305 310 315 320 Leu Lys Trp Lys Asn Ile Glu Thr Phe Thr Cys Asp Thr Gln Asn Ile 325 330 335 Thr Tyr Arg Phe Gln Cys Gly Asn Met Ile Phe Asp Asn Lys Glu Ile 340 345 350 Lys Leu Glu Asn Leu Glu Pro Glu His Glu Tyr Lys Cys Asp Ser Glu 355 360 365 Ile Leu Tyr Asn Asn His Lys Phe Thr Asn Ala Ser Lys Ile Ile Lys 370 375 380 Thr Asp Phe Gly Ser Pro Gly Glu Pro Gln Ile Ile Phe Cys Arg Ser 385 390 395 400 Glu Ala Ala His Gln Gly Val Ile Thr Trp Asn Pro Pro Gln Arg Ser 405 410 415 Phe His Asn Phe Thr Leu Cys Tyr Ile Lys Glu Thr Glu Lys Asp Cys 420 425 430 Leu Asn Leu Asp Lys Asn Leu Ile Lys Tyr Asp Leu Gln Asn Leu Lys 435 440 445 Pro Tyr Thr Lys Tyr Val Leu Ser Leu His Ala Tyr Ile Ile Ala Lys 450 455 460 Val Gln Arg Asn Gly Ser Ala Ala Met Cys His Phe Thr Thr Lys Ser 465 470 475 480 Ala Pro Pro Ser Gln Val Trp Asn Met Thr Val Ser Met Thr Ser Asp 485 490 495 Asn Ser Met His Val Lys Cys Arg Pro Pro Arg Asp Arg Asn Gly Pro 500 505 510 His Glu Arg Tyr His Leu Glu Val Glu Ala Gly Asn Thr Leu Val Arg 515,520,525 Asn Glu Ser His Asn Cys Asp Phe Arg Val Lys Asp Leu Gln Tyr 530 535 540 Ser Thr Asp Tyr Thr Phe Lys Ala Tyr Phe His Asn Gly Asp Tyr Pro 545 550 555 560 Gly Glu Pro Phe Ile Leu His Set Thr Ser Tyr Asn Ser Lys Ala 565,570,575 Leu Wings Where Leu Where Where Leu Wrote Val Thr Ser Wrote Leu 580,585,590 Lion Val Val Lion Tyr Lys And Tyr Asp Lion His Lys Arg Ser Cys 595,600,605 Asn Leu Asp Glu Gln Gln Glu Leu Val Glu Arg Asp Asp Glu Lys Gln 610 615 620 Many Asn Val Glu Pro and His Ala Asp and Many Many Glu Thr Tyr 625 630 635 640 Lys Arg Lys Ile Ala Asp Glu Gly Arg Leu Phe Leu Ala Glu Phe Gln 645,650,655 Ser Ile Pro Arg Val Phe Ser Lys Phe Pro Ile Lys Glu Ala Arg Lys 660 665 670 Pro Phe Asn Gln Asn Lys Asn Arg Tyr Val Asp Ile Leu Pro Tyr Asp 675 680 685 Tyr Asn Arg Val Glu Leu Ser Glu Ile Asn Gly Asp Ala Gly Ser Asn 690 695 700 Tyr Ile Asn Ala Ser Tyr Ile Asp Gly Phe Lys Glu Pro Arg Lys Tyr 705 710 715 720 Ile Ala Ala Gln Gly Pro Arg Asp Glu Thr Val Asp Asp Phe Trp Arg 725 730 735 Met Ile Trp Glu Gln Lys Ala Thr Val Ile Val Met Val Thr Arg Cys 740 745 750 Glu Glu Gly Asn Arg Asn Lys Cys Ala Glu Tyr Trp Pro Ser Met Glu 755 760 765 Glu Gly Thr Arg Ala Phe Gly Asp Val Val Val Lys Ile Asn Gln His 770 775 780 Lys Arg Cys Pro Asp Tyr Ile Ile Gln Lys Leu Asn Ile Val Asn Lys 785 790 795 800 Lys Glu Lys Ala Thr Gly Arg Glu Val Thr His Ile Gln Phe Thr Ser 805 810 815 Trp Pro Asp His Gly Val Pro Glu Asp Pro His Leu Leu Leu Lys Leu 820 825 830 Arg Arg Arg Val Asn Ala Phe Ser Asn Phe Phe Ser Gly Pro Ile Val 835 840 845 Val His Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Tyr Ile Gly Ile 850 855 860 Asp Ala Met Leu Glu Gly Leu Glu Ala Glu Asn Lys Val Asp Val Tyr 865 870 875 880 Gly Tyr Val Val Lys Leu Arg Arg Gln Arg Cys Leu Met Val Gln Val 885 890 895 Glu Ala Gln Tyr Ile Leu Ile His Gln Ala Leu Val Glu Tyr Asn Gln 900 905 910 Phe Gly Glu Thr Glu Val Asn Leu Ser Glu Leu His Pro Tyr Leu His 915 920 925 Asn Met Lys Lys Arg Asp Pro Pro Ser Glu Pro Ser Pro Leu Glu Ala 930 935 940 Glu Phe Gln Arg Leu Pro Ser Tyr Arg Ser Trp Arg Thr Gln His Ile 945 950 955 960 Gly Asn Gln Glu Glu Asn Lys Ser Lys Asn Arg Asn Ser Asn Val Ile 965 970 975 Pro Tyr Asp Tyr Asn Arg Val Pro Leu Lys His Glu Leu Glu Met Ser 980 985 990 Lys Glu Ser Glu His Asp Ser Asp Glu Ser Ser Asp Asp Asp Ser Asp 995 1000 1005 Ser Glu Glu Pro Ser Lys Tyr Ile Asn Ala Ser Phe Ile Met Ser 1010 1015 1020 Tyr Trp Lys Pro Glu Val Met Ile Ala Ala Gln Gly Pro Leu Lys 1025 1030 1035 Glu Thr Ile Gly Asp Phe Trp Gln Met Ile Phe Gln Arg Lys Val 1040 1045 1050 Lys Val Ile Val Met Leu Thr Glu Leu Lys His Gly Asp Gln Glu 1055 1060 1065 Ile Cys Ala Gln Tyr Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp 1070 1075 1080 Ile Glu Val Asp Leu Lys Asp Thr Asp Lys Ser Ser Thr Tyr Thr 1085 1090 1095 Leu Arg Val Phe Glu Leu Arg His Ser Lys Arg Lys Asp Ser Arg 1100 1105 1110 Thr Val Tyr Gln Tyr Gln Tyr Thr Asn Trp Ser Val Glu Gln Leu 1115 1120 1125 Pro Ala Glu Pro Lys Glu Leu Ile Ser