Method for inducing or improving wound healing property of mesenchymal stem cell

Abstract

To provide a method for inducing or improving wound healing property of mesenchymal stem cell population.SOLUTION: A method includes a step of culturing a mesenchymal stem cell population in culture solution including DMEM (Dulbecco's modified eagle's medium), F12 (ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum). It is preferable that the mesenchymal stem cell population be selected from the group consisting of a mesenchymal stem cell population of umbilical cord, a placenta mesenchymal stem cell population, a mesenchymal stem cell population of umbilical cord-placenta junction part, a mesenchymal stem cell population of umbilical blood, a mesenchymal stem cell population of bone marrow, and a mesenchymal stem cell population derived from fatty tissue.SELECTED DRAWING: Figure 29

Description

[Technical field]

[0001] FIELD OF THEINVENTION This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 656,531, filed April 12, 2018, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] The present invention relates to a method for inducing or improving wound healing properties of a mesenchymal stem cell population. The present invention is also directed to a cell culture medium suitable for inducing or improving wound healing properties of mesenchymal stem cells and / or for isolating a mesenchymal stem cell population. The present invention is also directed to pharmaceutical compositions and uses of the isolated mesenchymal stem cell population. The present invention is also directed to a method for treating a disease or disorder comprising administering to a subject in need thereof the mesenchymal stem cell population of the present invention or a pharmaceutical composition containing such a mesenchymal stem cell population. The present invention is also directed to a highly homogeneous and well-defined mesenchymal stem cell population, for example from the umbilical cord or placenta. [Background technology]

[0003] 2. Background of the Invention Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord were first reported in US Patent Application No. 2006 / 0078993 (leading to issued US 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 therefore considered to be a good 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 capabilities of human mesenchymal stem cells (MSCs) derived from the amniotic membrane of the umbilical cord (umbilical cord lining (CL-MSC)), umbilical cord blood (CB-MSC), placenta (P-MSC), and Wharton's gelatinous substance (WJ-MSC) (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 low immunogenicity and enhanced proliferation and migration capabilities, making these cells the most promising potential for cell-based therapy. Future studies should therefore focus on the best disease models in which CL-MSCs could be administered.

[0005] Although amniotic mesenchymal stem cells can be easily obtained using the protocols described in US Patent Application No. 2006 / 0078993 and International Patent Application 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 are highly homogenous and therefore can be used in clinical trials.In addition, it would be advantageous to have methods at hand to induce or improve the wound healing properties of mesenchymal stem cell populations in general.

[0006] It is therefore an object of the present invention to provide a method for inducing or improving the wound healing properties of a mesenchymal stem cell population. It is also an object of the present invention to isolate a population of mesenchymal stem cells from the amniotic membrane of the umbilical cord that meets this need. It is therefore also an object of the present invention to provide a highly homogenous population of mesenchymal stem cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application No. 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 [Non-patent literature]

[0008] [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 Summary of the Invention

[0009] This object is achieved by a method, a mesenchymal stem cell population, a respective pharmaceutical composition and a cell culture medium having the features of the independent claims.

[0010] In a first aspect, the present invention provides a method of inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). The mesenchymal stem cell population can be an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, an umbilical cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population, or an adipose tissue derived mesenchymal stem cell population.

[0011] In a second aspect, the invention provides an isolated mesenchymal stem cell population, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90, and CD105. Preferably, the isolated mesenchymal stem cell population lacks expression of the following markers: CD34, CD45, and HLA-DR. In embodiments of this second aspect, 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. In addition, in these embodiments of the second aspect, 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 preferably lacks expression of the markers CD34, CD45, and HLA-DR. The mesenchymal stem cell population may be obtained by the method of inducing or improving wound healing properties of the first aspect. Thus, the method of the first aspect may also be a method of isolating a mesenchymal stem cell population.

[0012] In a third aspect, the present invention provides a pharmaceutical composition comprising a mammalian cell (of the second aspect) of the invention.

[0013] In a fourth aspect, the present invention provides a method for producing a culture medium for inducing or improving wound healing properties of a mesenchymal stem cell population or for isolating a mesenchymal stem cell population, the method comprising the steps of: i. 250 ml of DMEM ii. M171 118ml iii. 118 ml of DMEM / F12 iv. 12.5 ml Fetal Bovine Serum (FBS) to obtain a final concentration of 2.5% (v / v) The method includes the step of mixing the above.

[0014] In a fifth aspect, the present invention provides a cell culture medium obtainable by the method of the fourth aspect.

[0015] In a sixth aspect, the present invention provides a method of isolating a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a culture medium prepared by the method of the fourth aspect.

[0016] In a seventh aspect, the present invention provides a method for producing a composition comprising the steps of: - 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 provides a cell culture medium comprising:

[0017] In an eighth aspect, the present invention provides a use of a cell culture medium of the seventh aspect for inducing or improving wound healing properties of a mesenchymal stem cell population or for isolating a mesenchymal stem cell population. [The present invention 1001] 1. A method of inducing or improving wound healing properties of a mesenchymal stem cell population, comprising culturing the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). [The present invention 1002] The method of the present invention 1001, wherein the mesenchymal stem cell population is selected from the group consisting of a mesenchymal stem cell population from the umbilical cord, a mesenchymal stem cell population from the placenta, a mesenchymal stem cell population from the umbilical cord-placenta junction, a mesenchymal stem cell population from umbilical cord blood, a mesenchymal stem cell population from bone marrow, and a mesenchymal stem cell population derived from adipose tissue. [The present invention 1003] The method of the present invention 1002, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of amniotic membrane (AM) mesenchymal stem cell population, perivascular (PV) mesenchymal stem cell population, Wharton's gelatin (WJ) mesenchymal stem cell population, umbilical cord amniotic membrane mesenchymal stem cell population, and umbilical cord mixed mesenchymal stem cell population (MC). [The present invention 1004] Any of the methods of the present invention 1001 to 1003, wherein the culture medium contains DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v). [The present invention 1005] The method of the present invention 1004, wherein the culture medium comprises DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v). [The present invention 1006] The method of the present invention 1005, wherein the culture medium comprises 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). [The present invention 1007] The method according to any one of claims 1001 to 1006, wherein the culture medium further contains epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. [The present invention 1008] The method of claim 1007, wherein the culture medium comprises EGF at a final concentration of about 10 ng / ml. [The present invention 1009] The method according to any one of claims 1001 to 1008, wherein the culture medium contains insulin at a final concentration of about 1 µg / ml to 10 µg / ml. [The present invention 1010] The method of any one of claims 10 to 15, wherein the culture medium comprises insulin at a final concentration of about 5 µg / ml. [The present invention 1011] The method of any of claims 1001 to 1010, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). [The present invention 1012] The method of any one of claims 1001 to 1011, wherein the culture medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). [The present invention 1013] The method of claim 1012 or 1013, wherein the culture medium comprises adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 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. [The present invention 1014] Any of the methods of the present inventions 1001 to 1013, wherein culturing a mesenchymal stem cell population in a culture medium defined in any of the present inventions 1001 to 1013 results in increased expression and / or secretion of at least one of angiopoietin 1 (Ang-1), TGF-β (specifically TGF-β1), VEGF, and HGF by the mesenchymal stem cell population, as compared to a reference culture medium that does not contain any of DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum). [The present invention 1015] 1014. The method of claim 1014, wherein the reference medium consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS. [The present invention 1016] Any of the methods of the present invention, wherein the mesenchymal stem cell population is isolated from its natural environment prior to culturing in the culture medium as defined in any of the present inventions 1001 to 1013. [The present invention 1017] The method according to any one of claims 1001 to 1015, comprising a step of isolating a mesenchymal stem cell population from a native tissue environment by culturing the native tissue in a culture medium defined in any one of claims 1001 to 1013. [The present invention 1018] The method of claim 1017, wherein the tissue is umbilical cord tissue. [The present invention 1019] The method of the present invention, wherein the umbilical cord tissue is selected from the group consisting of whole umbilical cord tissue, tissue comprising the amniotic membrane of the umbilical cord, tissue comprising Wharton's gel, tissue comprising the amniotic membrane, amniotic membrane and Wharton's gel, isolated umbilical cord blood vessels, Wharton's gel separated from other components of umbilical cord tissue, and isolated amniotic membrane of the umbilical cord. [The present invention 1020] The method of claim 1017, wherein the tissue comprises or is amniotic tissue of the placenta. [The present invention 1021] The method according to any one of claims 1017 to 1020, wherein the umbilical cord tissue is a piece from the entire umbilical cord, a piece from the amniotic membrane of the umbilical cord, or a piece from the amniotic membrane of the placenta. [The present invention 1022] The method of any of claims 1019 to 1021, comprising culturing the umbilical cord tissue or the amniotic tissue of the placenta until cell proliferation of the mesenchymal stem cell population in the amniotic membrane reaches about 70 to about 80% confluency. [The present invention 1023] The method of claim 1022, further comprising a step of removing the mesenchymal stem cell population from the culture vessel used for the culture. [The present invention 1024] The method of claim 1023, wherein the step of removing the mesenchymal stem cell population from the culture vessel is carried out by enzymatic treatment. [The present invention 1025] The method of claim 1024, wherein the enzymatic treatment comprises trypsin treatment. [The present invention 1026] The method according to any one of claims 1023 to 1025, wherein the mesenchymal stem cell population is transferred to a culture vessel for subculturing for subculturing. [The present invention 1027] The method according to any one of claims 1001 to 1016, wherein the mesenchymal stem cell population is transferred to a culture vessel for subculture for culture. [The present invention 1028] Mesenchymal cell populations were cultured or subcultured at 1.0 x 10 6 The method of any one of claims 1026 to 1027, wherein the cells are suspended at a concentration of 100 cells / ml. [The present invention 1029] The method of the present invention 1028, wherein the mesenchymal stem cell population is subcultured in a culture medium defined in any one of the present inventions 1001 to 1013. [The present invention 1030] The method of claim 1029, wherein the mesenchymal stem cell population is passaged until the mesenchymal stem cells reach about 70 to about 80% confluency. [The present invention 1031] The method of any of claims 1026 to 1030, wherein the culturing or subculturing is carried out in a self-contained bioreactor. [The present invention 1032] The method of claim 1031, wherein the bioreactor is selected from the group consisting of a parallel plate bioreactor, a hollow fiber bioreactor, and a microfluidic bioreactor. [The present invention 1033] Any of the methods of the present invention, wherein the culturing is carried out in a CO2 cell culture incubator at a temperature of 37°C. [The present invention 1034] The method of the present invention 1033, comprising a step of removing the mesenchymal stem cell population from the culture vessel used for (passage) culture. [The present invention 1035] The method of claim 1034, wherein the step of removing the mesenchymal stem cell population from the culture vessel is carried out by enzymatic treatment. [The present invention 1036] The method of claim 1035, wherein the enzymatic treatment comprises trypsin treatment. [The present invention 1037] The method of claim 1036, further comprising the step of collecting the isolated mesenchymal stem cell population. [The present invention 1038] Any of the methods of the present invention, 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 express the markers CD73, CD90, and CD105. [The present invention 1039] Any of the methods of the present invention, 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 1040] Any of the methods of 1038 or 1039, wherein about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR. [The present invention 1041] Any of the aforementioned methods of the invention further comprising the step of storing the isolated stem / progenitor cell population for further use. [The present invention 1042] The method of claim 1041, wherein the preserving step is performed by cryopreservation. [The present invention 1043] 1. An isolated mesenchymal stem cell population, wherein at least about 90% or more of the cells of said stem cell population express each of the markers CD73, CD90, and CD105. [The present invention 1044] 1043. A mesenchymal stem cell population of the present invention, wherein at least about 90% or more of the cells of the stem cell population lack expression of the markers CD34, CD45, and HLA-DR. [The present invention 1045] The mesenchymal stem cell population of the present invention, 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, and lack expression of each of CD34, CD45, and HLA-DR. [The present invention 1046] The mesenchymal stem cell population of any of claims 1043 to 1045 of the present invention, which is selected from the group consisting of a mesenchymal stem cell population from an umbilical cord, a placenta mesenchymal stem cell population, an umbilical cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population, and adipose tissue-derived mesenchymal stem cell population. [The present invention 1047] The mesenchymal stem cell population of any of the present inventions 1043 to 1046, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's gelatin (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed mesenchymal stem cell population (MC). [The present invention 1048] A mesenchymal stem cell population according to any one of claims 1043 to 1047, obtainable by the method defined in any one of claims 1001 to 1042. [The present invention 1049] A mesenchymal stem cell population according to any one of claims 1043 to 1048, obtained by the method defined in any one of claims 1001 to 1042. [The present invention 1050] A pharmaceutical composition comprising an isolated mesenchymal stem population as defined in any one of claims 1043 to 1047, wherein at least about 90% or more of the cells of said stem cell population express each of the markers CD73, CD90, and CD105, and lack expression of each of the markers CD34, CD45, and HLA-DR. [The present invention 1051] The pharmaceutical composition of the present invention 1050 adapted for systemic or local application. [The present invention 1052] The pharmaceutical composition of any one of claims 1050 to 1051, further comprising a pharma- ceutically acceptable excipient. [The present invention 1053] To induce or improve the wound healing properties of a mesenchymal stem cell population, - DMEM at a concentration of approximately 55-65% (v / v), - F12 at a concentration of approximately 5-15% (v / v), - M171 at a concentration of approximately 15-30% (v / v), and - FBS at a concentration of approximately 1-8% (v / v) Use of a cell culture medium comprising: [The present invention 1054] The use of the present invention 1053, wherein the cell culture medium contains DMEM at a concentration of about 57.5 to 62.5% (v / v), F12 at a concentration of about 7.5 to 12.5% ​​(v / v), M171 at a concentration of about 17.5 to 25.0% (v / v), and FBS at a concentration of about 1.75 to 3.5% (v / v). [The present invention 1055] The use of the present invention 1054, wherein the cell culture medium comprises DMEM at a concentration of about 61.8% (v / v), F12 at a concentration of about 11.8% (v / v), M171 at a concentration of about 23.6% (v / v), and FBS at a concentration of about 2.5% (v / v). [The present invention 1056] 1053-1055. Use of a cell culture medium as defined in any of claims 1053-1055 for the isolation of a mesenchymal stem cell population. [The present invention 1057] The use of any of the present inventions 1053 to 1056, wherein the mesenchymal stem cell population is selected from the group consisting of a mesenchymal stem cell population from an umbilical cord, a mesenchymal stem cell population from a placenta, a mesenchymal stem cell population from umbilical cord blood, a mesenchymal stem cell population from bone marrow, and a mesenchymal stem cell population derived from adipose tissue. [The present invention 1058] The use of the present invention 1057, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of amniotic membrane (AM) mesenchymal stem cell population, perivascular (PV) mesenchymal stem cell population, Wharton's gelatin (WJ) mesenchymal stem cell population, umbilical cord amniotic membrane mesenchymal stem cell population, and umbilical cord mixed mesenchymal stem cell population (MC). [Brief description of the drawings]

[0018] The invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and drawings.

