Methods for inducing or improving the wound healing properties of mesenchymal stem cells

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 Invention This application claims priority to U.S. Provisional Patent Application No. 62 / 656,531, filed on 12 April 2018, the contents of which are incorporated herein by reference in their entirety for all purposes.

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

[0003] Background of the Invention Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord were first reported in U.S. Patent Application No. 2006 / 0078993 (Patent Document 1) (leading to registered U.S. Patents No. 9,085,755 (Patent Document 2), No. 9,737,568 (Patent Document 3), and No. 9,844,571 (Patent Document 4)) and the corresponding International Patent Application WO2006 / 019357 (Patent Document 5). Since then, umbilical cord tissue has attracted attention as a source of pluripotent cells; stem cells isolated from the umbilical cord, and specifically from the amniotic membrane of the umbilical cord (also referred to as "cord lining stem cells"), are widely available and are therefore considered an excellent alternative source of cells for regenerative medicine. See Jeschke et al. Umbilical Cord Lining Membrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities and Differences; The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 21-27 (Non-Patent Literature 1).

[0004] Subsequent studies compared the phenotype, proliferation rate, migration, immunogenicity, and immunomodulatory capacity of human mesenchymal stem cells (MSCs) derived from umbilical cord amniotic membrane (umbilical cord lining (CL-MSC)), umbilical cord blood (CB-MSC), placenta (P-MSC), and Wharton's gelatinous tissue (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 (Non-Patent Literature 2)). 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 exhibit low immunogenicity and enhanced proliferative and migratory capabilities, suggesting they represent the most promising potential for cell-based therapies. Therefore, future research should focus on the best disease models in which CL-MSCs can be administered.

[0005] While amniotic mesenchymal stem cells can be readily obtained using the protocols described in U.S. Patent Application No. 2006 / 0078993 (Patent Document 1) and International Patent Application WO2006 / 019357 (Patent Document 5), having a method at hand to isolate a population of these umbilical cord-lined MSCs that is highly homogeneous and therefore usable in clinical trials is advantageous for clinical trials using these umbilical cord-lined MSCs. In addition, having a method at hand to induce or improve the wound healing properties of mesenchymal stem cell populations in general is advantageous.

[0006] Therefore, an object of the present invention is to provide a method for inducing or improving the wound healing properties of a mesenchymal stem cell population. Another object is to isolate a population of mesenchymal stem cells from the amniotic membrane of the umbilical cord that satisfies this requirement. Thus, providing a highly homogeneous population of mesenchymal stem cells is also an object of the present invention. [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 [Overview of the project]

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

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

[0011] In a second aspect, the present 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 the expression of the following markers: CD34, CD45, and HLA-DR. In multiple 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. Additionally, 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 lack the expression of the markers CD34, CD45, and HLA-DR. The mesenchymal stem cell population can be obtained by the method of inducing or improving the wound healing properties of the first aspect. Thus, the method of the first aspect can also be a method of isolating a mesenchymal stem cell population.

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

[0013] In a fourth aspect, the present invention provides a method for preparing a culture medium for inducing or improving the wound healing properties of a mesenchymal stem cell population or for isolating a mesenchymal stem cell population, the method comprising obtaining a culture medium with a final volume of 500 ml by i. 250 ml of DMEM ii. 118 ml of M171 iii. 118 ml of DMEM / F12 iv. 12.5 ml of fetal bovine serum (FBS) to obtain a final concentration of 2.5% (v / v) including the step of mixing.

[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 for 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 - 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) and provides a cell culture medium containing the same.