Met Ile Gln Val Val Lys 1130 1135 1140 Gln Lys Leu Pro Gln Lys Asn Ser Ser Glu Gly Asn Lys His His 1145 1150 1155 Lys Ser Thr Pro Leu Leu Ile His Cys Arg Asp Gly Ser Gln Gln 1160 1165 1170 Thr Gly Ile Phe Cys Ala Leu Leu Asn Leu Leu Glu Ser Ala Glu 1175 1180 1185 Thr Glu Glu Val Val Asp Ile Phe Gln Val Val Lys Ala Leu Arg 1190 1195 1200 Lys Ala Arg Pro Gly Met Val Ser Thr Phe Glu Gln Tyr Gln Phe 1205 1210 1215 Leu Tyr Asp Val Ile Ala Ser Thr Tyr Pro Ala Gln Asn Gly Gln 1220 1225 1230 Val Lys Lys Asn Asn His Gln Glu Asp Lys Ile Glu Phe Asp Asn 1235 1240 1245 Glu Val Asp Lys Val Lys Gln Asp Ala Asn Cys Val Asn Pro Leu 1250 1255 1260 Gly Ala Pro Glu Lys Leu Pro Glu Ala Lys Glu Gln Ala Glu Gly 1265 1270 1275 Ser Glu Pro Thr Ser Gly Thr Glu Gly Pro Glu His Ser Val Asn 1280 1285 1290 Gly Pro Ala Ser Pro Ala Leu Asn Gln Gly Ser 1295 1300 <210> 6 <211> 254 <212> PRT <213> human <400> 6 Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu Ser Trp Ala Ile Lys Glu Glu His Val Ile 20 25 30 Ile Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met 35 40 45 Phe Asp Phe Asp Gly Asp Glu Ile Phe His Val Asp Met Ala Lys Lys 50 55 60 Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu 65 70 75 80 Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu Glu 85 90 95 Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr Asn Val Pro Pro 100 105 110 Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg Glu Pro Asn 115 120 125 Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val Val Asn Val 130 135 140 Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Thr Gly Val Ser Glu Thr 145 150 155 160 Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe His Tyr Leu 165 170 175 Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg Val Glu His 180 185 190 Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe Asp Ala Pro 195 200 205 Ser Pro Leu Pro Glu Thr Thr Glu Asn Val Val Cys Ala Leu Gly Leu 210 215 220 Thr Val Gly Leu Val Gly Ile Ile Ile Gly Thr Ile Phe Ile Ile Lys 225 230 235 240 Gly Val Arg Lys Ser Asn Ala Ala Glu Arg Arg Gly Pro Leu 245 250 <210> 7 <211> 503 <212> PRT <213> human <400> 7 Met Glu Ala Ala Val Ala Ala Pro Arg Pro Arg Leu Leu Leu Leu Val 1 5 10 15 Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr 20 25 30 Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys 35 40 45 Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys 50 55 60 Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg 65 70 75 80 Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr 85 90 95 Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro 100 105 110 Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Ala Ala 115 120 125 Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser Leu Met Leu Met 130 135 140 Val Tyr Ile Cys His Asn Arg Thr Val Ile His His Arg Val Pro Asn 145 150 155 160 Glu Glu Asp Pro Ser Leu Asp Arg Pro Phe Ile Ser Glu Gly Thr Thr 165 170 175 Leu Lys Asp Leu Ile Tyr Asp Met Thr Thr Ser Gly Ser Gly Ser Gly 180 185 190 Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Arg Thr Ile Val Leu Gln 195 200 205 Glu Ser Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg Gly Lys Trp 210 215 220 Arg Gly Glu Glu Val Ala Val Lys Ile Phe Ser Ser Arg Glu Glu Arg 225 230 235 240 Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met Leu Arg His 245 250 255 Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn Lys Asp Asn Gly Thr 260 265 270 Trp Thr Gln Leu Trp Leu Val Ser Asp Tyr His Glu His Gly Ser Leu 275 280 285 Phe Asp Tyr Leu Asn Arg Tyr Thr Val Thr Val Glu Gly Met Ile Lys 290 295 300 Leu Ala Leu Ser Thr Ala Ser Gly Leu Ala His Leu His Met Glu Ile 305 310 315 320 Val Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser 325 330 335 Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu 340 345 350 Gly Leu Ala Val Arg His Asp Ser Ala Thr Asp Thr Ile Asp Ile Ala 355 360 365 Pro Asn His Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu 370 375 380 Asp Asp Ser Ile Asn Met Lys His Phe Glu Ser Phe