[0019] [Figure 1-1] 1 shows Lonza's technical information sheet for Dulbecco's Modified Eagle's Medium, including the catalog number of the DMEM used to make an example of the medium of the invention (PTT-6) in the experimental section. [Figure 1-2] See description of Figure 1-1. [Diagram 2] 1 shows Lonza's technical information sheet for Ham's F12 medium. [Diagram 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] 1 shows the Life Technologies Corporation technical information sheet for M171 medium, including the catalog number of the M171 medium used to make an example of the medium of the present invention (PTT-6) in the experimental section. [Figure 4-2] See description of Figure 4-1. [Diagram 5] A list of the components used in the experimental section to make medium PTT-6 is provided, including their commercial suppliers and catalog numbers. [Figure 6A]6A-C show the results of flow cytometry experiments in which mesenchymal stem cells isolated from umbilical cord were analyzed for expression of 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 the mesenchymal stem cells in each medium. In these experiments, three culture media were used: a) 90% (v / v / DMEM supplemented with 10% FBS (v / v), b) culture medium PTT-4, described in US patent application US 2008 / 0248005 and corresponding international patent application WO2007 / 046775 (see paragraph

[0183] of WO2007 / 046775), consisting of 90% (v / v) CMRL1066 and 10% (v / v) FBS, and c) culture medium PTT-6 of the present invention, the composition of which is described herein. In this flow cytometric 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, FIG. 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, FIG. 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 FIG. 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]7A-B show 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 pluripotent 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 of the umbilical cord 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. FIG. 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 FIG. 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 set-up is shown, the dark grey wells are standards reconstituted with PTT-4 medium and the corresponding samples from MSCs cultured in PTT-4; the light grey wells are standards reconstituted with PTT-6 medium and the corresponding samples from MSCs cultured in PTT-6. Samples in italics are control supernatants tested as part of replicates of pooled samples. [Figure 9] Singleplex measurements of TGFβ1 are shown. As can be seen, cultures CL-MSC and WJ-MSC produce more TGFβ1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced more or less the same amount of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from triplicate measurements. [Figure 10]FIG. 10A shows a multiplex measurement of PDGF-AA. As can be seen, the cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC cultures produce more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations from triplicate measurements. FIG. 10B shows a multiplex measurement of VEGF. As can be seen, the cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC cultures produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations from triplicate measurements. FIG. 10C shows a multiplex measurement of Ang-1. As can be seen, the cultures CL-MSC and WJ-MSC cultures produce much more Ang-1 when grown in PTT-6 than when grown in PTT-4. Cultured AT-MSC and BM-MSC produced essentially no Ang-1. All error bars are standard deviations from triplicate measurements. [Figure 11] Figure 1 shows multiplex measurements of HGF. As can be seen, cultures CL-MSC and WJ-MSC produce much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC essentially did not produce any HGF. All error bars are standard deviations from triplicate measurements. [Figure 12] 1 shows multiplex measurements of PDGF-AA. As can be seen, cultures CL-MSC and WJ-MSC produce more PDGF-AA when grown in PTT-4 than when grown in PTT-6. Cultures AT-MSC and BM-MSC produced the same amount of PDGF-AA in both cultures. All error bars are standard deviations from triplicate measurements. [Figure 13]FIG. 13A shows multiplex measurements of VEGF. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC cultures produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations from triplicate measurements. FIG. 13B shows multiplex measurements of Ang-1. As can be seen, cultures CL-MSC and WJ-MSC cultures produce much more Ang-1 when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC did not produce essentially any Ang-1. All error bars are standard deviations from triplicate measurements. FIG. 13C shows multiplex measurements of HGF. As can be seen, cultures CL-MSC and WJ-MSC cultures produce much more HGF when grown in PTT-6 than when grown in PTT-4. Cultured AT-MSC and BM-MSC essentially did not produce any HGF. All error bars are standard deviations from triplicate measurements. [Figure 14] Figure 1 shows multiplex measurements of bFGF. As can be seen, cultures CL-MSC and WJ-MSC produce more bFGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC produced the same amount of bFGF when grown in PTT-4 and PTT-6. All error bars are standard deviations from triplicate measurements. [Figure 15]The measurements of TGFβ1 over five different experiments (170328, 170804, 170814, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) measured for the TGFβ standard curve throughout the experiments is shown in the lower graph on the left. The MFI of the TGFβ standard curve obtained in PTT-4 and PTT-6 medium is shown in the upper graph. The lower graph on the right shows that cultures CL-MSC and WJ-MSC produced more TGFβ1 when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produced the same amount of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from different measurements in experiments 170328, 170804, 170814, 180105, 180226. [Figure 16] The measurements of Ang-1 over six different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) measured for the Ang-1 standard curve throughout the experiments is shown in the lower graph on the left. The MFI of the Ang-1 standard curve obtained in PTT-4 and PTT-6 medium is shown in the upper graph. The lower graph on the right shows that cultures CL-MSC and WJ-MSC produced more Ang-1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced essentially the same amount of Ang-1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from different measurements in experiments 170602, 170511, 170414, 170224, 180105, 180226. [Figure 17]The measurements of PDGF-BB over six different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) measured for the PDGF-BB standard curve across the experiments is shown in the bottom graph on the left. The MFI of the PDGF-BB standard curve obtained with PTT-4 and PTT-6 medium is shown in the top graph. Of note, no PDGF-BB was detected in any of the experiments. [Figure 18] The measurements of PDGF-AA over six different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) measured for the PDGF-AA standard curve throughout the experiments is shown in the bottom graph on the left. The MFI of the PDGF-AA standard curve obtained in PTT-4 and PTT-6 medium is shown in the top graph. The bottom graph on the right shows that cultures CL-MSC, AT-MSC, and BM-MSC, as well as WJ-MSC cultures, produce slightly more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations from measurements in experiments 170602, 170511, 170414, 170224, 180105, 180226. [Figure 19] The measurements of IL-10 over six different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) measured for the IL-10 standard curve across the experiments is shown in the bottom graph on the left. The MFI of the IL-10 standard curve obtained with PTT-4 and PTT-6 medium is shown in the top graph. Of note, no IL-10 was detected in any of the experiments. [Figure 20]The measurements of VEGF over six different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) measured for the VEGF standard curve throughout the experiments is shown in the lower graph on the left. The MFI of the VEGF standard curve obtained in PTT-4 and PTT-6 medium is shown in the upper graph. The lower graph on the right shows that the cultures CL-MSC, AT-MSC, and BM-MSC, and WJ-MSC produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations from the different measurements of experiments 170602, 170511, 170414, 170224, 180105, 180226. [Figure 21] The measurements of HGF over six different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) measured for the HGF standard curve throughout the experiments is shown in the bottom graph on the left. The MFI of the HGF standard curve obtained in PTT-4 and PTT-6 medium is shown in the top graph. The bottom graph on the right shows that cultures CL-MSC and WJ-MSC produced more HGF when grown in PTT-6 than when grown in PTT-4. On the other hand, cultures AT-MSC and BM-MSC did not produce as much HGF as the other cultures. All error bars are standard deviations from different measurements in experiments 170602, 170511, 170414, 170224, 180105, 180226. [Figure 22] Singleplex measurement of TGFβ1. The mean fluorescence intensity (MFI) measured for the standard TGFβ1 curve throughout the experiment is shown in the graph on the left. As can be seen from the graph on the right, CL-MSCs, WJ-MSCs, and placental MSCs all produce more TGFβ1 when grown in PTT-6 than when grown in PTT-4 or DMEM / F12 (referred to only as DMEM in FIG. 22). [Figure 23]Measurement of PDGF-BB in analyzed supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT-6, PTT-4, or DMEM / F12 is summarized. The mean fluorescence intensity (MFI) measured for a PDGF-BB standard curve throughout the experiments is shown in the graph on the left. Of note, no PDGF-BB was detected in any of the experiments. [Figure 24] Measurement of IL-10 in analyzed supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT-6, PTT-4, or DMEM / F12 is summarized. Mean fluorescence intensity (MFI) measured for the VEGF standard curve throughout the experiment is shown in the graph on the left. S6 shows the lowest standard used in the assay. Any samples below this are considered below detection. As can be seen in the graph on the right, CL-MSCs, WJ-MSCs, and placental MSCs all produced detectable levels of IL-10 when grown in PTT-6, while little or no IL-10 was detected when MSCs were grown in PTT-4 or DMEM / F12. [Diagram 25] Measurement of VEGF in analyzed supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT-6, PTT-4, or DMEM / F12 is summarized. The mean fluorescence intensity (MFI) measured for the VEGF standard curve throughout the experiment is shown in the graph on the left. S1 shows the highest standard used in the assay. Any samples above this are considered estimated (too concentrated). As can be seen in the graph on the right, CL-MSCs, WJ-MSCs, and placental MSCs all produce much higher levels of VEGF when MSCs are grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12. [Figure 26]Summarizing the multiplex measurement of bFGF. The mean fluorescence intensity (MFI) measured for the PDGF-AA standard curve throughout the experiment is shown in the graph on the left. As can be seen from the graph on the right, cultured CL-MSCs and WJ-MSCs produce more bFGF when grown in PTT-6 than when grown in PTT-4. As can be seen, CL-MSCs, WJ-MSCs, and placental MSCs all produce much lower levels of bFGF when grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12. [Figure 27] Summarizing the measurement of PDGF-AA. The mean fluorescence intensity (MFI) measured for the PDGF-AA standard curve throughout the experiment is shown in the graph on the left. S6 shows the lowest standard used in the assay. Any samples below this are considered below detection. As can be seen, CL-MSCs, WJ-MSCs, and placental MSCs all produce higher levels of PDGF-AS when MSCs are grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12. [Figure 28] Summarizing Ang-1 measurements: Mean fluorescence intensity (MFI) measured for the Ang-1 standard curve throughout the experiment is shown in the graph on the left. S1 shows the highest standard used in the assay. Any samples above this are considered estimated (too concentrated). The graph on the right shows that CL-MSCs, WJ-MSCs, and placental MSCs all produce much higher levels of Ang-1 when MSCs are grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12. [Figure 29] Summarizing the HGF measurements, the mean fluorescence intensity (MFI) measured for the HGF standard curve throughout the experiment is shown in the graph on the left. The graph on the right shows that CL-MSCs, WJ-MSCs, and placental MSCs all produced much higher levels of Ang-1 when MSCs were grown in PTT-6 compared to when MSCs were grown in PTT-4 or DMEM / F12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description of the Invention As explained above, in a first aspect, the present invention is directed to a method of inducing or improving wound healing properties of a mesenchymal stem cell population, comprising culturing the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). It has been surprisingly found in the present application that the use of such a medium has the effect of inducing or improving wound healing properties of a wide range of mesenchymal stem cell populations, independent of the native environment / compartment of the mesenchymal stem cell population. Without wishing to be bound by theory, it is believed that the induction or improvement of wound healing properties of the mesenchymal stem cell population is caused by the ability of the medium of the present invention to increase the expression and / or secretion of at least one, two, three, or all four of angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF by the mesenchymal stem cell population. The medium (PTT-4) used in US Patent Application US 2008 / 0248005 and corresponding International Patent Application WO2007 / 046775 for the isolation of the umbilical cord amniotic membrane mesenchymal stem cell population, which expression / secretion of Angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF by the umbilical cord amniotic membrane mesenchymal stem cell population was shown to have superior wound healing properties in US Patent Application US 2008 / 0248005 and corresponding International Patent Application WO2007 / 046775 (see Examples 23-26 of WO 2007 / 046775, which show that such umbilical cord amniotic membrane mesenchymal stem cell population (UCMC) alleviates 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)). Please refer to the experimental section showing that the number of mesenchymal stem cells is increased by culturing in the culture medium PTT-6 of the present invention as compared to culturing such mesenchymal stem cell populations in the culture medium PTT-6 of the present invention.As shown herein in the experimental section, culturing in a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum) increases the amount of Angiopoietin 1 (Ang-1), TGF-β1, VEGF, and / or HGF in mesenchymal stem cell populations in the amniotic membrane of the umbilical cord as well as in other compartments of the umbilical cord such as Wharton's gelatin or in (adjacent) compartments such as the placenta. Thus, the present application is believed to provide generally applicable teachings for inducing or improving the wound healing properties of a given mesenchymal stem cell population by culturing the mesenchymal stem cell population in a medium of the invention such as medium PTT-6.