[0017] In an eighth aspect, the present invention provides the use of the cell culture medium of the seventh aspect for inducing or improving the wound healing properties of a mesenchymal stem cell population or for isolating a mesenchymal stem cell population. [Invention 1001] A method for inducing or improving the wound healing properties of a mesenchymal stem cell population, comprising the step of culturing the mesenchymal stem cell population in a culture medium containing DMEM (Dulbeccoo's modified Eagle medium), F12 (Ham F12 medium), M171 (Medium 171), and FBS (fetal bovine serum). [Invention 1002] The method of the present invention 1001, wherein the mesenchymal stem cell population is selected from the group consisting of umbilical cord mesenchymal stem cell population, placental mesenchymal stem cell population, umbilical cord-placental junction mesenchymal stem cell population, umbilical cord blood mesenchymal stem cell population, bone marrow mesenchymal stem cell population, and adipose tissue-derived mesenchymal stem cell population. [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 gelatinous (WJ) mesenchymal stem cell population, amniotic membrane mesenchymal stem cell population of the umbilical cord, and mixed mesenchymal stem cell population (MC) of the umbilical cord. [Invention 1004] A method according to any one of the present invention 1001 to 1003, wherein the culture medium comprises 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 FBS at a final concentration of approximately 1-8% (v / v). [Invention 1005] The method of the present invention 1004, wherein the culture medium comprises 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). [Invention 1006] The method of the present invention 1005, wherein the culture medium comprises DMEM at a final concentration of approximately 61.8% (v / v), F12 at a final concentration of approximately 11.8% (v / v), M171 at a final concentration of approximately 23.6% (v / v), and FBS at a final concentration of approximately 2.5% (v / v). [Invention 1007] A method according to any one of the present invention 1001 to 1006, wherein the culture medium further contains epidermal growth factor (EGF) at a final concentration of approximately 1 ng / ml to approximately 20 ng / ml. [Invention 1008] The method of the present invention 1007, wherein the culture medium contains EGF at a final concentration of approximately 10 ng / ml. [Invention 1009] A method according to any one of the present invention 1001 to 1008, wherein the culture medium contains insulin at a final concentration of approximately 1 μg / ml to 10 μg / ml. [Invention 1010] The method of the present invention 1009, wherein the culture medium contains insulin at a final concentration of approximately 5 μg / ml. [Invention 1011] A method according to any one of the present invention 1001 to 1010, wherein the culture medium further comprises at least one of the supplementing substances adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). [Invention 1012] Any method of the present invention 1001 to 1011, wherein the culture medium contains all three of the following: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). [Invention 1013] The method of the present invention 1012 or 1013, wherein the culture medium comprises adenine at a final concentration of approximately 0.01 to approximately 0.1 μg / ml, hydrocortisone at a final concentration of approximately 0.1 to approximately 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of approximately 0.5 to approximately 5 ng / ml. [Invention 1014] A method according to any one of the inventions 1001 to 1013, wherein culturing a population of mesenchymal stem cells in a culture medium defined in any one of the inventions 1001 to 1013 increases the 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 compared to a reference culture medium that does not contain DMEM (Dulbecc's modified Eagle medium), F12 (Ham F12 medium), M171 (Medium 171), and FBS (fetal bovine serum). [Invention 1015] The method of the present invention 1014, wherein the reference medium consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS. [Invention 1016] Any method of the present invention, wherein a population of mesenchymal stem cells is isolated from its natural environment before being cultured in a culture medium as defined in any one of the above-described items 1001 to 1013. [Invention 1017] A method of any of the present inventions 1001 to 1015, comprising the step of isolating a population of mesenchymal stem cells from a natural tissue environment by culturing natural tissue in a culture medium defined in any of the present inventions 1001 to 1013. [Invention 1018] The method of the present invention 1017, wherein the tissue is umbilical cord tissue. [Invention 1019] The method of the present invention 1018, wherein the umbilical cord tissue is selected from the group consisting of the entire umbilical cord, the tissue containing the amnion of the umbilical cord, the tissue containing Wharton's gelatinous substance, the tissue containing the amnion, the amnion and Wharton's gelatinous substance, isolated umbilical cord vessels, Wharton's gelatinous substance separated from other components of the umbilical cord tissue, and isolated amnion of the umbilical cord. [Invention 1020] The method of the present invention 1017, wherein the tissue includes or is amniotic tissue of the placenta. [Invention 1021] The method according to any of the above 1017 to 1020 of the present invention, wherein the umbilical cord tissue is a fragment from the entire umbilical cord, a fragment from the amniotic membrane of the umbilical cord, or a fragment from the amniotic membrane of the placenta. [Invention 1022] A method according to any one of the present invention 1019 to 1021, comprising the step of culturing umbilical cord tissue or placental amniotic tissue until the cell proliferation of the amniotic mesenchymal stem cell population reaches a concentration density of approximately 70 to approximately 80%. [Invention 1023] The method of the present invention 1022, comprising the step of removing a population of mesenchymal stem cells from a culture vessel used for cultivation. [Invention 1024] The method of the present invention 1023, wherein the step of removing the mesenchymal stem cell population from the culture vessel is performed by enzymatic treatment. [Invention 1025] The method of the present invention 1024, wherein the enzymatic treatment includes trypsin treatment. [Invention 1026] A method according to any of items 1023 to 1025 of the present invention, wherein a population of mesenchymal stem cells is transferred to a culture vessel for subculturing. [Invention 1027] A method according to any of items 1001 to 1016 of the present invention, wherein a population of mesenchymal stem cells is transferred to a culture vessel for subculturing for cultivation. [Invention 1028] Mesenchymal cell populations are required for culture or subculture at a rate of 1.0 × 10⁶ 6 The method of the present invention 1026 or 1027, wherein the cells are suspended at a concentration of cells / ml. [Invention 1029] The method of the present invention 1028, wherein a population of mesenchymal stem cells is subcultured in a culture medium defined in any of the present inventions 1001 to 1013. [Invention 1030] The method of the present invention 1029, wherein a population of mesenchymal stem cells is subcultured until the concentration density of mesenchymal stem cells reaches approximately 70-80%. [Invention 1031] Any method of the present invention 1026 to 1030, wherein culture or subculturing is performed in a self-contained bioreactor. [Invention 1032] The method of the present invention 1031, wherein the bioreactor is selected from the group consisting of a parallel plate bioreactor, a hollow fiber bioreactor, and a microfluidic bioreactor. [Invention 1033] Any of the methods of the present invention, wherein the culture is performed in a CO2 cell culture incubator at a temperature of 37°C. [Invention 1034] The method of the present invention 1033, comprising the step of removing a population of mesenchymal stem cells from a culture vessel used for (subculturing). [Invention 1035] The method of the present invention 1034, wherein the step of removing a population of mesenchymal stem cells from a culture vessel is performed by enzymatic treatment. [Invention 1036] The method of the present invention 1035, wherein the enzymatic treatment includes trypsin treatment. [Invention 1037] The method of the present invention 1036, further comprising the step of collecting an isolated population of mesenchymal stem cells. [Invention 1038] Any method 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, and about 99% or more of the isolated mesenchymal stem cells express the markers CD73, CD90, and CD105. [Invention 1039] Any method 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, or 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). [Invention 1040] The method of either invention 1038 or 1039, wherein approximately 97% or more, approximately 98% or more, or approximately 99% or more of the isolated mesenchymal stem cells express CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR. [Invention 1041] Any method of the present invention further comprising the step of storing an isolated population of stem / progenitor cells for further use. [Invention 1042] The method of the present invention 1041, wherein the preservation step is carried out by cryopreservation. [Invention 1043] An isolated population of mesenchymal stem cells, wherein at least about 90% or more of the cells in the population of mesenchymal stem cells express the markers CD73, CD90, and CD105, respectively. [Invention 1044] A mesenchymal stem cell population according to the present invention 1043, wherein at least about 90% or more of the cells in the stem cell population lack the expression of markers CD34, CD45, and HLA-DR. [Invention 1045] A mesenchymal stem cell population of the present invention 1044, 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, and about 99% or more cells of the isolated mesenchymal stem cell population express CD73, CD90, and CD105 respectively, and lack expression of CD34, CD45, and HLA-DR respectively. [Invention 1046] A mesenchymal stem cell population selected from the group consisting of umbilical cord mesenchymal stem cell populations, placental mesenchymal stem cell populations, umbilical cord blood mesenchymal stem cell populations, bone marrow mesenchymal stem cell populations, and adipose tissue-derived mesenchymal stem cell populations, any of the mesenchymal stem cell populations described in invention 1043 to 1045. [Invention 1047] A mesenchymal stem cell population of the umbilical cord selected from the group consisting of mesenchymal stem cell populations of the amnion (AM), mesenchymal stem cell populations of the perivascular (PV), mesenchymal stem cell populations of Wharton's gelatinous tissue (WJ), mesenchymal stem cell populations of the amnion of the umbilical cord, and mixed mesenchymal stem cell populations (MC) of the umbilical cord, any of the mesenchymal stem cell populations of the present invention 1043 to 1046. [Invention 1048] A population of mesenchymal stem cells according to any of the methods defined in any of the inventions 1001 to 1042, wherein the population is according to any of the inventions 1043 to 1047. [Invention 1049] A population of mesenchymal stem cells obtained by any of the methods defined in any of the inventions 1001 to 1042, as described in any of the inventions 1043 to 1048. [Invention 1050] A pharmaceutical composition comprising an isolated mesenchymal stem population as defined in any of invention 1043 to 1047, wherein at least about 90% or more of the cells in the stem cell population express each of the markers CD73, CD90, and CD105, and lack the expression of each of the markers CD34, CD45, and HLA-DR. [Invention 1051] A pharmaceutical composition of the present invention 1050, suitable for systemic or topical application. [Invention 1052] A pharmaceutical composition according to the present invention 1050 or 1051, further comprising pharmaceutically acceptable excipients. [Invention 1053] To induce or improve the wound healing properties of mesenchymal stem cell populations, - DMEM with a concentration of approximately 55-65% (v / v), - F12 with 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 cell culture medium containing [the specified ingredient]. [Invention 1054] Use of Invention 1053, wherein the cell culture medium contains DMEM at a concentration of approximately 57.5-62.5% (v / v), F12 at a concentration of approximately 7.5-12.5% ​​(v / v), M171 at a concentration of approximately 17.5-25.0% (v / v), and FBS at a concentration of approximately 1.75-3.5% (v / v). [Invention 1055] Use of Invention 1054, wherein the cell culture medium contains approximately 61.8% (v / v) concentration of DMEM, approximately 11.8% (v / v) concentration of F12, approximately 23.6% (v / v) concentration of M171, and approximately 2.5% (v / v) concentration of FBS. [Invention 1056] Use of a cell culture medium as defined in any of invention 1053 to 1055 for the isolation of a mesenchymal stem cell population. [Invention 1057] The use of any of the inventions 1053 to 1056, wherein the mesenchymal stem cell population is selected from the group consisting of umbilical cord mesenchymal stem cell populations, placental mesenchymal stem cell populations, umbilical cord blood mesenchymal stem cell populations, bone marrow mesenchymal stem cell populations, and adipose tissue-derived mesenchymal stem cell populations. [Invention 1058] Use of 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 gelatinous ostium (WJ) mesenchymal stem cell population, amniotic membrane mesenchymal stem cell population of the umbilical cord, and mixed mesenchymal stem cell population (MC) of the umbilical cord. [Brief explanation of the drawing]

[0018] The present invention will be better understood by referring to the detailed description, in conjunction with non-limiting embodiments and drawings.