Lys Arg Ala Asp 385 390 395 400 Ile Tyr Ala Met Gly Leu Val Phe Trp Glu Ile Ala Arg Arg Cys Ser 405 410 415 Ile Gly Gly Ile His Glu Asp Tyr Gln Leu Pro Tyr Tyr Asp Leu Val 420 425 430 Pro Ser Asp Pro Ser Val Glu Glu Met Arg Lys Val Val Cys Glu Gln 435 440 445 Lys Leu Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys Glu Ala Leu 450 455 460 Arg Val Met Ala Lys Ile Met Arg Glu Cys Trp Tyr Ala Asn Gly Ala 465 470 475 480 Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ser Gln Leu Ser 485 490 495 Gln Gln Glu Gly Ile Lys Met 500 <210> 8 <211> 232 <212> PRT <213> human <400> 8 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135 140 Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr 145 150 155 160 Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp 165 170 175 Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 180 185 190 His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn 195 200 205 Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr 210 215 220 Cys Arg Cys Asp Lys Pro Arg Arg 225 230 <210> 9 <211> 1089 <212> PRT <213> human <400> 9 Met Gly Thr Ser His Pro Ala Phe Leu Val Leu Gly Cys Leu Leu Thr 1 5 10 15 Gly Leu Ser Leu Ile Leu Cys Gln Leu Ser Leu Pro Ser Ile Leu Pro 20 25 30 Asn Glu Asn Glu Lys Val Val Gln Leu Asn Ser Ser Phe Ser Leu Arg 35 40 45 Cys Phe Gly Glu Ser Glu Val Ser Trp Gln Tyr Pro Met Ser Glu Glu 50 55 60 Glu Ser Ser Asp Val Glu Ile Arg Asn Glu Glu Asn Asn Ser Gly Leu 65 70 75 80 Phe Val Thr Val Leu Glu Val Ser Ser Ala Ser Ala Ala His Thr Gly 85 90 95 Leu Tyr Thr Cys Tyr Tyr Asn His Thr Gln Thr Glu Glu Asn Glu Leu 100 105 110 Glu Gly Arg His Ile Tyr Ile Tyr Val Pro Asp Pro Asp Val Ala Phe 115 120 125 Val Pro Leu Gly Met Thr Asp Tyr Leu Val Ile Val Glu Asp Asp Asp 130 135 140 Ser Ala Ile Ile Pro Cys Arg Thr Thr Asp Pro Glu Thr Pro Val Thr 145 150 155 160 Leu His Asn Ser Glu Gly Val Val Pro Ala Ser Tyr Asp Ser Arg Gln 165 170 175 Gly Phe Asn Gly Thr Phe Thr Val Gly Pro Tyr Ile Cys Glu Ala Thr 180 185 190 Val Lys Gly Lys Lys Phe Gln Thr Ile Pro Phe Asn Val Tyr Ala Leu 195 200 205 Lys Ala Thr Ser Glu Leu Asp Leu Glu Met Glu Ala Leu Lys Thr Val 210 215 220 Tyr Lys Ser Gly Glu Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn 225 230 235 240 Glu Val Val Asp Leu Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys 245 250 255 Gly Ile Thr Met Leu Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val 260 265 270 Tyr Thr Leu Thr Val Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr 275 280 285 Glu Cys Ala Ala Arg Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys 290 295 300 Val Thr Ile Ser Val His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr 305 310 315 320 Phe Ser Gln Leu Glu Ala Val Asn Leu His Glu Val Lys His Phe Val 325 330 335 Val Glu Val Arg Ala Tyr Pro Pro Pro Arg Ile Ser Trp Leu Lys Asn 340 345 350 Asn Leu Thr Leu Ile Glu Asn Leu Thr Glu Ile Thr Thr Asp Val Glu 355 360 365 Lys Ile Gln Glu Ile Arg Tyr Arg Ser Lys Leu Lys Leu Ile Arg Ala 370 375 380 Lys Glu Glu Asp Ser Gly His Tyr Thr Ile Val Ala Gln Asn Glu Asp 385 390 395 400 Ala Val Lys Ser Tyr Thr Phe Glu Leu Leu Thr Gln Val Pro Ser Ser 405 410 415 Ile Leu Asp Leu Val Asp Asp His His Gly Ser Thr Gly Gly Gln Thr 420 425 430 Val Arg Cys Thr Ala Glu Gly Thr Pro Leu Pro Asp Ile Glu Trp Met 435 440 445 Ile Cys Lys Asp Ile Lys Lys Cys Asn Asn Glu Thr Ser Trp Thr Ile 450 455 460 Leu Ala Asn Asn Val Ser Asn Ile Ile Thr Glu Ile His Ser Arg Asp 465 470 475 480 Arg Ser Thr Val Glu Gly Arg Val Thr Phe Ala Lys Val Glu Glu Thr 485 490 495 Ile Ala Val Arg Cys Leu Ala Lys Asn Leu Leu Gly Ala Glu Asn Arg 500 505 510 Glu Leu Lys Leu Val Ala Pro Thr Leu Arg Ser Glu Leu Thr Val Ala 515 520 525 Ala Ala Val Leu Val Leu Leu Val Ile Val Ile Ile Ser Leu Ile Val 530 535 540 Leu Val Val Ile Trp Lys Gln Lys Pro Arg Tyr Glu Ile Arg Trp Arg 545 550 555 560 Val Ile Glu Ser Ile Ser Pro Asp Gly His Glu Tyr Ile Tyr Val Asp 565 570 575 Pro Met Gln Leu Pro Tyr Asp Ser Arg Trp Glu Phe Pro Arg Asp Gly 580 585 590 