[0021] In this regard, the present invention's finding that increasing the combined amounts of Ang-1, TGF-β1, VEGF, and / or HGF produced by a mesenchymal stem cell population improves or enhances the wound healing properties of this stem cell population also leads to mimicking the wound healing properties of a stem cell population with a composition / solution containing three or four of Ang-1, TGF-β1, VEGF, or HGF as the only wound healing proteins.

[0022] In this connection, it is noted that the involvement of the proteins Angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF in wound healing processes 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" or Bitto et al, "Angiopoietin-1 gene transfer improves the impaired wound healing of the genetically diabetic mice without increasing VEGF expression", Clinical Science May 14, 2008, 114 (12) 707-718. In the study by Li et al., the angiopoietin-1 gene was inserted into bone marrow mesenchymal stem cells, and the results showed that "Ang1-MSCs significantly promoted wound healing with increased epidermal and dermal regeneration and enhanced vascularization compared to MSCs, Ad-Ang1, or sham treatment." Of note, the authors of Li et al. stated that mesenchymal stem cells (MSCs) alone do not produce sufficient Ang-1, and for this reason, the authors inserted the Ang-1 gene into MSCs to generate genetically modified cells. In contrast to the Li study, it was surprisingly found in the present application that the culture of "native" mesenchymal stem cells in a culture medium such as PTT-6, for example, umbilical cord tissue mesenchymal stem cells (i.e., mesenchymal stem cell populations cultured in PTT-6), produces elevated levels of Ang-1, thus providing conditions that render mesenchymal stem cells suitable for wound healing or further improve their wound healing properties.This means that instead of genetically modifying naturally occurring mesenchymal stem cells to induce their wound healing properties (which is not only laborious but is not a preferred option for therapeutic applications due to the inherent risks of gene therapy), the present invention offers the advantage that the wound healing properties of naturally occurring mesenchymal stem cells are induced or enhanced by "simply" culturing the mesenchymal stem cell population in the culture medium of the present invention, which approach is simpler, safer and also more cost-effective.

[0023] Returning to other proteins, 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):470418; or Conway et al, "Hepatocyte growth factor regulation: An integral part of why wounds become chronic". Wound Rep Reg (2007) 15 683-692.

[0024] 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.

[0025] 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 TGFb 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.

[0026] In this context, it is also noted that the present invention has the further surprising advantage that culture in the culture medium of the present invention results in the isolation of mesenchymal stem cell populations, such as the amniotic membrane mesenchymal stem cell population of the umbilical cord, of which more than 90% or even 99% or more of the cells are positive for the three mesenchymal stem cell markers CD73, CD90, while at the same time these stem cells lack expression of CD34, CD45 and HLA-DR (see experimental section), which means that 99% or even more of the cells of this population express the stem cell markers CD73, CD90 and CD105, whilst not expressing the markers CD34, CD45 and HLA-DR. Such highly homogeneous and well-defined cell populations are ideal candidates for clinical trials and cell-based therapies, since they fully fulfill the generally accepted criteria for human mesenchymal stem cells to be used in cell therapy, as defined, for example, by 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., "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. P. 75-79. Also, 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 provides the additional advantage of providing the amount of stem cells required for therapeutic applications, such as for use in wound healing, in a cost-effective manner.In addition, all components used to make the culture medium of the present invention are commercially available with GMP quality.Therefore, the present invention opens a route for GMP production of highly homogeneous mesenchymal stem cell populations, such as the mesenchymal stem cell populations of the amniotic membrane of umbilical cord or the mesenchymal stem cell populations of Wharton's gel.

[0027] The mesenchymal stem cell population that is rendered suitable for wound healing (either by inducing wound healing properties in a population that did not have wound healing properties before undergoing the culture process of the present invention or by improving wound healing properties) can be any suitable mesenchymal stem cell known in the art, such as an adult stem cell population or a neonatal stem cell population. The mesenchymal stem cell population can be derived from any mammalian tissue or compartment / body site that is known to contain mesenchymal stem cells. In illustrative examples, the mesenchymal stem cell population can be a mesenchymal stem cell population from the umbilical cord (which are examples of neonatal stem cells), a mesenchymal stem cell population from the placenta (also a further example of neonatal stem cells), a mesenchymal stem cell population from the umbilical cord-placenta junction (a further example of neonatal stem cell population), a mesenchymal stem cell population from umbilical cord blood (yet another example of neonatal stem cells), a mesenchymal stem cell population from bone marrow (which can be an adult stem cell population), or a mesenchymal stem cell population from adipose tissue (yet another example of an adult stem cell population).

[0028] The umbilical cord mesenchymal stem cell population may be from any compartment of the umbilical cord tissue that contains mesenchymal stem cells. The mesenchymal stem cell population may be the amniotic membrane (AM) mesenchymal stem cell population, the perivascular (PV) mesenchymal stem cell population, the Wharton's gelatin (WJ) mesenchymal stem cell population of the umbilical cord, as well as a mixed umbilical cord mesenchymal stem cell population (MC), which means a population of mesenchymal stem cells that contains stem cells from two or more of these compartments. Mesenchymal stem cells from these compartments and their isolation are known to those skilled in the art and are described, for example, in Subramanian et al. "Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton's Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells", PLoS ONE 10(6): e0l27992, 2015, and references cited therein, Van Pham et al. "Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications", Cell Tissue Bank (2016) 17:289-302, 2016. A mixed mesenchymal stem cell population of umbilical cord can be obtained, for example, by removing arteries and veins from umbilical cord tissue, cutting the remaining tissue and Wharton's gel into small pieces, and culturing the umbilical cord tissue (by tissue explant) in the culture medium of the present invention.A mixed mesenchymal stem cell population of the umbilical cord can also be obtained by culturing the whole umbilical cord tissue with intact umbilical vessels as tissue explants under conditions as described by Schugar et al. "High harvest yield, high expansion, and phenotype stability of CD 146 mesenchymal stromal cells from whole primitive human umbilical cord tissue. Journal of biomedicine & biotechnology. 2009; 2009:78952" (culture in serum-supplemented DMEM containing 10% fetal bovine serum, 10% horse serum, and 1% penicillin / streptomycin). In this regard, it is noted that the mesenchymal stem cell population of the umbilical cord-placenta junction can be isolated as described in Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fet al. Placenta." J Vis Exp. 2017; (122): 55224.

[0029] In accordance with the above, it is noted herein that the mesenchymal stem cell population that is cultured in the present invention in a culture medium containing DMEM (Dulbecco's modified Eagle medium), F12 (Ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum) to induce or improve its wound healing properties can be isolated from its natural environment before being cultured in the culture medium of the present invention. Such an approach is particularly used for mesenchymal stem cell populations that cannot be easily isolated by tissue explant, such as mesenchymal stem cell populations from umbilical cord blood or bone marrow. However, this approach can also be adopted for mesenchymal stem cell populations from umbilical cord, placenta, or adipose tissue. Such stem cell populations, for example Wharton's gelatinous mesenchymal stem cell populations, can be first isolated as described above by Subramanian et al, 2015, PLoS ONE, or International Patent Application WO 2004 / 072273 "Progenitor Cells From Wharton's Jelly Of Human Umbilical Cord" and then subjected to culture of the isolated mesenchymal stem cell population in the culture medium of the present invention comprising DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum).Also, placental mesenchymal stem cell populations can be isolated from the placenta and then cultured in the culture medium of the invention, for example as described in European Patent Application EP1 288 293, Talwadekar et al, "Cultivation and Cryopreservation of Cord Tissue MSCs with Cord Blood AB Plasma" Biomed Res J 2014;1(2):126-136, Talwadekar et al, "Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts - a paired sample study" Scientific Reports 5:15784 (2015), or Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017; (122): 55224. Similarly, adipose tissue-derived mesenchymal stem cell populations can be isolated as described in Schneider et al, "Adipose-derived mesenchymal stem cells from liposuction and resected fat are feasible sources for regenerative medicine" Eur J Med Res. 2017; 22: 17, and references cited therein, and then cultured in the culture medium of the present invention (see also the Experimental Section).As a further example, a mesenchymal stem cell population at the umbilical cord-placenta junction can also be first isolated and then cultured in the culture medium of the present invention, as described in Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017; (122): 55224.

[0030] Alternatively, and in particular for mesenchymal stem cells that can be isolated by tissue explants, the mesenchymal stem cell population can be directly isolated from its native tissue environment by culturing the native tissue in the cell culture medium of the present invention. Such methodology is particularly suitable for culturing mesenchymal stem cell populations from umbilical cord tissue, placental tissue (placental tissue can include or be, for example, the amniotic membrane of the placenta), or umbilical cord-placental junction.

[0031] In this connection, it is noted that the culture medium of the present invention also allows the mesenchymal stem cell population (also referred to herein as "mesenchymal stem cell") to be isolated from its natural environment.Thus, the culture medium of the present invention also allows the mesenchymal stem cell population to be isolated under conditions that allow cell proliferation of mesenchymal stem / progenitor cells without differentiation of mesenchymal stem / progenitor cells.

[0032] In one embodiment, the culture medium of the present invention allows the isolation of mesenchymal stem cell population from amniotic membrane under conditions that allow cell proliferation of mesenchymal stem / progenitor cells without differentiation of mesenchymal stem / progenitor cells. Thus, after isolating mesenchymal stem cells from amniotic membrane as described herein, the isolated mesenchymal stem / progenitor cell population has the ability to differentiate into multiple cell types, as described, for example, in US Patent Application No. 2006 / 0078993, US Patent No. 9,085,755, International Patent Application No. WO2006 / 019357, US Patent No. 8,287,854, or WO2007 / 046775. As described, for example, in US Patent Application No. 2006 / 0078993, the mesenchymal stem cells of the amniotic membrane of the umbilical cord have a spindle shape, express the genes POU5f1, Bmi-1, 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 cells, including, but not limited to, skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, adipocytes, mucin-producing cells, cells derived from endocrine glands, such as insulin-producing cells (e.g., beta islet cells), or neuroectodermal cells. The stem cells isolated in the present invention can be differentiated in vitro for later 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 of the present invention can be used in an undifferentiated state for cell-based therapy, for example, for wound healing purposes, such as treatment of burns or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells of the present invention may serve to promote wound healing by interacting with the surrounding diseased tissue, or may also differentiate into the respective skin cells (see, for example, WO2007 / 046775 again).

[0033] It is noted here that, in accordance with the above disclosure, such mesenchymal stem cell populations described herein can be isolated and cultured from (i.e., derived from) any umbilical cord tissue, so long as the umbilical cord tissue contains the amniotic membrane (also referred to as "umbilical cord lining"). Thus, the mesenchymal stem cell populations can be isolated from (a small piece from) the whole umbilical cord, as described in the experimental section of this application. Thus, this umbilical cord tissue may contain any other tissue / component of the umbilical cord in addition to the amniotic membrane. For example, as shown in FIG. 16 of US Patent Application 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. In addition, 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, which is mostly made of mucopolysaccharides. Thus, the umbilical cord tissue used in the present invention may also include this one vein, two arteries, and Wharton's gel. The use of such an entire (intact) part of the umbilical cord has the advantage that it is not necessary to separate the amniotic membrane from other components of the umbilical cord. This reduces the isolation steps, which in turn makes the method of the present invention simpler, faster, less error-prone, and more economical - all of which are important aspects of GMP production required for therapeutic application of mesenchymal stem cells. Thus, the isolation of mesenchymal stem cells can start from tissue explants, after which the isolated mesenchymal stem cells can be subsequently subcultured (cultured) if larger amounts 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 other components of the umbilical cord and culturing the amniotic membrane in the culture medium of the present invention. The culture can also be performed on tissue explants, optionally followed by subculture of the isolated mesenchymal stem cells.

[0034] In this context, the term "tissue explant" or "tissue explant method" is used in its usual sense in the art to refer to a method in which once collected tissue (e.g., placental tissue or umbilical cord tissue) or a piece of tissue 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 new dishes by micropropagation (subculture), as also described herein. In this context, in terms of generating cells for therapeutic purposes, it is noted that in the first step of isolating / obtaining the mesenchymal stem cell population of the present invention, e.g., umbilical cord mesenchymal stem cells such as amniotic mesenchymal stem cells or Wharton's gelatinous mesenchymal stem cells, a master cell bank of isolated mesenchymal stem cells can be obtained, and in subsequent subcultures, a working cell bank can be obtained. When the mesenchymal stem cell population of the present invention (specifically, the population of mesenchymal stem cells, in which at least about 97% or more, 98% or more, or 99% or more of the cells express each of the markers CD73, CD90, and CD105, and lack the expression of each of the markers CD34, CD45, and HLA-DR) is used for clinical trials or as an approved treatment, the cell population of the working cell bank is typically used for this purpose. Both the stem cell population at the isolation stage (which may constitute the master cell bank) and the stem cell population at the subculture stage (which may constitute the working cell bank) can be stored, for example, in a cryopreserved form.