[0019] [Figure 1-1] The Lonza technical information sheet for Dulbecco's Modified Eagle Medium is shown in the Experimental section, including the catalog number of the DMEM used to prepare an example of the culture medium (PTT-6) of the present invention. [Figure 1-2] See the explanation in Figure 1-1. [Figure 2] This shows Lonza's technical information sheet regarding Ham F12 culture medium. [Figure 3] The Lonza technical information sheet for DMEM:F12 (1:1) medium, including the catalog number of the DMEM:F12 (1:1) medium used to prepare an example of the culture medium (PTT-6) of the present invention in the Experiment section, is shown below. [Figure 4-1] The technical information sheet from Life Technologies Corporation regarding M171 medium, including the catalog number of M171 medium used to prepare an example of the culture medium (PTT-6) of the present invention in the experimental section, is shown below. [Figure 4-2] See the explanation in Figure 4-1. [Figure 5] The following is a list of the components used in the experiment to prepare culture medium PTT-6, including their commercial suppliers and catalog numbers. [Figure 6A]Figures 6A-C show the results of flow cytometry experiments analyzing the expression of mesenchymal stem cells (MSC) markers CD73, CD90, and CD105 in MSCs isolated from the umbilical cord. For these experiments, MSCs were isolated from MSCs by culturing MSCs in three different culture media, and then the MSCs were subcultured in each medium. In these experiments, the following three culture media were used: a) 90% (v / v / DMEM) supplemented with 10% FBS (v / v), b) culture medium PTT-4 as described in U.S. Patent Application US 2008 / 0248005 and the 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, whose composition is described herein. In this flow cytometry analysis, two different samples of umbilical cord-lined mesenchymal stem cell (CLMC) populations were analyzed for each of the three culture media used. The results are shown in Figures 6A-6C. More specifically, Figure 6A shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue 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 and culture from umbilical cord tissue 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 and culture from umbilical cord tissue in PTT-6. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 7A]Figures 7A-B show the results of flow cytometry experiments analyzing the expression of stem cell markers CD73, CD90, and CD105, CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D-related), which are used to determine the suitability of pluripotent human mesenchymal stem cells for cell therapy, in mesenchymal stem cells isolated from umbilical cord, and comparing this with the expression of these markers in bone marrow mesenchymal stem cells. For this experiment, mesenchymal stem cells from the amniotic membrane of the umbilical cord were isolated from umbilical cord tissue by culturing 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 a standard protocol. Figure 7A shows the percentage of isolated mesenchymal umbilical cord-lining stem cells that express the stem cell markers CD73, CD90, and CD105 and lack the expression of CD34, CD45, and HLA-DR after isolation and culture from umbilical cord tissue in PTT-6 medium. Figure 7B shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90, and CD105 and lack the expression of CD34, CD45, and HLA-DR. [Figure 7B] See the explanation in Figure 7A. [Figure 8] The experimental setup is shown, with dark gray wells containing standard materials reconstituted using PTT-4 medium and corresponding samples from MSCs cultured in PTT-4; and light gray wells containing standard materials reconstituted using PTT-6 medium and corresponding samples from MSCs cultured in PTT-6. Italicized samples are control supernatants tested as part of a repeat test of storage samples. [Figure 9] This shows singleplex measurements of TGFβ1. As can be seen, CL-MSC and WJ-MSC cultures 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 the standard deviation from the triplicate measurements. [Figure 10]Figure 10A shows the multiplex measurement of PDGF-AA. As can be seen, 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 the standard deviation from the triple measurement. Figure 10B shows the multiplex measurement of VEGF. As can be seen, 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 the standard deviation from the triple measurement. Figure 10C shows the multiplex measurement of Ang-1. As can be seen, CL-MSC and WJ-MSC cultures produce significantly more Ang-1 when grown in PTT-6 than when grown in PTT-4. The AT-MSC and BM-MSC cultures essentially produced no Ang-1. All error bars represent the standard deviation from the triplicate measurements. [Figure 11] The multiplex measurements of HGF are shown. As can be seen, CL-MSC and WJ-MSC cultures produce significantly more HGF when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produced virtually no HGF. All error bars represent the standard deviation from the triplicate measurements. [Figure 12] This shows multiplex measurements of PDGF-AA. As can be seen, CL-MSC and WJ-MSC cultures produce more PDGF-AA when grown in PTT-4 than when grown in PTT-6. AT-MSC and BM-MSC cultures produced the same amount of PDGF-AA in both cultures. All error bars represent the standard deviation from the triplicate measurements. [Figure 13]Figure 13A shows the multiplex measurement of VEGF. As can be seen, 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 the triple measurements. Figure 13B shows the multiplex measurement of Ang-1. As can be seen, CL-MSC and WJ-MSC cultures produce significantly more Ang-1 when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produced virtually no Ang-1. All error bars are standard deviations from the triple measurements. Figure 13C shows the multiplex measurement of HGF. As can be seen, CL-MSC and WJ-MSC cultures produce significantly more HGF when grown in PTT-6 than when grown in PTT-4. The AT-MSC and BM-MSC cultures essentially produced no HGF. All error bars represent the standard deviation from the triplicate measurements. [Figure 14] Multiplex measurements of bFGF are shown. As can be seen, CL-MSC and WJ-MSC cultures produce more bFGF when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produced the same amount of bFGF when grown in PTT-4 and PTT-6. All error bars represent the standard deviation from the triplicate measurements. [Figure 15]This section summarizes the measurements of TGFβ1 across 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 left graph. The MFI for the TGFβ standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. The lower right graph shows that CL-MSC and WJ-MSC cultures 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, and 180226. [Figure 16] This section summarizes the measurements of Ang-1 across 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 left graph. The MFI for the Ang-1 standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. The lower right graph shows that CL-MSC and WJ-MSC cultures 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 represent the standard deviation from different measurements in experiments 170602, 170511, 170414, 170224, 180105, and 180226. [Figure 17]This report summarizes the measurements of PDGF-BB across 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 lower left graph. The MFI for the PDGF-BB standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. Notably, PDGF-BB was not detected in any of the experiments. [Figure 18] This section summarizes the measurements of PDGF-AA across 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 left graph. The MFI for the PDGF-AA standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. The lower right graph shows that the cultures CL-MSC, AT-MSC, BM-MSC, and WJ-MSC produced slightly more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are the standard deviation from the measurements in experiments 170602, 170511, 170414, 170224, 180105, and 180226. [Figure 19] This section summarizes the measurements of IL-10 across six different experiments (170602, 170511, 170414, 170224, 180105, 180226). The mean fluorescence intensity (MFI) measured for the IL-10 standard curve throughout the experiments is shown in the lower left graph. The MFI for the IL-10 standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. Notably, IL-10 was not detected in any of the experiments. [Figure 20]This section summarizes VEGF measurements across 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 left graph. The MFI for the VEGF standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. The lower right graph shows that the cultures CL-MSC, AT-MSC, BM-MSC, and WJ-MSC produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are the standard deviations from the different measurements in experiments 170602, 170511, 170414, 170224, 180105, and 180226. [Figure 21] This section summarizes HGF measurements across 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 lower left graph. The MFI for the HGF standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. The lower right graph 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 the standard deviations from different measurements in experiments 170602, 170511, 170414, 170224, 180105, and 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, all CL-MSCs, WJ-MSCs, and placental MSCs 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). [Figure 23]This report summarizes the measurement of PDGF-BB in the 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 PDGF-BB standard curve throughout the experiments is shown in the graph on the left. Notably, PDGF-BB was not detected in any of the experiments. [Figure 24] This report summarizes the measurement of IL-10 in the 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 left-hand graph. S6 indicates the lowest standard used in the assay. Any sample below this is considered subdetectable. As can be seen from the right-hand graph, all CL-MSCs, WJ-MSCs, and placental MSCs produced detectable levels of IL-10 when grown in PTT-6, while IL-10 was little to no detectable when MSCs were grown in PTT-4 or DMEM / F12. [Figure 25] This report summarizes the measurement of VEGF in the 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 left-hand graph. S1 indicates the highest standard substance used in the assay. Any sample exceeding this is considered estimated (too high concentration). As can be seen from the right-hand graph, all CL-MSCs, WJ-MSCs, and placental MSCs produce significantly higher levels of VEGF when grown in PTT-6 compared to when the MSCs are grown in PTT-4 or DMEM / F12. [Figure 26]This summarizes the 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 the MSCs are grown in PTT-4 or DMEM / F12. [Figure 27] The measurement of PDGF-AA is summarized below. The mean fluorescence intensity (MFI) measured for the PDGF-AA standard curve throughout the experiment is shown in the graph on the left. S6 indicates the lowest standard used in the assay. Any sample below this is considered subdetectable. As can be seen, CL-MSCs, WJ-MSCs, and placental MSCs all produce higher levels of PDGF-AA when grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12. [Figure 28] The measurement of Ang-1 is summarized below. 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 substance used in the assay. Any sample exceeding this is considered estimated (too high concentration). The graph on the right shows that CL-MSCs, WJ-MSCs, and placental MSCs all produce significantly higher levels of Ang-1 when grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12. [Figure 29] The measurements of HGF are summarized below. 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 produce much higher levels of Ang-1 when grown in PTT-6 compared to when MSCs grow in PTT-4 or DMEM / F12. [Modes for carrying out the invention]