Leu Val Leu Gly Arg Val Leu Gly Ser Gly Ala Phe Gly Lys Val Val 595 600 605 Glu Gly Thr Ala Tyr Gly Leu Ser Arg Ser Gln Pro Val Met Lys Val 610 615 620 Ala Val Lys Met Leu Lys Pro Thr Ala Arg Ser Ser Glu Lys Gln Ala 625 630 635 640 Leu Met Ser Glu Leu Lys Ile Met Thr His Leu Gly Pro His Leu Asn 645 650 655 Ile Val Asn Leu Leu Gly Ala Cys Thr Lys Ser Gly Pro Ile Tyr Ile 660 665 670 Ile Thr Glu Tyr Cys Phe Tyr Gly Asp Leu Val Asn Tyr Leu His Lys 675 680 685 Asn Arg Asp Ser Phe Leu Ser His His Pro Glu Lys Pro Lys Lys Glu 690 695 700 Leu Asp Ile Phe Gly Leu Asn Pro Ala Asp Glu Ser Thr Arg Ser Tyr 705 710 715 720 Val Ile Leu Ser Phe Glu Asn Asn Gly Asp Tyr Met Asp Met Lys Gln 725 730 735 Ala Asp Thr Thr Gln Tyr Val Pro Met Leu Glu Arg Lys Glu Val Ser 740 745 750 Lys Tyr Ser Asp Ile Gln Arg Ser Leu Tyr Asp Arg Pro Ala Ser Tyr 755 760 765 Lys Lys Lys Ser Met Leu Asp Ser Glu Val Lys Asn Leu Leu Ser Asp 770 775 780 Asp Asn Ser Glu Gly Leu Thr Leu Leu Asp Leu Leu Ser Phe Thr Tyr 785 790 795 800 Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Lys Asn Cys Val His 805 810 815 Arg Asp Leu Ala Ala Arg Asn Val Leu Leu Ala Gln Gly Lys Ile Val 820 825 830 Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Met His Asp Ser Asn 835 840 845 Tyr Val Ser Lys Gly Ser Thr Phe Leu Pro Val Lys Trp Met Ala Pro 850 855 860 Glu Ser Ile Phe Asp Asn Leu Tyr Thr Thr Leu Ser Asp Val Trp Ser 865 870 875 880 Tyr Gly Ile Leu Leu Trp Glu Ile Phe Ser Leu Gly Gly Thr Pro Tyr 885 890 895 Pro Gly Met Met Val Asp Ser Thr Phe Tyr Asn Lys Ile Lys Ser Gly 900 905 910 Tyr Arg Met Ala Lys Pro Asp His Ala Thr Ser Glu Val Tyr Glu Ile 915 920 925 Met Val Lys Cys Trp Asn Ser Glu Pro Glu Lys Arg Pro Ser Phe Tyr 930 935 940 His Leu Ser Glu Ile Val Glu Asn Leu Leu Pro Gly Gln Tyr Lys Lys 945 950 955 960 Ser Tyr Glu Lys Ile His Leu Asp Phe Leu Lys Ser Asp His Pro Ala 965 970 975 Val Ala Arg Met Arg Val Asp Ser Asp Asn Ala Tyr Ile Gly Val Thr 980 985 990 Tyr Lys Asn Glu Glu Asp Lys Leu Lys Asp Trp Glu Gly Gly Leu Asp 995 1000 1005 Glu Gln Arg Leu Ser Ala Asp Ser Gly Tyr Ile Ile Pro Leu Pro 1010 1015 1020 Asp Ile Asp Pro Val Pro Glu Glu Glu Asp Leu Gly Lys Arg Asn 1025 1030 1035 Arg His Ser Ser Gln Thr Ser Glu Glu Ser Ala Ile Glu Thr Gly 1040 1045 1050 Ser Ser Ser Ser Thr Phe Ile Lys Arg Glu Asp Glu Thr Ile Glu 1055 1060 1065 Asp Ile Asp Met Met Asp Asp Ile Gly Ile Asp Ser Ser Asp Leu 1070 1075 1080 Val Glu Asp Ser Phe Leu 1085 <210> 10 <211> 498 <212> PRT <213> human <400> 10 Met Thr Val Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 10 15 Ile Gly Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30 Tyr Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 Glu His Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60 Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser 65 70 75 80 Gln Lys Leu Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp 85 90 95 Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met 100 105 110 Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu 115 120 125 Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155 160 Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165 170 175 Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser 180 185 190 Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu 195 200 205 Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr 210 215 220 Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala 225 230 235 240 Thr Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp 245 250 255 Thr Val His Asn Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu 260 265 270 Lys Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp 275 280 285 Val Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile 290 295 300 Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn 305 310 315 320 Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp 325 330 335 Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser 340 345 350 Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln 355 360 