[0035] As described above, the present method of inducing or improving the wound healing properties of a mesenchymal stem cell population (and optionally simultaneously isolating mesenchymal stem cells from tissues such as Wharton's gel or the amniotic membrane of the umbilical cord) has the advantage that all components used in the culture medium of the present invention are available in GMP quality, thus offering the possibility for mesenchymal stem cells to be isolated under GMP conditions for subsequent therapeutic administration.

[0036] By "inducing or improving the wound healing properties of a mesenchymal stem cell population" herein is meant the ability of the culture medium to increase or initiate (induce) the expression and / or secretion of at least one of the proteins Ang-1, TGF-β1, VEGF, and HGF by the mesenchymal stem cell population. As explained above, all four of these proteins are known to be involved in wound healing. "Inducing or improving wound healing properties" is assessed relative to the culture of the mesenchymal stem cell population in a reference (culture) medium, such as the medium PTT-4 (consisting of 90% (v / v) CMRL1066 and 10% (v / v) FBS) used in U.S. Patent Application US 2008 / 0248005 and corresponding International Patent Application WO2007 / 046775 for the isolation and culture of the mesenchymal stem cell population from the amniotic membrane of the umbilical cord, which was shown in U.S. Patent Application US 2008 / 0248005 and corresponding International Patent Application WO2007 / 046775 to have superior wound healing properties. The wound healing properties of the mesenchymal stem cell population are increased if the mesenchymal stem cell population secretes at least one of the four marker proteins Ang-1, TGF-β1, VEGF, HGF in greater amounts (corresponding to higher secretion levels or higher concentrations) into the supernatant / culture medium when cultured in the culture medium of the present invention compared to the culture of the mesenchymal stem cell population in a reference medium. If no (detectable) secretion of any of these four marker proteins is observed by the mesenchymal stem cell population during culture in the reference medium, while detectable secretion of at least one of the four markers is observed during or after the culture of the mesenchymal stem cell population in the culture medium of the present invention, the wound healing properties of the stem cell population are induced. The wound healing properties of the mesenchymal stem cell population are also improved if the expression or secretion of at least two or at least three or all of the four marker proteins Ang-1, TGF-β1, VEGF, and HGF is increased compared to the culture of the stem cell population in the reference medium. Secretion of the four marker proteins into the culture medium (and thus production of these factors by the stem cell population) can be measured / determined in any suitable manner, for example by measuring the amount of protein using commercially available antibodies / immunoassays (see Experimental Section).Such measurements can be performed in an automated fashion, for example using systems such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).

[0037] "DMEM" refers to Dulbecco's Modified Eagle's Medium, developed in 1969 and a modification of Basal Medium Eagle (BME) (see FIG. 1 showing the data sheet for DMEM available from Lonza). The first DMEM formulation contained 1000 mg / L glucose and was first reported for the culture of embryonic mouse cells. Since then, DMEM has become a standard medium for cell culture and is commercially available from a variety of sources, such as ThermoFisher Scientific (catalog number 11965-084), Sigma Aldrich (catalog number D5546), or Lonza, to name just a few suppliers. Thus, 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 DMEM medium from Sigma Aldrich catalog number D5546, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0038] "F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed to be used with serum in combination with hormones and transferrin to culture a wide variety of mammalian cells and hybridoma cells (see FIG. 2 showing the data sheet of 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.

[0039] "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham's F12 medium (see FIG. 3 showing the data sheet of DMEM:F12 (1:1) medium from Lonza). DMEM / F12 (1:1) medium is also a widely used basal medium 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.

[0040] "M171" refers to medium 171, which was developed as a basal medium for culturing the proliferation of normal human mammary epithelial cells (see FIG. 4 showing the data sheet of M171 medium from Life Technologies Corporation). This basal medium is also widely used and is 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.

[0041] "FBS" refers to fetal bovine serum (also called fetal calf serum), i.e., the blood fraction remaining after natural blood clotting followed by centrifugation to remove any remaining red blood cells. 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 more growth factors, and allows 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 provenance tracking, authenticity of labeling, and proper standardization and oversight. Suppliers of FBS that are ISIA members 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.

[0042] Turning now to the culture medium of the present invention, the culture medium may comprise DMEM at a final concentration of about 55-65% (v / v), F12 at a final concentration of about 5-15% (v / v), M171 at a final concentration of about 15-30% (v / v), and FBS at a final concentration of about 1-8% (v / v) for inducing or improving wound healing properties of mesenchymal stem cells or for isolating or culturing mesenchymal stem cells. As used herein, the "% (v / v)" values ​​refer to the volume of the individual components 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), then one liter of culture medium will contain about 550-650 ml DMEM.

[0043] In other embodiments, the culture medium may comprise a final concentration of about 57.5-62.5% (v / v) DMEM, a final concentration of about 7.5-12.5% ​​(v / v) F12, a final concentration of about 17.5-25.0% (v / v) M171, and a final concentration of about 1.75-3.5% (v / v) FBS. In further embodiments, the culture medium may comprise a final concentration of about 61.8% (v / v) DMEM, a final concentration of about 11.8% (v / v) F12, a final concentration of about 23.6% (v / v) M171, and a final concentration of about 2.5% (v / v) FBS.

[0044] In addition to the above components, the culture medium may contain supplements that are advantageous for 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.

[0045] The culture medium of the present invention may also contain insulin. When 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.

[0046] The culture medium may further include 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 include all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may include adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 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.

[0047] In one embodiment of the method of the present invention, tissues such as umbilical cord tissue or placenta can be cultured until an appropriate number of (primary) mesenchymal stem cells, e.g., umbilical cord lining stem cells, Wharton's gelatin or placental stem cells, proliferate from the tissue. In a typical embodiment, umbilical cord tissue is cultured until cell proliferation of mesenchymal stem cells of each tissue reaches about 70 to about 80% confluency. It is noted that the term "confluency" or "confluence" is used herein in its ordinary sense in the art of cell culture and refers to an estimate / indication of the number of adherent cells in a culture dish or flask with reference to the percentage of the surface covered by cells. For example, 50 percent confluency means that approximately half of the surface is covered and there is still room for cells to proliferate. 100 percent confluency means that the surface is completely covered by cells and there is no room left for cells to proliferate as a monolayer.

[0048] Once an adequate number of primary cells (mesenchymal stem cells) are obtained from each tissue by tissue explant, the mesenchymal stem cells are removed from the culture vessel used for the culture. By doing so, a master cell bank can be obtained that contains (primary) isolated mesenchymal stem cells, for example, from umbilical cord or placenta. Typically, such mesenchymal stem cells are adherent cells, so the recovery of cells is carried out using standard enzyme treatment. For example, the enzyme treatment can include trypsin treatment, as described in International Patent Application No. 2006 / 0078993, International Patent Application WO2006 / 019357, or International Patent Application WO2007 / 046775, which means that the proliferating cells can be recovered by trypsin treatment (0.125% trypsin / 0.05% EDTA) for further expansion. When the recovered mesenchymal stem cells are used, for example, to make a master cell bank, the cells can also be cryopreserved and stored for further use, as described herein below.

[0049] Once collected, the mesenchymal stem cells can be transferred to a culture vessel for subculture. Subculture or culture (both terms are used interchangeably hereafter) is also performed when a mesenchymal stem cell population previously isolated from its natural environment is used (as explained above, such isolated stem cells used in the method of the present invention can be derived from umbilical cord blood, bone marrow, or adipose tissue, but also from umbilical cord tissue or placental tissue). 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. Mesenchymal cells can be seeded for this purpose, for example, at 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 the culture medium of the present invention) at a concentration of about 1.0×10 cells / ml for subculturing. In one embodiment, the cells can be suspended in an appropriate medium for subculturing (most conveniently the culture medium of the present invention) at a concentration of about 1.0×10 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 for subculturing the mesenchymal stem cell populations of the invention is the Xuri Cell Expansion System available from GE Heathcare. Cultivation of mesenchymal stem cells in an automated system such as the Quantum® Cell Expansion System is particularly useful when working cell banks for therapeutic applications are to be generated under GMP conditions and large numbers of cells are required.

[0050] The subculture of the mesenchymal stem cells of the present invention is carried out in the culture medium of the present invention. Thus, the culture medium of the present invention can be used both for the isolation of mesenchymal stem cell populations, for example, from the amniotic membrane of the placenta, from the amniotic membrane, or from the Wharton's gel of the umbilical cord, and for the subsequent culture of the isolated primary cells by subculture. Similarly for subculture, the mesenchymal stem cells can be cultured until an appropriate amount of cells has proliferated. In an illustrative embodiment, the mesenchymal stem cells are subcultured until they reach about 70 to about 80% confluency.

[0051] The isolation / culture of the mesenchymal stem cell population can be carried out under standard conditions for culturing mammalian cells. Typically, the method of the present invention for isolating a mesenchymal stem cell population is typically carried out under conditions (temperature, atmosphere) that are 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 each typically cultured at 37°C in a normal atmosphere containing 5% CO2. In this regard, it is noted that the mesenchymal cell population in the present invention can be derived from any mammalian species, such as mouse, rat, guinea pig, pig, rabbit, goat, horse, dog, cat, sheep, monkey, or human, and in one embodiment, mesenchymal stem cells of human origin are preferred.

[0052] Once a desired / suitable number of mesenchymal stem cells are obtained from the culture or subculture, they are harvested by removing them from the culture vessel used for subculture. The harvesting of mesenchymal stem cells is typically performed by enzymatic treatment, again including trypsinization of the cells. The isolated mesenchymal stem cells are then collected and either used directly or stored for further use. Typically, the storage is performed 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 dimethylsulfoxide (DMSO) or glycerol, which retard the formation of ice crystals in the cells of the umbilical cord.

[0053] The isolated population of mesenchymal stem cells obtained by the culture and / or isolation method of the present invention 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, 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, about 99% or more of the isolated mesenchymal stem cells can lack the 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 expresses CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.

[0054] Thus, consistent with the above disclosure, the present invention is also directed to a mesenchymal stem cell population, such as a placental mesenchymal stem cell population, or an umbilical cord mesenchymal stem cell population (e.g., isolated from Wharton's gel or the amniotic membrane of the umbilical cord), in which at least about 90% or more of the cells 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, about 99% or more of the cells of 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). In addition, 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: 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 lacks expression of CD34, CD45, and HLA-DR while expressing CD73, CD90, and CD105. Such a highly homogenous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord is reported herein for the first time and fulfills the criteria for mesenchymal stem cells to be used in cell therapy (see also the Experimental Section and, for example, Sensebe et al. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra). In this connection, it is noted that this mesenchymal stem cell population can be obtained by the isolation method of the invention, but can also be obtained, if desired, by different methods, such as cell sorting.In one embodiment of such a mesenchymal stem cell population of the umbilical cord of the present invention, in which at least about 91% or more of the cells of the stem cell population express CD73, CD90, and CD105, respectively, and lack expression of CD34, CD45, and HLA-DR, mesenchymal stem cell populations isolated from the amniotic membrane of the umbilical cord are excluded.

[0055] Consistent with the above, the present invention is also directed to a pharmaceutical composition comprising a mesenchymal stem cell population as described herein, wherein at least about 90% or more of the cells of the stem cell population express each of the markers CD73, CD90, and CD105, and optionally lack expression of CD34, CD45, and HLA-DR. The pharmaceutical composition may include any pharma- ceutical acceptable excipient and may be formulated for any desired pharmaceutical administration method. The pharmaceutical composition may be adapted for, for example, systemic or local application. In a related aspect, the present invention also provides a pharmaceutical composition containing three or four of Ang-1, TGF-β1, VEGF, or HGF as the sole wound healing proteins. Such pharmaceutical compositions may be formulated as liquids or as lyophilized materials / lyophilized preparations, for example, by using a pharma- ceutical suitable buffer, such as 0.9% saline, Ringer's solution, or phosphate buffered saline (PBS).

[0056] In a further aspect, the present invention is directed to a method of producing a culture medium for inducing or improving wound healing properties and / or for isolating a mesenchymal stem cell population, the method comprising the steps of: i. 250 ml of DMEM ii. M171 118ml 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.

[0057] 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).

[0058] 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

[0059] In these embodiments, it is noted herein that the above volumes of these components i-vi when mixed will result in a final volume of 499.675 ml of culture medium. If no further components are added to the culture medium, the remaining 0.325 ml (to make a total volume of 500 ml) can be any of components i-iv, meaning, for example, any of DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of the stock solution of EGF or insulin can of course be adjusted so that the total volume of the culture medium is 500 ml. In addition, it is also noted that components i-vi do not necessarily have to be added in the order in which they are listed, and it is of course possible to mix these components using 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 give final concentrations as 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).

[0060] 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 of culture medium 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).

[0061] Consistent with the above disclosure, the present invention is also directed to a cell culture medium obtainable or obtainable by the methods of making a medium described herein.

[0062] In addition, the present invention also relates to a method of 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 methods described herein.

[0063] Thus, the present invention also provides a method for producing a method for the treatment of atopic dermatitis. - 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 a cell culture medium comprising:

[0064] In certain embodiments of the culture medium described herein, the medium comprises 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 comprise 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).

[0065] In addition, 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.

[0066] The cell culture medium of the present invention 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 comprises all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). When present, the culture medium may comprise 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.

[0067] In an embodiment of the cell culture medium, 500 ml of the cell culture medium of the present invention is i. 250 ml of DMEM ii. M171 118ml iii. 118 ml of DMEM / F12 iv. Fetal Bovine Serum (FBS) 12.5 ml (final concentration 2.5%) Includes.

[0068] 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 It may further include.