[0020] Detailed description of the invention As described above, in the first phase, the present invention relates to a method for inducing or improving the wound healing properties of a mesenchymal stem cell population, the method comprising the step of culturing a mesenchymal stem cell population in a culture medium containing DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). It has been surprisingly found in this application that the use of such media has the effect of inducing or improving the wound healing properties of a wide range of mesenchymal stem cell populations, regardless of the natural environment / enclosure of the mesenchymal stem cell population. While we do not wish to be bound by theory, it is thought that the induction or improvement of the wound healing properties of the mesenchymal stem cell population is caused by the ability of the media of the present invention to increase the expression and / or secretion of at least one, two, three, or all of angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF by the mesenchymal stem cell population. The expression / secretion of angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF by the mesenchymal stem cell population of the umbilical cord amniotic membrane has been shown to have superior wound healing properties in U.S. Patent Application US 2008 / 0248005 and the corresponding International Patent Application W02007 / 046775 (see Examples 23-26 of W02007 / 046775, which show that such a mesenchymal stem cell population of the umbilical cord amniotic membrane (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)). The culture medium (PTT-4) used in U.S. Patent Application US 2008 / 0248005 and International Patent Application W02007 / 046775 for the isolation of the mesenchymal stem cell population of the umbilical cord amniotic membrane. See the section on experiments demonstrating the increase in mesenchymal stem cell populations cultured in the PTT-6 culture medium of the present invention compared to cultured in other mediums.As shown in the Experiments section of this specification, culturing in a medium containing DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum) increases the levels of angiopoietin 1 (Ang-1), TGF-β1, VEGF, and / or HGF not only in the mesenchymal stem cell population of the amniotic membrane of the umbilical cord, but also in the mesenchymal stem cell populations of other compartments of the umbilical cord, such as Wharton's gelatinous tissue, or (adjacent) compartments, such as the placenta. Therefore, this application is considered to provide generally applicable teachings for inducing or improving the wound healing properties of a given mesenchymal stem cell population by culturing it in the medium of the present invention, such as Medium PTT-6.

[0021] In this regard, the findings of the present invention that an increase in the combination of 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 lead to mimicking the wound healing properties of the stem cell population with a composition / solution containing three or four of Ang-1, TGF-β1, VEGF, or HGF as the sole wound healing protein.

[0022] In this regard, it should be noted that the involvement of the proteins angiopoietin-1 (Ang-1), TGF-β1, VEGF, and HGF in the wound healing process is known to those skilled in the art. For the involvement of angiopoietin-1 in wound healing, see, for example, Li et al. Stem Cell Research & Therapy 2013, 4:113 "Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing" 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 Li et al.'s study, 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 angiogenesis, compared to MSCs, Ad-Ang1, or sham treatments." Notably, the authors of Li et al. stated that mesenchymal stem cells (MSCs) alone do not produce sufficient Ang-1, and for this reason, they created genetically modified cells by inserting the Ang-1 gene into MSCs. In contrast to Li's study, it was surprisingly found in this application that culturing "natural" mesenchymal stem cells in a culture medium such as PTT-6, for example, umbilical cord tissue mesenchymal stem cells (i.e., the mesenchymal stem cell population cultured in PTT-6), produces elevated levels of Ang-1, thereby providing conditions that make the mesenchymal stem cells suitable for wound healing or further improve their wound healing properties.This means that the present invention offers the advantage that, instead of genetically modifying naturally occurring mesenchymal stem cells to induce their wound-healing properties (which is not only laborious but also not a preferred option for therapeutic application due to the inherent risks of gene therapy), the wound-healing properties of naturally occurring mesenchymal stem cells are induced or enhanced by simply culturing a population of mesenchymal stem cells in the culture medium of the present invention. This approach is simpler, safer, and 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 βl-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 more information on the role of vascular endothelial growth factor (VEGF) in wound healing, specifically 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 information on the involvement of transforming growth factor beta (including TGF-β1, TGF-β2, and TGF-β3) in wound healing, specifically 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 regard, it should also be noted that the present invention has the further remarkable advantage that culturing in the culture medium of the present invention results in the isolation of mesenchymal stem cell populations, such as mesenchymal stem cell populations of the amniotic membrane of the umbilical cord, in which more than 90% or even 99% or more of the cells are positive for three mesenchymal stem cell markers CD73 and CD90, while at the same time these stem cells lack expression of CD34, CD45, and HLA-DR (see the Experiments section), which means that 99% or even more of the cells in this population express the stem cell markers CD73, CD90, and CD105, while not expressing the markers CD34, CD45, and HLA-DR. Such extremely homogeneous and clearly defined cell populations are ideal candidates for clinical trials and cell-based therapies, as they fully meet 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. Furthermore, by using bioreactors such as the Quantum cell proliferation system, it is possible to obtain a large number of mesenchymal stem cells, such as 300 million to 700 million per run (see also the section on experiments).Therefore, the present invention offers the further advantage of providing, in a cost-effective manner, the amount of stem cells necessary for therapeutic applications such as use in wound healing. In addition, all components used to prepare the culture medium of the present invention are commercially available in GMP quality. Thus, the present invention opens a route for GMP production of highly homogeneous mesenchymal stem cell populations, for example, placental tissue or umbilical cord tissue, such as mesenchymal stem cell populations of the amniotic membrane of the umbilical cord or mesenchymal stem cell populations of Wharton's gelatinous ostoma.

[0027] The mesenchymal stem cell population that is made suitable for wound healing (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) may be any suitable mesenchymal stem cells known in the art, such as adult stem cell populations or neonatal stem cells. The mesenchymal stem cell population may originate from any mammalian tissue or compartment / body part known to contain mesenchymal stem cells. In practice, the mesenchymal stem cell population may be an umbilical cord mesenchymal stem cell population (these are examples of neonatal stem cells), a placental mesenchymal stem cell population (likewise a further example of neonatal stem cells), an umbilical cord-placental junction mesenchymal stem cell population (a further example of neonatal stem cell populations), an umbilical cord blood mesenchymal stem cell population (yet another example of neonatal stem cells), a bone marrow mesenchymal stem cell population (which may be an adult stem cell population), or adipose tissue-derived mesenchymal stem cell population (yet yet another example of an adult stem cell population).

[0028] The umbilical cord mesenchymal stem cell population may consist of (or originate from) any compartment of the umbilical cord tissue containing mesenchymal stem cells. The mesenchymal stem cell population may be not only the mesenchymal stem cell population of the amnion (AM), the mesenchymal stem cell population of the perivascular (PV), the mesenchymal stem cell population of Wharton's gelatinous tissue (WJ), and the mesenchymal stem cell population of the amnion of the umbilical cord, but also the mixed mesenchymal stem cell population of the umbilical cord (MC), which means a population of mesenchymal stem cells containing stem cells from two or more of these compartments. The mesenchymal stem cells of these compartments and their isolation are known to those skilled in the art, for example, as described 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 the reference 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 the umbilical cord can be obtained, for example, by removing arteries and veins from umbilical cord tissue, cutting the remaining tissue and Wharton's jelly into small pieces, and culturing the umbilical cord tissue in the culture medium of the present invention (by tissue explantation).Mixed mesenchymal stem cell populations from the umbilical cord can also be obtained by culturing the entire umbilical cord tissue with intact umbilical vessels as an explant under conditions such as those 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% equine serum, and 1% penicillin / streptomycin). In this regard, it should be noted that mesenchymal stem cell populations from the umbilical cord-placental junction can be isolated as described by 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] Accordingly, it is noted herein that, in order to induce or improve their wound healing properties, the mesenchymal stem cell populations cultured in the present invention in a culture medium containing DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum) may be isolated from their natural environment before being cultured in the culture medium of the present invention. Such an approach is specifically used for mesenchymal stem cell populations that cannot be readily isolated by tissue explants, such as umbilical cord blood mesenchymal stem cell populations or bone marrow mesenchymal stem cell populations. However, this approach may also be employed for umbilical cord mesenchymal stem cell populations, placental mesenchymal stem cell populations, or adipose tissue-derived mesenchymal stem cell populations. 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 by 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, which includes DMEM (Dulbeccoo's modified Eagle medium), F12 (Ham's F12 medium), M171 (Medium 171), and FBS (fetal bovine serum).Furthermore, the placental mesenchymal stem cell population can be isolated from the placenta and subsequently cultured in the culture medium of the present invention, as described, for example, 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 and subsequently cultured in the culture medium of the present invention, 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 the references cited therein (see also the Experiments section).As a further example, mesenchymal stem cell populations from the umbilical cord-placental junction can also be initially 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 specifically 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 a methodology is particularly suitable for culturing mesenchymal stem cell populations derived from umbilical cord tissue, placental tissue (placental tissue may, for example, include or be the amniotic membrane of the placenta), or the umbilical cord-placental junction.