365 Arg Gln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg 370 375 380 Ala Tyr Ser Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn 385 390 395 400 Tyr Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser 405 410 415 Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn 420 425 430 Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp 435 440 445 Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala 450 455 460 Gly Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys 465 470 475 480 Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu 485 490 495 Asp Phe <210> 11 <211> 728 <212> PRT <213> Humana <400> 11 Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu 1 5 10 15 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 145 150 155 160 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170 175 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205 Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220 His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 225 230 235 240 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250 255 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365 Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380 Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met 385 390 395 400 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp 405 410 415 Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His 435 440 445 Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu 465 470 475 480 Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val 485 490 495 Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg 500 505 510 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp 515 520 525 Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr 530 535 540 Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys 545 550 555 560 Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575 Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605 Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615 620 Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu 625 630 635 640 Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu 645 650 655 Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp 660 665 670 Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu 675 680 685 Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 700 Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile 705 710 715 720 Leu Thr Tyr Lys Val Pro Gln Ser 725 <210> 12 <211> 241 <212> PRT <213> human <400> 12 Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met 20 25 30 Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu 35 40 45 His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met 50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln 115 120 125 Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg 145 150 155 160 Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170 175 Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 180 185 190 Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val 195 200 205 Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg 210 215 220 Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly 225 230 235 240 Ala <210> 13 <211> 178 <212> PRT <213> human <400> 13 Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val 1 5 10 15 Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His 20 25 30 Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe 35 40 45 Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu 50 55 60 Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 65 70 75 80 Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro 85 90 95 Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu 100 105 110 Gly Glu Asn With Lys Thr With Arg With Arg With Arg With Arg With Cys His Arg 115 120 125 Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn 130 135 140 Phe Asn Lys Leu Gln Glu Lys Gly And Tyr Lys Ala Met Ser Glu 145 150 155 160 Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 165 170 175 Arg Asn