[0069] Both insulin and EGF can be added to the culture medium using storage solutions of choice such that the total volume of the culture medium does not exceed 500 ml.

[0070] In a particular example, components i-vi of the culture medium of 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-vi as shown in Figure 5 is also referred to as "PTT-6" in this specification. In this regard, it is again noted that components i-vi and any other components such as antibiotics from any other commercial supplier can be used in making the medium of the present invention.

[0071] In addition, 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 adenine or about 0.05 to about 0.1 μg / ml adenine, 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 hydrocortisone, 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.

[0072] Finally, the present invention also provides a method of treating a non-human mammal (such as a cat, dog, horse, etc., to name just a few) or a human patient suffering from a disease or condition, comprising administering to the non-human mammal or human patient a mesenchymal stem cell population or a pharmaceutical composition containing a stem cell population as disclosed herein. The disease may be any disease or condition, in particular any disease or condition in which wound healing is desired / required. The subject (patient or non-human mammal) may suffer from a wound caused by a burn, bite, trauma, surgery, or a disease such as a skin disease or a metabolic disorder. As an example of such a metabolic disorder, the patient may suffer from, for example, type I or type II diabetes and suffer from a chronic foot ulcer. To treat the subject, the mesenchymal stem cell population of the present invention may be administered in any suitable manner, including, for example, but not limited to, topical administration, implantation, or injection. In principle, any local administration method is meant herein. The administration of the mesenchymal stem cell population may be performed by a syringe. However, it is also possible to contact mesenchymal stem cells in cream, ointment, gel, suspension or any other suitable substance before applying mesenchymal stem cells to subject.The stem cell population can then be placed directly on wounds, such as burn or diabetic wounds (see International Patent Application WO2007 / 046775).After being applied to 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.Alternatively, the stem cell population can be implanted subcutaneously, such as directly under the skin, into body fat, or into the peritoneum.

[0073] The present invention also relates to unit dosages 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.

[0074] It is also contemplated that the unit dose comprises 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 comprises about 1000 cells to about 5 million cells. The unit dose can be applied at a dose of about 100,000 cells, 300,000 cells, or 500,000 cells. As described herein, the unit dose can be applied topically, particularly when used for wound healing. For example, the unit dose can be applied in a dose of about 100,000 cells, 300,000 cells, or 500,000 cells. 2 It may be applied topically for

[0075] Depending on the need, the unit dose can be applied once, twice, three times, or more per week. For example, the unit dose can 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. 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.

[0076] 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, preferably 1 cm 2 The unit dosage may alternatively be contained in a syringe.

[0077] In the unit dose of the present invention, the cells can be in contact with a pharma- ceutically acceptable carrier, for example, a liquid carrier. The carrier can be any known carrier, such as HypoThermosol™, Hypothermosol™-FRS, or PlasmaLyte. The culture medium of the present invention can also be used as a carrier for the (unit dose) of the mesenchymal stem cell population of the present invention. In that case, the mesenchymal stem cells can be separated from the carrier before administration. For example, the cells can be centrifuged and isolated before administration to a subject.

[0078] The methods and unit dosages of treatment of the present invention may include the use of viable cells. The viability of the mesenchymal stem cell population can be tested by known methods, for example, staining with trypan blue as described in the experimental section.

[0079] The present invention is further illustrated by the following non-limiting experimental examples.

[0080] The present invention is further illustrated by the following non-limiting experimental examples.

[0081] The sequences used herein are shown in Table 1 below.

[0082] Table 1: Protein sequences used herein TIFF2024069365000002.tif160170TIFF2024069365000003.tif245170TIFF20240693650 00004.tif245170TIFF2024069365000005.tif245170TIFF2024069365000006.tif180170 EXAMPLES

[0083] Experimental Example 1. Cryopreservation of umbilical cord tissue prior to isolation of mesenchymal stem cells 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.

[0084] 1.1 Washing of umbilical cord tissue samples: a. Remove the scalpel from its protective cover. b. Hold the cord firmly with forceps and cut it into 10 cm pieces with a scalpel. Place unusable tissue back into the original tissue cup. c. Transfer the 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. Holding the syringe at a 45° angle with one hand, 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 waste receptacle 300 ml transfer bag and dispose of it in the biohazard trash. h. Repeat the washing procedure, using a new culture dish for each wash if necessary. Ensure that all blood clots on the surface are removed. Additional Plasmalyte A can be used if tissue needs cleaning. i. Place the tissue into a new labeled tissue culture dish and continue dissecting the tissue. Add 20 ml of Plasmalyte A to the dish to keep the tissue from drying out during dissection. j. Cut the umbilical cord into equal approximately 1-cm segments for a total of 10 segments. 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 the 30 ml syringe and dispense directly onto the umbilical cord tissue piece. n. Hold the culture dish at an angle to collect all the Plasmalyte A used to wash the tissue on one side and remove it with a syringe and blunt needle. o. Repeat the wash once more - no blood clots should remain.

[0085] NOTE: If the umbilical cord is not frozen immediately, the umbilical cord tissue is maintained in Plasmalyte A until immediately prior to freezing.

[0086] 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 sterile technique. iii. Remove 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" with the date and time the solution was made. iv. With a 20 ml syringe, remove 15 ml of 5% Human Serum Albumin and transfer it into the labeled transfer bag. v. Add 5 ml of DMSO to the transfer bag. vi. Mix thoroughly and record the mixing of the freeze 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, pick 8 randomly selected sections and place them into each of four 4 ml cryovials. Pick 4 randomly selected sections and place them into one 1.8 ml cryovial. These sections should not contain any 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 with 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 the aerobic and anaerobic Bactec bottles. 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.

[0087] 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 PTT-6 (culture / growth medium), add the following in the order listed: i. 250 ml of DMEM ii. M171 118ml 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 insulin (final concentration 5 μg / ml).

[0088] The above volumes of components i-vi result in a final volume of 499.675 ml of culture. If no further components are added to the culture, the remaining 0.325 ml (to bring the total volume to 500 ml) can be any of components i-iv, meaning for example any of DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of a stock solution of EGF or insulin can of course be adjusted to bring the total volume of the culture to 500 ml. Alternatively, a stock solution of an antibiotic, 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 also be added to the culture in a volume of 0.325 ml, thereby bringing the total volume of the culture to 500 ml.

[0089] Label the bottle "PTT-6" with the date the medium was prepared, the operator's initials, and the phrase "Expiration Date" 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.

[0090] b. To make Rinse Medium (Hank's Balanced 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 as "Rinse Medium" with the operator's 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.

[0091] 2.2 Thawing of umbilical cord tissue for MSC collection: Thawing begins when the operator is ready to process the samples in the clean room. Do not thaw more than one vial at a time unless the vials are from the same donor. b. Wipe the water bath with disinfectant followed by 70% isopropanol and 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 rinsing 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 that the water bath temperature is 36-38°C. f. Remove vials of tissue from liquid nitrogen storage and rapidly thaw 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 and be used as a floating rack when thawing samples. g. Remove the vials from the water bath and spray them with the 70% isopropanol solution. A good time to remove the vials from the water bath is when you see small ice cubes 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.

[0092] 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 the biosafety cabinet. c. Conduct bioparticle counts while working in the biosafety cabinet. d. Assemble all necessary items into the biosafety cabinet, checking each packaging for damage and expiration dates. When handling syringes, serological pipettes, sterile forceps, scalpels, tissue plates, and needles, never touch any surfaces that will come in contact with sterile products. Only the exterior of syringe barrels, tubing, plunger tips, and / or needle caps or casings may be safely handled. Discard items if they are touched or if a surface has come into contact with a nonsterile surface. e. Record lot numbers and expiration dates (if applicable) of all reagents and materials used. f. Receive thawed vials by cleaning the vials with a lint-free wipe 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 5 ml aliquots 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 needle. Repeat this rinse process two more times. k. Add 2 mL of rinsing medium to the tissue to prevent it from drying out.

[0093] 2.4. Initiating MSC expansion from tissue: Label the bottom of a 6-well plate "Expansion 1" with the MSC lot number or umbilical cord tissue ID and the date expansion will begin. If using 60 mm tissue culture dishes, draw a grid on the bottom of the dish to divide the plate into 4 sections. b. Using sterile disposable forceps, place one piece of tissue measuring 3x3 mm to 5x5 mm into each well. If using 60 mm tissue culture dishes, center the tissue in each section and keep the tissues separated (>1 cm from each other). c. Fill each well with 3 ml of PTT-6. 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 medium daily, always equilibrate medium to room temperature before use. i. Aspirate the medium. ii. Add 3 ml of PTT-6. iii. 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 procedures as 4.a-4.e above, except label the plate as "Growth 2". Maintain cell growth in the "Growth 1" plate by adding 2 ml of PTT-6 to each well. Observe daily for confluency. Replace medium every 2-3 days (be sure to equilibrate 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 PTT-6 to each well. Observe daily for confluency. Replace medium every 2-3 days (be sure to equilibrate medium to room temperature before use). i. Discard the tissue when growth is observed in the "Growth 3" plate. If the tissue is very small and does not interfere with cell growth, discard the tissue when subculturing. j. Once the cells reach 40-50% confluency, monitor the cells daily to prevent overgrowth. k. Subculture the cells when they reach 70-80% confluency. Do not allow cells to grow beyond 80% confluency.

[0094] When tissue explants are about 1-3 mm in size and the culture of tissue explants / cells is performed in 175 mm square culture dishes, the average number of mesenchymal stem cells recovered from the explants is typically about 4,000-6,000 cells / explant. Thus, when mesenchymal stem cells are simultaneously grown from 48 explants, about 300,000 cells can be obtained at the time of harvest. These 300,000 mesenchymal stem cells harvested from the explants are then cultured in 175 cm square culture dishes 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 a bioreactor, as described in Example 2.7 below.

[0095] 2.5. Subculture of MSCs in cell culture dishes 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 PTT-6 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 migrate downwards by gravity flow. Gently tap the side of the plate to facilitate detachment. v. Add 1 ml of PTT-6. Pipette up and down gently then transfer cells to a 15 ml centrifuge tube. Use a clean pipette tip for each well. Pool 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 PTT-6. 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 live cells x 100 / (number of live cells + number of dead cells) v. Cell suspension at 1.0 x 10 6 Dilute to cells / ml: 1. “X” Volume = Total Viable Cells / 10 6 cells / ml 2. For example, if the total number of live cells 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 6 If the number of cells / ml is less than 150 mm, each 175 cm 2 2 x 10 cells in flask 6Determine the volume required to sow the pieces. 1. Cells 2×10 6 Capacity per cell = 2 x 10 cells 6 cells ÷ live 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 30 ml of PTT-6 in each 150 mm Petri dish or 175 cm 2 Seed the flasks. ix. Observe every 3 days for attachment, colony formation, and confluence. Once cells reach 40-50% confluence, observe cells daily to every 2 days to prevent over-expansion. Do not allow cells to expand 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.

[0096] 2.6 Cryopreservation of MSC cells a. Conduct bioparticle counts while working in the biosafety cabinet. b. When cells reached 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 PTT-6 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 migrate downwards by gravity flow. Gently tap the side of the Petri dish to help promote detachment. v. Add 10 ml of PTT-6 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 PTT-6. 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 the cells.

[0097] 2.7. Subculture (expansion) of MSCs in Quantum bioreactor (Terumo BTC, Inc.) It is also possible to expand MSCs using the Quantum bioreactor. The starting cell number for expansion in the Quantum bioreactor should be 20-30 million cells per run. The typical yield per run is 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.

[0098] Materials / Reagents: 1. Quantum Augmentation Set 2. Quantum Waste Bag 3. Quantum Media Bags 4. Quantum Inlet Bag 5. PTT-6 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 Combitip

[0099] 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 Tube Connectors 7. M4 repeating pipettor 8. RF Sealer

[0100] procedure: 1. Quantum Bioreactor Preparation a) Preparation of the Quantum bioreactor b) Coating of bioreactors: 1) Prepare 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 18g needle, transfer the fibronectin solution into a Ccell 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 of 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 PTT-6 media. 7) Using a sterile tubing connector, connect the media bag to the IC media line. 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) Adaptation of the bioreactor with culture medium 2. Cultivating cells in the Quantum bioreactor a) Cell loading and attachment using homogenous suspension: b) Cell feeding and cultivation 1) Select the medium flow rate to feed the cells. 2) Sample daily for lactate and glucose. 3) Adjust the media flow rate as lactate levels rise. The actual maximum tolerated lactate concentration is dictated by the flask culture from which the cells were derived. Ensure there is enough PTT-6 media in the media bag. Replace the PTT-6 media bag with a fresh PTT-6 media bag if necessary. 4) Once the flow rate has reached the desired value, measure the lactate level every 8-12 hours. If the lactate level does not decrease or if it continues to increase, harvest the cells. 3. Harvesting Cells from the Quantum Bioreactor a) Once 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, connect a new bag containing at least 1.7 liters of PBS to the "Wash" line using a sterile tubing connector. 3) Implement a take-back program. 4. Cell cryopreservation 1) Once the cells have harvested, transfer the cells to a 50 ml centrifuge tube and pellet the cells. 2) Resuspend using 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) Change the cell concentration to 2×10 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 cell suspension in cryopreservative to each 1.8 ml vial. Freeze using a controlled rate freezer as described in SOP D6.100 CB Cryopreservation using the CRF program. 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.

[0101] 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.

[0102] 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 the mesenchymal stem cells were subcultured in each medium.