[0031] In this regard, it should be noted that the culture medium of the present invention also makes it possible to isolate a mesenchymal stem cell population (also referred to herein as "mesenchymal stem cells") from its natural environment. Therefore, the culture medium of the present invention also makes it possible to isolate a mesenchymal stem cell population under conditions that enable cell proliferation of mesenchymal stem / progenitor cells without differentiation of the mesenchymal stem / progenitor cells.

[0032] In one embodiment, the culture medium of the present invention makes it possible to isolate a population of mesenchymal stem cells from the amniotic membrane under conditions that allow for the proliferation of mesenchymal stem / progenitor cells without differentiation of the mesenchymal stem / progenitor cells. Thus, after isolating mesenchymal stem cells from the amniotic membrane as described herein, the isolated population of mesenchymal stem / progenitor cells has the ability to differentiate into multiple cell types, as described in, for example, U.S. Patent Application No. 2006 / 0078993, U.S. Patent No. 9,085,755, International Patent Application No. WO2006 / 019357, U.S. Patent No. 8,287,854, or WO2007 / 046775. For example, as described in U.S. Patent Application No. 2006 / 0078993, mesenchymal stem cells from the amniotic membrane of the umbilical cord are spindle-shaped, express the genes POU5f1, Bmi-1, and leukemia suppressor (LIF), and secrete activin A and follistatin. The mesenchymal stem cells isolated in this invention can be differentiated into any type of mesenchymal cell, including, but is not limited to, cutaneous fibroblasts, chondrocytes, osteoblasts, tendinocytes, ligamentous fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes, mucin-producing cells, insulin-producing cells (e.g., β-islet cells), or neuroectoderm cells. The stem cells isolated in this invention can be differentiated in vitro for later use as differentiated cells for medical purposes. An example of such an approach is the differentiation of mesenchymal stem cells into insulin-producing β-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 this invention can be used in an undifferentiated state for cell-based therapies for wound healing purposes, such as the treatment of burns or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells of the present invention may help promote wound healing by interacting with the surrounding affected tissue, or they may differentiate into their respective skin cells (see, for example, WO2007 / 046775 again).

[0033] In accordance with the above disclosure, it should be noted here that such mesenchymal stem cell populations described herein can be isolated and cultured from any umbilical cord tissue (i.e., derived from any umbilical cord tissue), insofar as the umbilical cord tissue contains the amnion (also referred to as the “umbilical cord lining”). Thus, the mesenchymal stem cell population can be isolated from the entire umbilical cord (or a fragment thereof), as described in the Experiments section of this application. Thus, this umbilical cord tissue may contain any other tissue / components of the umbilical cord in addition to the amnion. For example, as shown in Figure 16 of U.S. Patent Application No. 2006 / 0078993 or International Patent Application WO2006 / 019357, the amnion 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 within Wharton's colloid, a gelatinous substance largely composed of mucopolysaccharides. Thus, the umbilical cord tissue used in this invention may also include this one vein, two arteries, and Wharton's colloid. The use of such an entire (intact) portion of the umbilical cord has the advantage that it is not necessary to separate the amnion from the other components of the umbilical cord. This reduces the isolation step, and consequently makes the method of this invention simpler, faster, less prone to error, and more economical—all important aspects of GMP production required for the therapeutic application of mesenchymal stem cells. Therefore, the isolation of mesenchymal stem cells can be started from the tissue explant, and then, if a larger quantity of mesenchymal stem cells is desired for use, for example, in clinical trials, the isolated mesenchymal stem cells can be subsequently subcultured (cultured). Alternatively, it is also possible to isolate mesenchymal umbilical cord-lining stem cells from the amnion by first separating the amnion from the other components of the umbilical cord and culturing the amnion in the culture medium of this invention. This culture can also be performed using tissue explants, and optionally, subsequent subculture of isolated mesenchymal stem cells is carried out.

[0034] In this regard, the terms “explant” or “explant method” are used in their usual sense in the art to refer to a method in which, once a tissue (e.g., placental tissue or umbilical cord tissue) or tissue fragment is recovered, it is placed in a cell culture dish containing a culture medium (growth medium), and stem cells migrate from the tissue to the surface of the dish over time. These primary stem cells can then be further increased by micropropagation (subculture), as also described herein, and transferred to a new dish. In this regard, it should be noted that, in terms of generating cells for therapeutic purposes, in the first step of isolating / obtaining the mesenchymal stem cell population of the present invention, for example, umbilical cord mesenchymal stem cells such as amniotic mesenchymal stem cells or Wharton's colloidal 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, a population of mesenchymal stem cells in which at least about 97% or more, 98% or more, or 99% or more cells express the markers CD73, CD90, and CD105, respectively, and lack the expression of the markers CD34, CD45, and HLA-DR, respectively) is used for clinical trials or as an approved therapy, a cell population from a working cell bank is typically used for this purpose. Both the isolated stem cell population (which may constitute a master cell bank) and the subcultured stem cell population (which may constitute a working cell bank) can be stored, for example, in cryopreservation form.

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

[0036] "Inducing or improving the wound healing properties of a mesenchymal stem cell population" means, as herein, the ability of a 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 a mesenchymal stem cell population. As described above, the involvement of all four of these proteins in wound healing is well known. "Inducing or improving wound healing properties" is evaluated by comparing the culture of mesenchymal stem cell populations in reference (culture) media such as PTT-4 (consisting of 90% (v / v) CMRL1066 and 10% (v / v) FBS), which was used in U.S. Patent Application US 2008 / 0248005 and International Patent Application W02007 / 046775 for the isolation and culture of mesenchymal stem cell populations of umbilical cord amniotic membrane, demonstrating superior wound healing properties in U.S. Patent Application US 2008 / 0248005 and the corresponding International Patent Application W02007 / 046775. Compared to culturing a mesenchymal stem cell population in a reference medium, if the mesenchymal stem cell population secretes at least one of the four marker proteins Ang-1, TGF-β1, VEGF, and HGF in greater quantities into the supernatant / culture medium (corresponding to higher secretion levels or higher concentrations), the wound healing properties of the mesenchymal stem cell population are enhanced. 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 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, at least three, or all of the four marker proteins Ang-1, TGF-β1, VEGF, and HGF is increased compared to culturing the stem cell population in the reference medium. The secretion of the four marker proteins into the culture medium (and consequently, the production of these factors by the stem cell population) can be measured / determined by any suitable method, for example, by measuring the amount of protein using a commercially available antibody / immunoassay (see the section on experiments).Such measurements can be performed in an automated manner using systems such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).

[0037] "DMEM" refers to Dulbecco's Modified Eagle Medium, a modified version of the basic Eagle medium (BME), developed in 1969 (see Figure 1, which shows the datasheet for DMEM available from Lonza). The first DMEM formulation contained 1000 mg / L of 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 various sources, including ThermoFisher Scientific (catalog no. 11965-084), Sigma Aldrich (catalog no. D5546), or Lonza, to name just a few suppliers. Therefore, any commercially available DMEM can be used in this invention. In a preferred embodiment, the DMEM used herein is DMEM medium available from Lonza under catalog no. 12-604F. This medium is DMEM supplemented with 4.5 g / L of glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is Sigma Aldrich catalog number D5546 DMEM medium, which contains 1000 mg / L glucose and sodium bicarbonate but does not contain L-glutamine.

[0038] "F12" medium refers to Ham F12 medium. This medium is also a standard cell culture medium and is a nutrient mixture originally designed to culture a wide variety of mammalian and hybridoma cells when used with serum in combination with hormones and transferrin (see Figure 2, which shows the datasheet for Ham F12 medium from Lonza). Any commercially available Ham F12 medium (for example, from just a few suppliers, ThermoFisher Scientific (catalog no. 11765-054), Sigma Aldrich (catalog no. N4888), or from Lonza) can be used in this invention. In a preferred embodiment, Ham F12 medium from Lonza is used.

[0039] "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham F12 culture medium (see Figure 3, which shows the datasheet for DMEM:F12 (1:1) medium from Lonza). DMEM / F12 (1:1) medium is also a widely used basic medium for supporting the growth of many different mammalian cells and is commercially available from various suppliers such as ThermoFisher Scientific (catalog no. 11330057), Sigma Aldrich (catalog no. D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in this invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM / F12 (1:1) medium available from Lonza under catalog no. 12-719F (DMEM:F12 with L-glutamine, 15 mM HEPES, and 3.151 g / L glucose).