Claims

1. 1. A composition comprising a mesenchymal stem cell population and a liquid carrier, the mesenchymal stem cell population is a mesenchymal stem cell population isolated from the amniotic membrane of an umbilical cord; At least 97% or more of the cells of the isolated mesenchymal stem cell population express each of CD73, CD90 and CD105, and lack expression of each of CD34, CD45 and HLA-DR; and the composition comprises between 500,000 cells and 20 million cells; The isolated mesenchymal stem cell population is a) Amniotic membrane as tissue explant DMEM at a final concentration of 55–65% (v / v), F12 at a final concentration of 5–15% (v / v), M171 at a final concentration of 15–30% (v / v), and Fetal bovine serum (FBS) at a final concentration of 1-8% (v / v) and culturing the culture medium in a culture medium comprising: b) harvesting cells grown from the amniotic membrane during the culturing process to provide primary isolated mesenchymal stem cells; and c) subculturing the primary isolated mesenchymal stem cells in a culture medium to provide a mesenchymal stem cell population. and wherein the compound is isolated by a method comprising: The composition.

2. 10. The composition of claim 1, comprising a mesenchymal stem cell population of 15 million cells, 10 million cells, 9 million cells, 8 million cells, 7 million cells, 6 million cells, 5 million cells, 4 million cells, 3 million cells, 2 million cells, or 1 million cells.

3. 3. The composition of claim 1 or 2, comprising 10 million cells.

4. 4. The composition of any one of claims 1 to 3, wherein the 500,000 to 20 million cells are contained in a 1 ml vial.

5. The liquid carrier is i) Trolox, keen on + 、 iii) K + 、 iv) Cl - 、 (v) H 2 PO 4 - 、 vi) HEPES, vii) lactobionate, viii) sucrose, ix) mannitol, x) glucose, xi) dextran-40, xii) adenosine, and xiii) Glutathione The composition of any one of claims 1 to 4, comprising:

6. The composition of any one of claims 1 to 5, wherein the liquid carrier comprises HypoThermosol®.

7. The composition of any one of claims 1 to 6, wherein the cells are viable cells.

8. The composition of any one of claims 1 to 7, wherein the isolated mesenchymal stem cell population is a human mesenchymal stem cell population.

9. The human mesenchymal stem cell population a) Amniotic membrane as tissue explant DMEM at a final concentration of 57.5–62.5% (v / v); F12 at a final concentration of 7.5–12.5% ​​(v / v); M171 at a final concentration of 17.5–25.0% (v / v), and Fetal bovine serum (FBS) at a final concentration of 1.75–3.5% (v / v) and culturing the compound in a culture medium comprising 9. The composition of claim 8, isolated by a method comprising:

10. The human mesenchymal stem cell population a) Amniotic membrane as tissue explant DMEM at a final concentration of 61.8% (v / v), F12 at a final concentration of 11.8% (v / v), M171 at a final concentration of 23.6% (v / v), and Final concentration of 2.5% (v / v) FBS and culturing the compound in a culture medium comprising 10. The composition of claim 9, isolated by a method comprising:

11. The composition according to any one of claims 1 to 10, which is cryopreserved.

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