[0103] In these experiments, three culture media were used: a) 90% (v / v / DMEM supplemented with 10% FBS (v / v), b) culture medium PTT-4, described in US Patent Application No. 2008 / 0248005 and corresponding International Patent Application WO2007 / 046775 (see paragraph

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

[0104] The following protocol was used for flow cytometric analysis.

[0105] material and method TIFF2024069365000007.tif195152

[0106] 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 and submerged in the respective media, which were 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 dishes by trypsinization (0.0125% trypsin / 0.05% EDTA) and used for flow cytometry experiments. b) Trypsinization of cells for experiments 1. Remove the medium from the cell culture plates. 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 rinse the plate 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 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 will 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; non-viable cells readily take up trypan blue and are therefore dark in color) 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 number / 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 3 tubes (CD73, CD90, CD105) and 2 tubes (negative control). ii. Primary antibody (Ab) staining Add 1 μl of primary antibody [0.5 mg / ml Ab] to 100 ul 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.

[0107] The results of the 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, 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. As can be seen from Fig. 6a, the population isolated using DMEM / 10% FBS as the medium culture has about 75% CD73+ cells, 78% 90+ cells, and 80% CD105+ cells (average value of two experiments), whereas after isolating / culturing umbilical cord tissue using PTT-4 medium (see Fig. 6b), the number of mesenchymal stem cells that are CD73 positive, CD90 positive, and CD105 positive is about 87% (CD73+ cells), 93% / CD90+ cells, and 86% (CD105+ cells) in the average value of two experiments. The purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention is at least 99.0% for all three markers (CD73, CD90, CD105), which means that the purity of this cell population is significantly higher than that of culture 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 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 can be the gold standard for such stem cell-based therapeutic approaches.

[0108] 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 100% pure and well-defined stem cell population that meets the criteria for realizing that mesenchymal stem cells are used in cell therapy (95% or more of the stem cell population expresses 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 are adherent to plastic in standard culture conditions and differentiate in vitro into osteoblasts, adipocytes, and chondroblasts, see US Patent No. 9,085,755, US Patent No. 8,287,854, or WO2007 / 046775, thus meeting the generally accepted criteria for the use of mesenchymal stem cells in cell therapy.

[0109] Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90 and CD105, and lacking the expression of CD34, CD45 and HLA-DR. As shown in Figure 7b, bone marrow mesenchymal stem cell population contains 94.3% live cells, of which 100% express each of CD73, CD90 and CD105 (see columns "CD73+CD90+" and "CD73+CD105+"), while only 62.8% of bone marrow stem cell population lacks the expression of CD45, and 99.9% of stem cell population lacks the expression of CD34 and HLA-DR (see columns "CD34-CD45-" and "CD34-HLA-DR-"). Thus, bone marrow mesenchymal stem cells, 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 be the gold standard for stem cell-based therapeutic approaches.

[0110] 4. Analysis of wound healing marker protein secretion in isolated mesenchymal stem cell populations cultured in the culture medium of the present invention Based on the highly significant results (that culturing in PTT-6 yields an essentially 100% pure and defined mesenchymal stem cell population), various isolated mesenchymal stem cell populations were cultured in PTT-6 and analyzed for secretion of wound healing marker proteins compared to culture in PTT-4 medium (which served as the reference medium).

[0111] More specifically, the following isolated mesenchymal stem cell populations were analyzed: - Mesenchymal stem cells of the amniotic membrane of the umbilical cord (umbilical cord lining MSCs / CL-MSCs). This population of CL-MSCs was isolated from tissue explants of the human umbilical cord lining membrane (cultured in DMEM supplemented with 10% fetal bovine serum, DMEM / 10% FBS) as described in Example 2 of WO2007 / 046775. - Wharton's gelatinous mesenchymal stem cells (WJ-MSCs). This population of WJ-MSCs was isolated from tissue explants of Wharton's gelatinous from human umbilical cord (cultured in DMEM containing 4,500 mg / mL glucose and 2 mM L-glutamine, supplemented with 10% human serum / FBS and antibiotic solution) as described in Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017; (122): 55224. - Adipose tissue-derived mesenchymal stem cells (AT-MSCs). This population of AT-MSCs was isolated from adipose tissue of skin tissue donated after abdominoplasty by tissue explant (culture in DMEM supplemented with 5% penicillin / streptomycin and 10% FBS) as described in Schneider et al, "Adipose-derived mesenchymal stem cells from liposuction and resected fat are feasible sources for regenerative medicine" Eur J Med Res. 2017; 22: 17. - Bone marrow mesenchymal stem cells (BM-MSCs). This population of BM-MSCs was kindly provided by the AO Foundation, Davos, Switzerland. - Placental Mesenchymal Stem Cells (PT-MSCs). This population of PT-MSCs was isolated from the placenta as described in Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017; (122): 55224.

[0112] Culture protocol for culturing isolated MSCs Five million MSCs from each source were plated in 100 mm tissue culture dishes in DMEM / F12 / 10% FCS for 24 hours. The medium was discarded and PTT-6 / PTT-4 was added to the cultures for 24 hours. The medium was discarded and the cells were washed with PBS. ·10ml DMEM was added to the cultures for 24 hours. The medium was discarded and 5ml DMEM was added to the culture. After 24 h of incubation, conditioned medium was collected and centrifuged to remove cell debris, and the supernatant was aliquoted into tubes for storage at -80°C and subsequent analysis of marker protein secretion by cytokine assays.

[0113] Cytokine assays in PTT-6 versus PTT-4 medium supernatants from MSCs of CL-MSC, WJ-MSC, bone marrow MSC, and adipose MSC origin Cytokine detection was performed in MSC supernatants. Measurements and analysis were performed using Luminex 200 and Xponent software.

[0114] The purpose of this experiment was to measure the relative levels of multiplex (PDGF-AA, PDGF-BB, IL-10, VEGF, Ang-1, and HGF), TGFβ1 singleplex, and bFGF2 singleplex cytokines in cell culture supernatants as follows (MSC, mesenchymal stem cells; CL, umbilical cord lining; WJ, Wharton's gelatinous matter; AT, adipose tissue; BM, bone marrow): CL-MSCs cultured in PTT-4 WJ-MSCs cultured in PTT-4 AT-MSCs cultured in PTT-4 BM-MSCs cultured in PTT-4 CL-MSCs cultured in PTT-6 WJ-MSCs cultured in PTT-6 AT-MSCs cultured in PTT-6 BM-MSCs cultured in PTT-6

[0115] Each sample was tested in triplicate (3 wells) except for samples fed in PTT-4, which were tested in 6 wells. In addition, samples CR001A, CR001C, CR001D, and CR001G were included as positive controls to validate the cytokine assays (conditioned media from CR001A, CR001C, CR001D, and CR001G were not prepared by culturing cells in PTT-6 or PTT-4).

[0116] The goal of this experiment was to generate a cytokine profile of MSCs cultured in either PTT-4 or PTT-6 and to compare the profiles of MSCs from different tissue origins (umbilical cord lining vs. Wharton's gelatin vs. adipose tissue vs. bone marrow). This profile would reveal which stem cell populations grown in which media secrete more cytokines of interest to promote wound healing.

[0117] The plate setup for all plates is described in Figure 8. In what follows, the following acronyms are used: MSC, mesenchymal stem cells; CL, umbilical cord lining; WJ, Wharton's gelatin; AT, adipose tissue; BM, bone marrow.

[0118] Multiplex Analysis Multiplex Information: R&D Systems / Bio-techne catalog number LXSAHM. This kit has lot number L123680, expires on August 28, 2018, and corresponds to the following analytes: Ang-1, angiopoietin ·VEGF, vascular endothelial growth factor PDGF-AA, Platelet-derived growth factor (PDGF-AA refers to a disulfide-linked homodimer consisting of an A chain, and PDGF-BB consists of a B homodimer. R&D states that the PDGF-BB antibody will detect the PDGF-AB heterodimer as well) PDGF-BB ·HGF, hepatocyte growth factor IL-10, Interleukin-10

[0119] TGFβ1 Singleplex Information: R&D Systems / Bio-techne): Basic Kit, Catalog No. LTGM00, Lot No. P156217, received 2 / 27 / 2018, expiration date 8 / 30 / 2018. TGFβ1 component, catalog number LTGM100, lot number P161760, received 2 / 27 / 2018, expiration date 11 / 27 / 2019.

[0120] bFGF2 Simplex Information (used on March 19, 2018): eBioscience / Thermo: Basic Kit, catalog number EPX010-10420-901, lot number 172174000, expiration date 01 / 31 / 2020. bFGF2 component, catalog number EPX01A-12074-901, lot number 169751102, expiration date 12 / 31 / 2019.

[0121] bFGF2 Simplex Information (used on March 22, 2018): eBioscience / Thermo: Basic Kit, catalog number EPX010-10420-901, lot number 172174000, expiration date 01 / 31 / 2020. bFGF2 component, catalog number EPX01A-12074-901, lot number 166916102, expiration date 12 / 31 / 2019.

[0122] Multiplex Information: R&D Systems / Bio-techne catalog number LXSAHM. This kit has lot number L123999, expires September 25, 2018, and corresponds to the following analytes: Ang-1, angiopoietin ·VEGF, vascular endothelial growth factor PDGF-AA, Platelet-derived growth factor 2 PDGF-BB ·HGF, hepatocyte growth factor IL-10, Interleukin-10 ·bFGF, basic fibroblast growth factor

[0123] Data Entry The raw data is output in PDF and Excel formats. The Excel format data is used for data processing.

[0124] procedure Cytokine detection in MSC supernatants was performed according to the detailed protocol information. As part of this experiment, there is a single modification to the protocol: standard 8 in the multiplex kit is no longer used. The reason for the discontinuation of standard 8 is that the R&D Systems protocol itself uses only standards 1-6. Furthermore, standard 8 was validated in ClinImmune only for two of the six analytes that make up the multiplex: PDGF-BB and HGF. In the case of PDGF-BB, this analyte was not detected at all in the supernatant. In the case of HGF, this analyte falls in the middle area of ​​the standard curve. As the standards are reconstituted with growth medium, the standard curve was constructed with both PTT-6 and PTT-4. Test samples grown either in PTT-6 or in PTT-4 were extrapolated from the respective standard curves.

[0125] Results were extrapolated by the Luminex software from analyte-specific standard curves generated by the same software: analysis algorithm is set to Logistic 5P Weighted with weighted analysis using 1 / y2 for weighting.

[0126] sample 1. PTT-4 and PTT-6 media (not exposed to MSCs) 2. Supernatant of MSCs to be tested 3. Optional: Supernatants from CL-MSCs from different donors; CR001A, C, D, and G.

[0127] Summary of Experimental Results TGFβ1 singleplex assay A constant aliquot of 1 out of 3 was used and is shown in Figure 9. All error bars are standard deviations from triplicate measurements.

[0128] Figure 9: Singleplex measurements of TGFβ1. As can be seen, cultures CL-MSC and WJ-MSC produce more TGFβ1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced more or less the same amount of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from triplicate measurements.

[0129] First multiplex assay A fixed aliquot of 1 out of 3 was used. · PDGF-BB and IL-10 were not detected in any of the samples.

[0130] The data are shown in Figures 10 and 11.

[0131] FIG. 10: FIG. 10A Multiplex measurement of PDGF-AA. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC cultures produce more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations from triplicate measurements. FIG. 10B Multiplex measurement of VEGF. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC cultures produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations from triplicate measurements. FIG. 10C Multiplex measurement of Ang-1. As can be seen, cultures CL-MSC and WJ-MSC cultures produce much more Ang-1 when grown in PTT-6 than when grown in PTT-4. Cultured AT-MSC and BM-MSC produced essentially no Ang-1. All error bars are standard deviations from triplicate measurements.

[0132] Figure 11: Multiplex measurement of HGF. As can be seen, cultures CL-MSC and WJ-MSC produce much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC did not produce essentially any HGF. All error bars are standard deviations from triplicate measurements.

[0133] Multiplex assay (including bFGF) A fixed aliquot of 3 was used. The data are shown in Figures 12-14.

[0134] Figure 12: Multiplex measurement of PDGF-AA. As can be seen, cultures CL-MSC and WJ-MSC produce more PDGF-AA when grown in PTT-4 than when grown in PTT-6. Cultures AT-MSC and BM-MSC produced the same amount of PDGF-AA in both cultures. All error bars are standard deviations from triplicate measurements.

[0135] FIG. 13: FIG. 13A Multiplex measurement of VEGF. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC cultures produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations from triplicate measurements. FIG. 13B Multiplex measurement of Ang-1 multiplex assay. As can be seen, cultures CL-MSC and WJ-MSC cultures produce much more Ang-1 when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC did not produce essentially any Ang-1. All error bars are standard deviations from triplicate measurements. FIG. 13C. Multiplex measurement of HGF. As can be seen, cultures CL-MSC and WJ-MSC cultures produce much more HGF when grown in PTT-6 than when grown in PTT-4. Cultured AT-MSC and BM-MSC essentially did not produce any HGF. All error bars are standard deviations from triplicate measurements.

[0136] Figure 14: Multiplex measurement of bFGF. As can be seen, cultures CL-MSC and WJ-MSC produce more bFGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC produced the same amount of bFGF when cultured in PTT-4 and PTT-6. All error bars are standard deviations from triplicate measurements. It should be noted that the bFGF samples were very low in abundance, at or near the lower limit of detection.

[0137] Figures 15 to 21 show an overview of the data obtained across the different experiments.