[0040] "M171" refers to Culture Medium 171, developed as a basic culture medium for the proliferation of normal human mammary epithelial cells (see Figure 4, which shows the datasheet for M171 medium from Life Technologies Corporation). This basic medium is also widely used and commercially available from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in this invention. In a preferred embodiment, the M171 medium used herein is the M171 medium available from Life Technologies Corporation under catalog number M171500.

[0041] "FBS" refers to fetal bovine serum (also known as bovine fetal serum), that is, the blood fraction remaining after natural blood coagulation, 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 contains very low levels of antibodies, more growth factors, and allows for versatility in many different cell culture applications. It is preferable to obtain FBS from members of the International Serum Industry Association (ISIA), whose main focus is on the safety and safe use of serum and animal-derived products through proper origin tracing, truthfulness of labeling, and proper standardization and monitoring. ISIA member suppliers of FBS include, to name a few, Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production. In the currently preferred embodiment, FBS is obtained from GE Healthcare under catalog number A15-151.

[0042] Now, turning our attention to the culture medium of the present invention, the culture medium may contain, for the purpose of inducing or improving the wound healing properties of mesenchymal stem cells, or for the isolation or culture of mesenchymal stem cells, 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 FBS at a final concentration of approximately 1-8% (v / v). The value "% (v / v)" as used herein refers to the volume of each component relative to the final volume of the culture medium. For example, if DMEM is present in the culture medium at a final concentration of approximately 55-65% (v / v), it means that 1 liter of culture medium contains approximately 550-650 ml of DMEM.

[0043] In another embodiment, the culture medium may contain 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). In yet another embodiment, the culture medium may contain DMEM at a final concentration of approximately 61.8% (v / v), F12 at a final concentration of approximately 11.8% (v / v), M171 at a final concentration of approximately 23.6% (v / v), and FBS at a final concentration of approximately 2.5% (v / v).

[0044] In addition to the above components, the culture medium may contain supplements that are advantageous for culturing mesenchymal umbilical cord-lining stem cells. The culture medium of the present invention may contain, for example, epidermal growth factor (EGF). If 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. If present, insulin may be present at a final concentration of approximately 1 μg / ml to 10 μg / ml. In some of these embodiments, the culture medium may contain insulin at a final concentration of approximately 5 μg / ml.

[0046] The culture medium may further contain at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may contain all three: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may contain adenine at a final concentration of about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0047] In one aspect of the method of the present invention, tissue such as umbilical cord tissue or placenta can be cultured until an appropriate number of (primary) mesenchymal stem cells, such as umbilical cord-lining stem cells, Wharton's gelatinous tissue, or placental stem cells, proliferate from the tissue. In a typical embodiment, umbilical cord tissue is cultured until the proliferation of mesenchymal stem cells in each tissue reaches a concentration density of about 70–80%. It should be noted herein that the terms “concentration density” or “cluster” are used in their usual sense in the art of cell culture and mean an estimate / indicator of the number of adherent cells in a culture dish or flask, referring to the percentage of the surface covered by cells. For example, 50 percent concentration means that about half of the surface is covered and there is still room for cells to proliferate. 100 percent concentration 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 appropriate number of primary cells (mesenchymal stem cells) have been obtained from each tissue by tissue explantation, the mesenchymal stem cells are removed from the culture vessel used for cultivation. This allows for the creation of a master cell bank containing, for example, isolated (primary) mesenchymal stem cells from the umbilical cord or placenta. Typically, since such mesenchymal stem cells are adherent cells, cell recovery is performed using standard enzymatic treatment. For example, the enzymatic treatment may include trypsin treatment, as described in International U.S. Patent Application No. 2006 / 0078993, International Patent Application WO2006 / 019357, or International Patent Application WO2007 / 046775, which means that proliferating cells can be recovered by trypsin treatment (0.125% trypsin / 0.05% EDTA) for further growth. If the recovered mesenchymal stem cells are to be used, for example, to create a master cell bank, the cells may also be cryopreserved and stored for further use, as described below herein.

[0049] Once recovered, the mesenchymal stem cells can be transferred to a culture vessel for subculturing. Subculturing or culture (both terms are used interchangeably hereafter herein) is also carried out when a population of mesenchymal stem cells previously isolated from its natural environment is used (as described above, such isolated stem cells used in the methods of the present invention may originate from umbilical cord blood, bone marrow, or adipose tissue, as well as from umbilical cord tissue or placental tissue). Subculturing can also be initiated from frozen primary cells, i.e., from a master cell bank. For subculturing, any appropriate 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, about 0.5 × 10⁶ 6 cells / ml ~ approx. 5.0×10 6 The cells can be suspended at a concentration of cells / ml in a suitable medium for subculturing (most conveniently, the culture medium of the present invention). In one embodiment, the cells are subculturified in a medium of about 1.0 × 10⁶. 6Suspend at a concentration of cells / ml. Subculturing can be carried out by culturing in a simple culture flask, or by culturing in a multi-layer system such as CellStack (Corning, Corning, NY, USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA), which can be stacked in an incubator. Alternatively, subculturing can also be carried out in a closed, self-contained system such as a bioreactor. Various designs of bioreactors are well known to those skilled in the art, and include, for example, parallel plate, hollow fiber, or microfluidic bioreactors. See, for example, Sensebe et al. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," mentioned above. An example of a commercially available hollow fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT, Inc.), which is used for increasing 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 population of the present invention is the Xuri Cell Expansion System, available from GE Heathcare. Culturing mesenchymal stem cells in automated systems such as the Quantum® Cell Expansion System is particularly effective when a working cell bank for therapeutic application should be generated under GMP conditions and a large number of cells are required.

[0050] The subculture of mesenchymal stem cells according to the present invention is carried out in the culture medium of the present invention. Therefore, the culture medium of the present invention can be used for both the isolation of mesenchymal stem cell populations from, for example, the amniotic membrane of the placenta, or the Wharton's gelatinous tissue of the umbilical cord, and the subsequent culture of the isolated primary cells by subculture. Similarly, with subculture, mesenchymal stem cells can be cultured until an appropriate number of cells have proliferated. In an illustrative embodiment, mesenchymal stem cells are subcultured until they reach a concentration density of about 70-80%.

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

[0052] Once the desired / appropriate number of mesenchymal stem cells have been obtained from culture or subculture, they are recovered by removing the mesenchymal stem cells from the culture vessel used for subculture. Recovery of mesenchymal stem cells is typically carried out by enzymatic treatment, including trypsin treatment of the cells, in this case as well. The isolated mesenchymal stem cells are then collected and either used immediately or stored for further use. Typically, storage is carried out by cryopreservation. The term "cryopreservation" is used herein in its usual sense to describe the process by which mesenchymal stem cells are preserved by cooling them to a sub-zero temperature, such as (typically) -80°C or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be carried out as is known to those skilled in the art and may involve the use of cryoprotective agents such as dimethyl sulfoxide (DMSO) or glycerol, which slow the formation of ice crystals in the umbilical cord cells.

[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 a typical embodiment 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, and 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, and 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, approximately 97% or more, approximately 98% or more, or approximately 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] Accordingly, consistent with the above disclosure, the present invention also targets a mesenchymal stem cell population, e.g., a placental mesenchymal stem cell population, or an umbilical cord mesenchymal stem cell population (e.g., isolated from Wharton's gelatinous tissue or the amniotic membrane of the umbilical cord), wherein at least about 90% or more of the cells in the stem cell population express each of the following markers: CD73, CD90, and CD105. In a preferred embodiment, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, and about 99% or more of the cells in the isolated mesenchymal stem cell population are CD73+, CD90+, and CD105+, meaning that this proportion 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, and about 99% or more of 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 isolated mesenchymal stem cell populations lack expression of CD34, CD45, and HLA-DR, while expressing CD73, CD90, and CD105. Such a highly homogeneous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord is reported herein for the first time and meets the criteria for mesenchymal stem cells to be used in cell therapy (see the Experiments section, and also see, for example, Sensebe et al. "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review," also mentioned above). In this regard, it should be noted that this mesenchymal stem cell population can be obtained by the isolation method of the present invention, but may also be obtained by other methods such as cell sorting, if desired.In one embodiment of such a mesenchymal stem cell population of the umbilical cord according to the present invention, in which at least about 91% or more of the cells in the stem cell population express CD73, CD90, and CD105 respectively, and lack the expression of CD34, CD45, and HLA-DR, the mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord is excluded.