[0138] Figure 15: Summarizing the measurements of TGFβ1 over five different experiments (170328, 170804, 170814, 180105, 180226). The mean fluorescence intensity (MFI) measured for the TGFβ standard curve throughout the experiments is shown in the lower graph on the left. The MFI of the TGFβ standard curve obtained in PTT-4 and PTT-6 medium is shown in the upper graph. The lower graph on the right shows that cultures CL-MSC and WJ-MSC produced more TGFβ1 when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produced the same amount of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from different measurements in experiments 170328, 170804, 170814, 180105, 180226.

[0139] Figure 16: Summarizing the measurements of Ang-1 over six different experiments (170602, 170511, 170414, 170224, 180105, 180226). The mean fluorescence intensity (MFI) measured for the Ang-1 standard curve throughout the experiments is shown in the lower graph on the left. The MFI of the Ang-1 standard curve obtained with PTT-4 and PTT-6 medium is shown in the upper graph. The lower graph on the right shows that cultures CL-MSC and WJ-MSC produced more Ang-1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced essentially the same amount of Ang-1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from different measurements in experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0140] Figure 17: Summarizing the measurements of PDGF-BB over six different experiments (170602, 170511, 170414, 170224, 180105, 180226). The mean fluorescence intensity (MFI) measured for the PDGF-BB standard curve throughout the experiments is shown in the bottom graph on the left. The MFI of the PDGF-BB standard curve obtained with PTT-4 and PTT-6 medium is shown in the top graph. Of note, no PDGF-BB was detected in any of the experiments.

[0141] Figure 18: Summarizing the measurements of PDGF-AA over six different experiments (170602, 170511, 170414, 170224, 180105, 180226). The mean fluorescence intensity (MFI) measured for the PDGF-AA standard curve throughout the experiments is shown in the lower graph on the left. The MFI of the PDGF-AA standard curve obtained in PTT-4 and PTT-6 medium is shown in the upper graph. The lower graph on the right shows that cultures CL-MSC, AT-MSC, and BM-MSC, as well as WJ-MSC cultures, produce slightly more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations from measurements in experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0142] Figure 19: Summarizing the measurements of IL-10 over six different experiments (170602, 170511, 170414, 170224, 180105, 180226). The mean fluorescence intensity (MFI) measured for the IL-10 standard curve across the experiments is shown in the bottom graph on the left. The MFI of the IL-10 standard curve obtained with PTT-4 and PTT-6 medium is shown in the top graph. Of note, no IL-10 was detected in any of the experiments.

[0143] Figure 20: Summarizing the measurements of VEGF over six different experiments (170602, 170511, 170414, 170224, 180105, 180226). The mean fluorescence intensity (MFI) measured for the VEGF standard curve throughout the experiments is shown in the lower graph on the left. The MFI of the VEGF standard curve obtained in PTT-4 and PTT-6 medium is shown in the upper graph. The lower graph on the right shows that the cultures CL-MSC, AT-MSC, and BM-MSC, as well as WJ-MSC, produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations from the different measurements of experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0144] Figure 21: Summarizing the measurements of HGF over six different experiments (170602, 170511, 170414, 170224, 180105, 180226). The mean fluorescence intensity (MFI) measured for the HGF standard curve throughout the experiments is shown in the bottom graph on the left. The MFI of the HGF standard curve obtained in PTT-4 and PTT-6 medium is shown in the top graph. The bottom graph on the right shows that cultures CL-MSC and WJ-MSC produced more HGF when grown in PTT-6 than when grown in PTT-4. On the other hand, cultures AT-MSC and BM-MSC did not produce as much HGF as the other cultures. All error bars are standard deviations from different measurements in experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0145] Cytokine assays in PTT-6 versus PTT-4 medium or DMEM / F12 supernatants from CL-MSCs, WJ-MSCs, and MSCs of placental MSC origin Cytokine detection was performed in MSC supernatants. Measurements and analyses were performed as described above.

[0146] The purpose of this experiment was to measure the relative levels of multiplex (PDGF-AA, PDGF-BB, IL-10, VEGF, Ang-1, and HGF), TGFβ1 singleplex, and bFGF2 singleplex cytokines in cell culture supernatants. Supernatants were obtained from mesenchymal stem cells derived from umbilical cord lining (CL), Wharton's gelatin (WJ), and placenta. Mesenchymal stem cells were cultured in PTT-6, PPT-4, or DMEM / F12 media. CL-MSCs cultured in PTT-4 WJ-MSCs cultured in PTT-4 Placental MSCs cultured in PTT-4 CL-MSCs cultured in PTT-6 WJ-MSCs cultured in PTT-6 Placental MSCs cultured in PTT-6 CL-MSCs cultured in DMEM / F12 WJ-MSCs cultured in DMEM / F12

[0147] Each sample was tested in triplicate, except for the placental supernatant sample. The purpose of this experiment was to generate a cytokine profile of MSCs cultured in either PTT-4 or PTT-6, and to compare the profiles of MSCs from different tissue origins (umbilical cord lining vs. Wharton's gelatin vs. placental MSCs). Cytokine measurements were performed as described above. This profile will reveal which stem cell populations grown in which media secrete more cytokines of interest to promote wound healing.

[0148] Figure 22: Singleplex measurement of TGFβ1. The mean fluorescence intensity (MFI) measured for the standard TGFβ1 curve throughout the experiment is shown in the graph on the left. As can be seen in the graph on the right, CL-MSCs, WJ-MSCs, and placental MSCs all produce more TGFβ1 when grown in PTT-6 than when grown in PTT-4 or DMEM / F12 (referred to only as DMEM in Figure 22).

[0149] Figure 23: Summarizing the measurement of PDGF-BB in analyzed supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT-6, PTT-4, or DMEM / F12. The mean fluorescence intensity (MFI) measured for a PDGF-BB standard curve throughout the experiments is shown in the graph on the left. Of note, no PDGF-BB was detected in any of the experiments.

[0150] Figure 24: Summarizing the measurement of IL-10 in analyzed supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT-6, PTT-4, or DMEM / F12. The mean fluorescence intensity (MFI) measured for the VEGF standard curve throughout the experiment is shown in the graph on the left. S6 shows the lowest standard used in the assay. Any samples below this are considered below detection. As can be seen in the graph on the right, CL-MSCs, WJ-MSCs, and placental MSCs all produced detectable levels of IL-10 when grown in PTT-6, while little or no IL-10 was detected when MSCs were grown in PTT-4 or DMEM / F12.

[0151] Figure 25: Summarizing the measurement of VEGF in analyzed supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT-6, PTT-4, or DMEM / F12. The mean fluorescence intensity (MFI) measured for the VEGF standard curve throughout the experiment is shown in the graph on the left. S1 shows the highest standard used in the assay. Any samples above this are considered estimated (too concentrated). As can be seen in the graph on the right, CL-MSCs, WJ-MSCs, and placental MSCs all produce much higher levels of VEGF when MSCs are grown in PTT-6 compared to when they are grown in PTT-4 or DMEM / F12.

[0152] Figure 26: Summary of multiplex measurements of bFGF. The mean fluorescence intensity (MFI) measured for the PDGF-AA standard curve throughout the experiment is shown in the graph on the left. As can be seen from the graph on the right, cultured CL-MSCs and WJ-MSCs produce more bFGF when grown in PTT-6 than when grown in PTT-4. As can be seen, CL-MSCs, WJ-MSCs, and placental MSCs all produce much lower levels of bFGF when grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12.

[0153] Figure 27: Summarizing the measurement of PDGF-AA. The mean fluorescence intensity (MFI) measured for the PDGF-AA standard curve throughout the experiment is shown in the graph on the left. S6 shows the lowest standard used in the assay. Any samples below this are considered below detection. As can be seen, CL-MSCs, WJ-MSCs, and placental MSCs all produce higher levels of PDGF-AS when MSCs are grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12.

[0154] Figure 28: Summarizing Ang-1 measurements. The mean fluorescence intensity (MFI) measured for the Ang-1 standard curve throughout the experiment is shown in the graph on the left. S1 indicates the highest standard used in the assay. Any samples above this are considered estimated (too concentrated). The graph on the right shows that CL-MSCs, WJ-MSCs, and placental MSCs all produce much higher levels of Ang-1 when MSCs are grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12.

[0155] Figure 29: Summarizing the measurement of HGF. The mean fluorescence intensity (MFI) measured for the HGF standard curve throughout the experiment is shown in the graph on the left. The graph on the right shows that CL-MSCs, WJ-MSCs, and placental MSCs all produced much higher levels of Ang-1 when MSCs were grown in PTT-6 compared to when MSCs were grown in PTT-4 or DMEM / F12.

[0156] From the above experiments, the following can be concluded: When mesenchymal stem cells, specifically those isolated from the umbilical cord compartment or isolated from the placenta, are cultured in PTT-6 medium, the secretion of factors angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF by the mesenchymal stem cell population is significantly increased compared to their production levels in PTT-4 medium or in commercial culture media such as DMEM / F12. Notably, PTT-6 medium can increase the production / secretion of these factors regardless of the natural environment / compartment of the mesenchymal stem cell population.

[0157] Since PTT-6 medium induces secretion of Ang-1, TGF-β1, VEGF, and HGF (which are all known to be involved in wound healing as discussed herein) in the mesenchymal stem cell population, it is apparent that PTT-6 medium has the effect of inducing or improving the wound healing properties of a wide range of mesenchymal stem cell populations, regardless of the native environment / compartment of the mesenchymal stem cell population from which the mesenchymal stem cells were originally derived - it is again noted here that experiment 4 was performed with a cell population that was isolated from its native environment prior to culturing in PTT-6.

[0158] In addition, culturing mesenchymal stem cells in PTT-6 from tissue explants results in a highly homogenous mesenchymal stem cell population of the amniotic membrane of the umbilical cord (containing 97.5% viable cells, of which 100% expressed each of CD73, CD90, and CD105, while 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 rows "CD34-CD45-" and "CD34-HLA-DR-"). PTT-6 Since the culture of Wharton's gel mesenchymal stem cells in PTT-6 also has a positive effect on the production of cytokines Ang-1, TGF-β1, VEGF, and HGF, similar to the effect on the production of these cytokines in umbilical cord lining stem cells, it can be predicted that the culture of Wharton's gel in PTT-6 will also result in such a highly homogenous mesenchymal Wharton's gel stem cell population. It is therefore expected that tissue explants of other compartments of the umbilical cord, such as the culture of umbilical cord blood vessels, will produce similar homogenous perivascular cells. It is also expected that the culture of placental tissue, including the amniotic membrane of the placenta, in PTT-6 will result in a similar homogeneous placental mesenchymal stem cell population. Thus, the present invention provides a generally applicable methodology for obtaining a mesenchymal stem cell population, in which 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, and lack the expression of each of CD34, CD45, and HLA-DR.

[0159] The present invention is also characterized by the following items. 1. A method of inducing or improving wound healing properties of a mesenchymal stem cell population, comprising culturing the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). 2. The method of item 1, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, a mesenchymal stem cell population at the umbilical cord-placenta junction, a mesenchymal stem cell population from umbilical cord blood, a mesenchymal stem cell population from bone marrow, and a mesenchymal stem cell population derived from adipose tissue. 3. The method of item 2, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's gelatin (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed mesenchymal stem cell population (MC). 4. The method of any one of items 1 to 3, wherein the culture medium comprises DMEM at a final concentration of about 55-65% (v / v), F12 at a final concentration of about 5-15% (v / v), M171 at a final concentration of about 15-30% (v / v), and FBS at a final concentration of about 1-8% (v / v). 5. The method of item 4, wherein the culture 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). 6. The method of item 5, wherein the culture medium comprises 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). 7. The method of any one of items 1 to 6, wherein the culture medium further contains epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. 8. The method of item 7, wherein the culture medium contains EGF at a final concentration of about 10 ng / ml. 9. The method of any one of items 1 to 8, wherein the culture medium contains insulin at a final concentration of about 1 μg / ml to 10 μg / ml. 10. The method of item 9, wherein the culture medium contains insulin at a final concentration of about 5 μg / ml. 11. The method of any one of items 1 to 10, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 12. The method of any one of items 1 to 11, wherein the culture medium contains all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 13. The method of items 12 or 13, wherein the culture medium comprises adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 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. 14. The method of any one of items 1 to 13, wherein culturing a mesenchymal stem cell population in a culture medium defined in any one of items 1 to 13 increases expression and / or secretion of at least one of angiopoietin 1 (Ang-1), TGF-β (specifically TGF-β1), VEGF, and HGF by the mesenchymal stem cell population, as compared to a reference culture medium that does not contain all of DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum). 15. The method of item 14, wherein the reference medium consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS. 16. The method of any one of the preceding paragraphs, wherein the mesenchymal stem cell population is isolated from its native environment prior to culturing in the culture medium defined in any one of the preceding paragraphs 1 to 13. 17. The method of any one of items 1 to 15, comprising a step of isolating the mesenchymal stem cell population from a native tissue environment by culturing the native tissue in a culture medium defined in any one of items 1 to 13. 18. The method of item 17, wherein the tissue is umbilical cord tissue. 19. The method of item 18, wherein the umbilical cord tissue is selected from the group consisting of whole umbilical cord tissue, tissue comprising the amnion of the umbilical cord, tissue comprising Wharton's gel, tissue comprising the amnion, amnion and Wharton's gel, isolated umbilical cord blood vessels, Wharton's gel separated from other components of umbilical cord tissue, and isolated amnion of the umbilical cord. 20. The method of item 17, wherein the tissue comprises or is placental amniotic tissue. 21. The method of any one of items 17 to 20, wherein the umbilical cord tissue is a piece from the entire umbilical cord, a piece from the amniotic membrane of the umbilical cord, or a piece from the amniotic membrane of the placenta. 22. The method of any one of items 19 to 22, comprising culturing the umbilical cord tissue or placental amniotic tissue until cell proliferation of the amniotic mesenchymal stem cell population reaches about 70 to about 80% confluency. 23. The method of item 22, comprising a step of removing the mesenchymal stem cell population from the culture vessel used for the culture. 24. The method of item 23, wherein the step of removing the mesenchymal stem cell population from the culture vessel is carried out by enzymatic treatment. 25. The method of item 24, wherein the enzyme treatment comprises trypsin treatment. 26. The method according to any one of items 23 to 25, wherein the mesenchymal stem cell population is transferred to a culture vessel for subculture for subculture. 27. The method of any one of items 1 to 16, wherein the mesenchymal stem cell population is transferred to a subculture container for culture. 28. Mesenchymal cell populations are cultured or subcultured at 1.0 x 10 6 28. The method of item 26 or 27, wherein the cells are suspended at a concentration of 1000 cells / ml. 29. The method of item 28, wherein the mesenchymal stem cell population is subcultured in a culture medium defined in any one of items 1 to 13. 30. The method of item 29, wherein the mesenchymal stem cell population is subcultured until the mesenchymal stem cells reach about 70 to about 80% confluency. 31. The method of any one of items 26 to 30, wherein the culturing or subculturing is carried out in a self-contained bioreactor. 32. The method of item 31, wherein the bioreactor is selected from the group consisting of a parallel plate bioreactor, a hollow fiber bioreactor, and a microfluidic bioreactor. 33. The method of any one of the preceding items, wherein the culturing is carried out in a CO2 cell culture incubator at a temperature of 37°C. 34. The method of item 33, comprising a step of removing the mesenchymal stem cell population from the culture vessel used for (passage) culture. 35. The method of item 34, wherein the step of removing the mesenchymal stem cell population from the culture vessel is carried out by enzymatic treatment. 36. The method of item 35, wherein the enzyme treatment comprises trypsin treatment. 37. The method of item 36, further comprising the step of collecting the isolated mesenchymal stem cell population. 38. The method of any one of the preceding items, 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 express markers CD73, CD90, and CD105. 39. The method of any one of the preceding items, 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). 40. The method of any one of items 38 or 39, wherein about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR. 41. The method of any one of the preceding items, further comprising the step of storing the isolated stem / progenitor cell population for further use. 42. The method of claim 41, wherein the preserving step is carried out by cryopreservation. 43. An isolated mesenchymal stem cell population, wherein at least about 90% or more of the cells of said stem cell population express each of the markers CD73, CD90, and CD105. 44. The mesenchymal stem cell population of item 43, wherein at least about 90% or more of the cells of the stem cell population lack expression of the markers CD34, CD45, and HLA-DR. 45. The mesenchymal stem cell population of item 44, 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, and lack expression of each of CD34, CD45, and HLA-DR. 46. ​​The mesenchymal stem cell population according to any one of items 43 to 45, which is selected from the group consisting of a mesenchymal stem cell population from an umbilical cord, a mesenchymal stem cell population from a placenta, a mesenchymal stem cell population from umbilical cord blood, a mesenchymal stem cell population from bone marrow, and a mesenchymal stem cell population derived from adipose tissue. 47. The mesenchymal stem cell population according to any one of items 43 to 46, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's gelatin (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed mesenchymal stem cell population (MC). 48. A mesenchymal stem cell population according to any one of items 43 to 47, obtainable by the method defined in any one of items 1 to 42. 49. A mesenchymal stem cell population according to any one of items 43 to 48, obtained by the method defined in any one of items 1 to 42. 50. A pharmaceutical composition comprising an isolated mesenchymal stem population as defined in any one of items 43 to 47, wherein at least about 90% or more of the cells of the stem cell population express each of the markers CD73, CD90, and CD105, and lack expression of each of the markers CD34, CD45, and HLA-DR. 51. The pharmaceutical composition of item 50, adapted for systemic or local application. 52. The pharmaceutical composition of item 50 or 51, further comprising a pharma- ceutically acceptable excipient. 53. A method for producing a culture medium suitable for inducing or improving the wound healing properties of a mesenchymal stem cell population, comprising: i. 250 ml of DMEM ii. M171 118ml iii. 118 ml of DMEM / F12 iv. Fetal Bovine Serum (FBS) 12.5 ml (final concentration 2.5%) The method further comprising the step of mixing 54. v. 1 ml of EGF stock solution (5 μg / ml) to achieve a final concentration of 10 ng / ml vi. 0.175 ml of insulin stock solution (14.28 mg / ml) to achieve a final concentration of 5 μg / ml 54. The method of claim 53, further comprising the step of adding 55. The method of items 53 or 54, further comprising the step of adding one or more of the supplements adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) to the DMEM, thereby bringing the total volume of the culture medium to 500 ml. 56. The final concentration of supplements in DMEM is 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 to 5 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3), e.g., 1.36 ng / ml of 3,3',5-triiodo-L-thyronine sodium salt (T3) The method of item 55. 57. A cell culture medium obtainable by the method according to any one of Items 53 to 56. 58. A method for inducing or improving wound healing properties of mesenchymal stem cells, comprising culturing amniotic tissue in a culture medium prepared by the method defined in any one of items 53 to 56. 59. The method of item 58, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, an umbilical cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population, and adipose tissue-derived mesenchymal stem cell population. 60. The method of item 59, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's gelatin (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed mesenchymal stem cell population (MC). 61. - 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 A cell culture medium comprising: 62. The cell culture medium of item 61 containing DMEM at a final concentration of approximately 57.5-62.5% (v / v), F12 at a final concentration of approximately 7.5-12.5% ​​(v / v), M171 at a final concentration of approximately 17.5-25.0% (v / v), and FBS at a final concentration of approximately 1.75-3.5% (v / v). 63. The cell culture medium of item 62, containing a final concentration of about 61.8% (v / v) DMEM, a final concentration of about 11.8% (v / v) F12, a final concentration of about 23.6% (v / v) M171, and a final concentration of about 2.5% (v / v) FBS. 64. The cell culture medium according to any one of items 61 to 62, further comprising epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. 65. The cell culture medium according to any one of items 61 to 65, comprising EGF at a final concentration of about 10 ng / ml. 66. The cell culture medium of any one of items 61 to 65, comprising insulin at a final concentration of about 1 μg / ml to 10 μg / ml. 67. The cell culture medium of item 66 containing insulin at a final concentration of about 5 μg / ml. 68. The cell culture medium according to any one of items 61 to 67, further comprising at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 69. The cell culture medium of item 68 containing all three of the following: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 70. The cell culture medium of item 68 or 69, comprising adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 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. 71. 500 ml of cell culture medium is i. 250 ml of DMEM ii. M171 118ml iii. 118 ml of DMEM / F12 iv. Fetal Bovine Serum (FBS) 12.5 ml (final concentration 2.5%) 71. The cell culture medium of any one of items 61 to 70, comprising: 72. v. EGF at a final concentration of 10 ng / ml vi. Insulin at a final concentration of 5 μg / ml vi. Insulin 0.175 ml (final concentration 5 μg / ml) 72. The cell culture medium of item 71, further comprising: 73. The cell culture medium of item 71 or 72, further comprising adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 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. 74. Use of a cell culture medium as defined in any one of items 61 to 73 for inducing or improving wound healing properties of a mesenchymal stem cell population. 75. Use of a cell culture medium as defined in any one of items 61 to 73 for the isolation of a mesenchymal stem cell population. 76. Use of item 74 or 75, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, an umbilical cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population, and adipose tissue-derived mesenchymal stem cell population. 77. The method of item 76, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's gelatin (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed mesenchymal stem cell population (MC). 78. The use of any one of items 74 to 77, wherein at least about 90% or more of the cells of the mesenchymal stem cell population express each of the markers CD73, CD90, and CD105. 79. The use of item 78, wherein at least about 90% or more of the cells of the mesenchymal stem cell population lack expression of the markers CD34, CD45, and HLA-DR. 80. The use of item 79, 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, and lack expression of each of CD34, CD45, and HLA-DR. 81. A pharmaceutical composition containing three or four of Ang-1, TGF-β1, VEGF, or HGF as the only wound healing proteins. 82. The pharmaceutical composition of item 81, formulated as a liquid or as a lyophilized material / lyophilized preparation.

[0160] It will be readily apparent to one 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.

[0161] All patents and publications mentioned in this specification are indicative of the level 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.

[0162] The invention illustratively described herein can be suitably carried out in the absence of any element or elements, or limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing" and the like are to be read inclusively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description, not 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, and it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, although the invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention embodied therein disclosed herein may be left to those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention. The invention is 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 also 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. In addition, 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 members or subgroups of members of that Markush group. Further aspects of the invention will become apparent from the appended claims.

[0163] Sequence information SEQUENCE LISTING <110> CellResearch Corporation Pte. Ltd. <120> A method of inducing or improving wound healing properties of Mesenchymal stem cells <150> US 62 / 656,531 <151> 2018-04-12 <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> Humana <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 Lys 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 His Ser Thr Ser Tyr Asn Ser Lys Ala 565 570 575 Leu Ile Ala Phe Leu Ala Phe Leu Ile Ile Val Thr Ser Ile Ala Leu 580 585 590 Leu Val Val Leu Tyr Lys Ile Tyr Asp Leu His Lys 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 Leu Met Asn Val Glu Pro Ile His Ala Asp Ile Leu Leu 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> human <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> Humana <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 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 Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg 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 Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile 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. A method for preparing a conditioned medium from isolated mesenchymal stem cells, comprising: The isolated mesenchymal stem cells are umbilical cord amniotic stem cells or Wharton's gelatin (WJ) mesenchymal stem cells; The method comprises: culturing the isolated mesenchymal stem cells in a medium containing 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 FBS at a final concentration of 1-8% (v / v); - discarding the medium; - washing the cultured mesenchymal stem cells; - culturing the cultured mesenchymal stem cells in a medium containing DMEM; and Harvesting the conditioned medium Including, The method.

2. A conditioned medium obtained from isolated mesenchymal stem cells, comprising: The conditioned medium, wherein the isolated mesenchymal stem cells are umbilical cord amniotic stem cells or Wharton's gelatin (WJ) mesenchymal stem cells, and the conditioned medium is obtained by the method of claim 1.

3. 1. An isolated population of mesenchymal stem cells comprising: at least 97% or more of the cells of said stem cell population express each of the markers CD73, CD90, and CD105, and lack expression of each of CD34, CD45, and HLA-DR; and the mesenchymal stem cell population is selected from the group consisting of umbilical cord mesenchymal stem cell populations selected from the group consisting of perivascular (PV) mesenchymal stem cell populations, Wharton's gelatin (WJ) mesenchymal stem cell populations, and mixed umbilical cord mesenchymal stem cell populations (MC); or the mesenchymal stem cell population is selected from the group consisting of a placental mesenchymal stem cell population and a mesenchymal stem cell population of the umbilical cord-placenta junction; The mesenchymal stem cell population.

4. 4. The mesenchymal stem cell population of claim 3, wherein 98% or more of the isolated mesenchymal stem cells express the markers CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR.

5. 5. The mesenchymal stem cell population of claim 4, wherein 99% or more of the isolated mesenchymal stem cells express the markers CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR.

6. A pharmaceutical composition comprising an isolated mesenchymal stem cell population as defined in any one of claims 3 to 5.

7. 7. The pharmaceutical composition of claim 6, adapted for systemic or local application.

8. 8. The pharmaceutical composition of claim 6 or 7, further comprising a pharma- ceutically acceptable excipient.

9. To improve the wound healing properties of mesenchymal stem cell populations, - DMEM at a concentration of 55-65% (v / v), - F12 at a concentration of 5-15% (v / v), M171 at a concentration of 15–30% (v / v), and - FBS at a concentration of 1-8% (v / v) An in vitro use of a cell culture medium comprising The in vitro use, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and a mesenchymal stem cell population of the umbilical cord-placenta junction.

10. 10. The use according to claim 9, wherein the cell culture medium comprises DMEM at a concentration of 57.5-62.5% (v / v), F12 at a concentration of 7.5-12.5% ​​(v / v), M171 at a concentration of 17.5-25.0% (v / v), and FBS at a concentration of 1.75-3.5% (v / v).

11. 11. The use of claim 10, wherein the cell culture medium comprises DMEM at a concentration of 61.8% (v / v), F12 at a concentration of 11.8% (v / v), M171 at a concentration of 23.6% (v / v), and FBS at a concentration of 2.5% (v / v).

12. For the in vitro isolation of mesenchymal stem cell populations, - DMEM at a concentration of 55-65% (v / v), - F12 at a concentration of 5-15% (v / v), M171 at a concentration of 15–30% (v / v), and - FBS at a concentration of 1-8% (v / v) Use of a cell culture medium comprising: the mesenchymal stem cell population is selected from the group consisting of umbilical cord mesenchymal stem cell populations selected from the group consisting of perivascular (PV) mesenchymal stem cell populations, Wharton's gelatin (WJ) mesenchymal stem cell populations, and mixed umbilical cord mesenchymal stem cell populations (MC); or the mesenchymal stem cell population is selected from the group consisting of a placental mesenchymal stem cell population and a mesenchymal stem cell population of the umbilical cord-placenta junction; The above uses.

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