[0055] In accordance with the foregoing, the present invention also relates to a pharmaceutical composition comprising a population of mesenchymal stem cells as described herein, wherein at least about 90% or more of the cells in the stem cell population express the markers CD73, CD90, and CD105, respectively, and optionally lack the expression of CD34, CD45, and HLA-DR. The pharmaceutical composition may contain any pharmaceutically acceptable excipients and may be formulated for any desired method of pharmaceutical administration. The pharmaceutical composition may be adapted for, for example, systemic or topical application. In a relevant 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 protein. Such a pharmaceutical composition may be formulated as a liquid or as a lyophilized substance / lyophilized preparation by using a pharmaceutically suitable buffer such as 0.9% saline, Ringer's solution, or phosphate-buffered saline (PBS), for example.

[0056] In a further aspect, the present invention relates to a method for preparing a culture medium for inducing or improving wound healing properties and / or for isolating a population of mesenchymal stem cells, the method for obtaining a final volume of 500 ml of culture medium. i. DMEM 250 ml ii. M171 118 ml iii. DMEM / F12 118 ml iv. 12.5 ml of fetal bovine serum (FBS) to reach a final concentration of 2.5% (v / v) This includes the step of mixing the ingredients.

[0057] As explained above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham F12 culture medium. Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Thus, when using this method to prepare the culture medium, the final concentration (v / v) in a total volume of 500 ml is 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] The embodiment of this method for preparing the culture medium is: v. 1 ml of EGF preservation solution (5 μg / ml) to achieve a final EGF concentration of 10 ng / ml, and vi. 0.175 ml of insulin storage solution (14.28 mg / ml) to achieve a final insulin concentration of 5 μg / ml. The process further includes the step of adding [a certain substance].

[0059] In these embodiments, it is noted herein that the combined volumes of these components i-vi yield a final culture medium of 499.675 ml. If no further components are added to the culture medium, the remaining 0.325 ml (to make a total volume of 500 ml) may be any of components i-iv, meaning, for example, DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of the EGF or insulin storage solution 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 they are listed, and it is, of course, possible to mix these components in any order to achieve 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 obtain the final concentrations described herein, namely, a final concentration of approximately 55-65% (v / v) for DMEM, approximately 5-15% (v / v) for F12, approximately 15-30% (v / v) for M171, and approximately 1-8% (v / v) for FBS.

[0060] In another embodiment, the method further comprises the step of adding one or more of the supplements adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) to DMEM in a volume of 0.325 ml, thereby bringing the total volume of the culture medium to 500 ml. In this embodiment, the final concentrations of these supplements in DMEM may be as follows: Adenine at approximately 0.05-0.1 μg / ml, for example, adenine at approximately 0.025 μg / ml, Approximately 1-10 μg / ml of hydrocortisone, Approximately 0.5 to 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] In accordance with the above disclosure, the present invention also relates to cell culture media that can be obtained or obtained by the methods for preparing the media described herein.

[0062] In addition, the present invention also relates to a method for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, the method comprising the step of culturing amniotic tissue in a culture medium prepared by the method described herein.

[0063] Therefore, the present invention also, - DMEM with a final concentration of approximately 55-65% (v / v), - F12 with a final concentration of approximately 5-15% (v / v), - M171 with a final concentration of approximately 15-30% (v / v), and - FBS with a final concentration of approximately 1-8% (v / v) The target is cell culture media containing the following:

[0064] In certain embodiments of the culture medium described herein, the medium comprises 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). In other embodiments, the culture medium may comprise DMEM at a final concentration of approximately 61.8% (v / v), F12 at a final concentration of approximately 11.8% (v / v), M171 at a final concentration of approximately 23.6% (v / v), and FBS at a final concentration of approximately 2.5% (v / v).

[0065] In addition, the culture medium may further contain epidermal growth factor (EGF) at a final concentration of approximately 1 ng / ml to approximately 20 ng / ml. In a particular embodiment, the culture medium contains EGF at a final concentration of approximately 10 ng / ml. The culture medium described herein may further contain insulin at a final concentration of approximately 1 μg / ml to 10 μg / ml. In such an embodiment, the culture medium may contain insulin at a final concentration of approximately 5 μg / ml.

[0066] The cell culture medium of the present invention may further contain 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 contains all three: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). If present, the culture medium may contain adenine at a final concentration of about 0.01 to about 0.1 μg / ml or about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml or about 1 to about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

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

[0068] In a further aspect, the cell culture medium is v. EGF at a final concentration of 10 ng / ml, and vi. Insulin with a final concentration of 5 μg / ml This may further include:

[0069] Both insulin and EGF can be added to the culture medium using an optimal preservation solution, ensuring that the total volume of the culture medium does not exceed 500 ml.

[0070] In certain examples, components i-vi of the culture medium of the present invention are the components shown in Figure 5, meaning that they are available from each manufacturer using the catalog numbers shown in Figure 5. The culture medium obtained by mixing components i-vi as shown in Figure 5 is also referred to herein as "PTT-6". In this regard, it should be noted again that components i-vi and any other components, such as antibiotics, from any other commercial supplier may be used in preparing the culture medium of the present invention.

[0071] In addition, the cell culture medium of the present invention may contain adenine at a final concentration of approximately 0.01 to approximately 0.1 μg / ml or approximately 0.05 to approximately 0.1 μg / ml, hydrocortisone at a final concentration of approximately 0.1 to 10 μg / ml, approximately 0.5 to approximately 10 μg / ml, or approximately 1 to approximately 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of approximately 0.1 to approximately 5 ng / ml or approximately 0.5 to approximately 5 ng / ml.

[0072] Finally, the present invention also provides a method for treating a non-human mammal (to name just a few examples, cats, dogs, horses, etc.) or a human patient having a disease or suffering from a condition, the method comprising the step of administering to the non-human mammal or human patient a pharmaceutical composition containing a mesenchymal stem cell population or stem cell population as disclosed herein. The disease may be any disease or condition, specifically any disease or condition in which wound healing is desired / needed. The subject (patient or non-human mammal) may have a wound resulting from a disease such as a burn, bite, trauma, surgery, or a skin disease or metabolic disorder. As an example of such a metabolic disorder, the patient may have, for example, type 1 or type 2 diabetes, or chronic foot ulcers. To treat the subject, the mesenchymal stem cell population of the present invention can be administered by any suitable method, including, for example, topical administration, transplantation, or injection, but not limited to these. In principle, any topical administration method is as defined herein. Administration of the mesenchymal stem cell population can be done by syringe. However, before applying the mesenchymal stem cells to a target, it is also possible to contact the mesenchymal stem cells in a cream, ointment, gel, suspension, or any other suitable substance. The stem cell population may then be placed directly on a wound, for example, a burn or diabetic wound (see International Patent Application WO2007 / 046775). After application to a target, the mesenchymal stem cell population may 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 may be transplanted subcutaneously, for example directly under the skin, into body fat, or into the peritoneum.

[0073] The present invention also relates to unit doses of approximately 15 million cells, approximately 10 million cells, approximately 5 million cells, approximately 4 million cells, approximately 3 million cells, approximately 2 million cells, approximately 1 million cells, approximately 500,000 cells, approximately 250,000 cells, or less than 250,000 cells, including approximately 20 million cells of the mesenchymal stem cell population described herein.

[0074] It is also conceivable that the unit dose may contain approximately 10 million, 9 million, 8 million, 7 million, 6 million, 5 million, 4 million, 3 million, 2 million, 1 million, 500,000, 250,000, or 100,000 cells. Preferably, the unit dose contains approximately 10 million cells. It is further conceivable that the unit dose contains approximately 1,000 to 5 million cells. The unit dose can be applied in doses of approximately 100,000, 300,000, or 500,000 cells. As described herein, the unit dose may be applied topically, specifically when used for wound healing. For example, the unit dose may be cm 2 It can be applied locally in each case.

[0075] If necessary, the unit dose can be applied once, twice, three times, or more times per week. For example, the unit dose can be applied over one, two, three, four, five, six, seven, eight, nine, ten, eleven weeks, or longer. A unit dose containing approximately 100,000 cells, approximately 300,000 cells, or approximately 500,000 cells is preferably 1 cm 2 This can be applied twice a week for eight weeks.

[0076] The unit dose can be contained in any suitable container. For example, the unit dose can be contained in a 1 ml vial. In such a case, for example, 0.1 ml of the vial is preferably 1 cm 2 Each dose can be applied to the target individual. The unit dose may be contained in a syringe.

[0077] In the unit dose of the present invention, the cells may be in contact with a pharmaceutically acceptable carrier, such as a liquid carrier. The carrier may 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 this case, the mesenchymal stem cells may be separated from the carrier before administration. For example, the cells may be centrifuged and isolated before administration to the subject.

[0078] The treatment method and unit dose of the present invention may include the use of living cells. The viability of the mesenchymal stem cell population can be tested by known methods, for example, by staining with trypan blue as described in the Experiments 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 in this specification are shown in Table 1 below.

[0082] (Table 1) Sequences of proteins used in this specification TIFF0007876213000001.tif160170TIFF0007876213000002.tif245170TIFF00078762130 00003.tif245170TIFF0007876213000004.tif245170TIFF0007876213000005.tif180170 [Examples]

[0083] Experimental Examples 1. Cryopreservation of umbilical cord tissue before isolating mesenchymal stem cells. Umbilical cord tissue (the umbilical cord was donated with the mother's informed consent) was processed as follows to subsequently isolate mesenchymal stem cells from the amniotic membrane of the umbilical cord.

[0084] 1.1 Washing of umbilical cord tissue samples: a. Remove the surgical scalpel from its protective cover. b. Using forceps, firmly hold the umbilical cord and cut it into 10 cm long pieces using a surgical scalpel. Return any unusable tissue to the original tissue cup. c. Transfer the 10 cm long umbilical cord fragment to a new 150 mm culture dish. A 150 mm culture dish can also be used instead of a cup. d. Use the cover of the 150 mm culture dish as a place to put forceps and surgical scalpels. e. Dispense 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 Plasmalyte A using a 30 ml syringe and blunt needle. g. Collect used Plasmalyte A in a 300 ml transfer bag that will serve as a waste container, and dispose of it in a biohazard waste bin. h. Repeat the washing procedure using a new culture dish for each wash, as needed. Ensure that all blood clots on the surface have been removed. If tissue cleansing is required, additional Plasmalyte A may be used. i. Place the tissue into a new, labeled tissue culture dish and continue cutting the tissue. Add 20 ml of Plasmalyte A to the dish to prevent the tissue from drying out during cutting. j. Cut the umbilical cord into sections of approximately 1 cm in size, for a total of 10 sections. k. Each 1 cm section is further cut into smaller pieces of approximately 0.3 cm × 0.3 cm to 0.5 cm × 0.5 cm each. l. Remove all Plasmalyte A from the dish. m. Withdraw 25 ml of Plasmalyte A from the original Plasmalyte A bag using a 30 ml syringe and dispense it directly onto the umbilical cord tissue sample. n. Hold the culture dish at an angle and collect all the Plasmalyte A used for tissue washing on one side, then remove it with a syringe and blunt needle. o. Repeat the washing process. No blood clots should remain.

[0085] Note: If the umbilical cord is not to be frozen immediately, the umbilical cord tissue should be kept in Plasmalyte A until just before freezing.

[0086] 1.2 Cryopreservation of umbilical cord tissue: a. Prepare the cryopreservation solution: i. Prepare 50 ml of a frozen solution consisting of 60% Plasmalyte A, 30% 5% human serum albumin, and 10% dimethyl sulfoxide (DMSO). ii. Label the 150 ml transfer bag with "tissue freezing solution" and attach the plasma transfer set to the port using sterile techniques. iii. Remove 30 ml of Plasmalyte A from the original Plasmalyte A bag using a 30 ml syringe and transfer it to a transfer bag labeled "Tissue Freezing Solution" along with the date and time the solution was prepared. iv. Take 15 ml of 5% human serum albumin using a 20 ml syringe and transfer it to a labeled transfer bag. v. Add 5 ml of DMSO to the transfer bag. vi. Mix thoroughly and record the mixing of the frozen solution. b. Remove Plasmalyte A from the tissue before adding the freezing solution. c. Using a 60 ml syringe, draw out the entire 50 ml of frozen solution into the syringe and add approximately 30 ml of the frozen solution to a 150 mm cell culture dish containing umbilical cord tissue. Attach a blunt needle to the syringe and keep it sterile. d. Swirl the culture dish containing the tissue and frozen solution every minute for 10 minutes. e. Using forceps, select eight randomly chosen sections and place them into four 4 ml cryovials. Select four randomly chosen sections and place them into one 1.8 ml cryovial. These sections must not contain any blood clots. f. Fill each cryovial containing the umbilical cord tissue with the remaining frozen 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 Bactec Lytic / 10 - Anaerobic / F bottle and one Bactec Pluc Aerobic / F bottle with tissue ID labels. h. Using a syringe and blunt needle, remove 20 ml of the frozen solution from the culture dish, wipe the Bactec vial with an alcohol swab, replace the blunt needle with an 18 g needle, and inoculate 10 ml each into the aerobic and anaerobic Bactec bottles. i. Activate the controlled speed freezer. j. After the controlled-speed freezing is complete, leave the unit 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 culture medium for processing MSCs from umbilical cord tissue: a. To prepare 500 ml of PTT-6 (culture medium / growth medium), add the following in the order listed: i. DMEM 250 ml ii. M171 118 ml iii. DMEM F12 118 ml iv. FBS 12.5 ml (final concentration 2.5%) v. EGF 1 ml (final concentration 10 ng / ml) vi. Insulin 0.175 ml (final concentration 5 μg / ml).

[0088] The above volumes of components i-vi 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) may be any of components i-iv, meaning, for example, DMEM, M171, DMEM / F12, or FBS. Alternatively, the concentration of the EGF or insulin preservation solution can be adjusted so that the total volume of the culture medium is 500 ml. Alternatively, an antibiotic preservation solution such as penicillin-streptomycin-amphotericin can be added to make a final volume of 500 ml. It is also possible to add one or more of the supplements adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) in 0.325 ml volumes to the culture medium to make a total volume of 500 ml of culture medium.

[0089] vii. Label the bottle with "PTT-6" along with the date of preparation of the culture medium, the operator's initials, and the words "Expiration Date" followed by the expiration date. The expiration date is the earlier of either the earliest expiration date of any of the components or one month after the preparation date.

[0090] b. To prepare rinse medium (Hanks buffer solution (HBSS) free of 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 with the operator's initials and the date the medium was prepared, along with the label "Rinse Medium". c. Test all media for sterility using Bactec Lytic / 10 - Anaerobic / F (Becton Dickinson & Company) and Bactec Plus + Aerobic / F (Becton Dickinson & Company). Pour 20 ml of the prepared media into each bottle.

[0091] 2.2 Thawing of umbilical cord tissue for MSC recovery: a. Thawing should begin when the operator is ready to process the sample in the cleanroom. Do not thaw more than one vial at a time, except when the vials originate from the same donor. b. Wipe the water bath with disinfectant and then with 70% isopropanol, and fill it with 1 L of sterile water. Heat the water bath to 36-38°C. c. Prepare 10 ml of rinse medium consisting of 70%–90% PlasmaLyte A under a biosafety cabinet in a cleanroom. Sterile filter this solution using a 0.2-μm syringe filter attached to a 10 ml syringe, and keep the solution refrigerated until use. d. Attach a processing label to the 50 ml conical tube. e. Ensure the water bath temperature is between 36 and 38°C. f. Remove the tissue vials from liquid nitrogen storage and rapidly thaw them in a 37°C water bath filled with 1 L of sterile water. The vial holder of the Mr. Frosty Nalgene Cryo 1°C freezing container can be used as a floating rack to hold the vials in place and thaw the samples. g. Remove the vials from the water bath and spray them with a 70% isopropanol solution. The appropriate time to remove the vials from the water bath is when you can see small ice crystals floating inside the vials—this suggests that the internal temperature of the vials is below 37°C. h. Place the vial in the pass-through and inform the cleanroom technician.

[0092] 2.3 Preparation for tissue processing: a. Umbilical cord tissue processing must be performed in an environmental monitoring (EM) cleanroom. The room and hood must be thoroughly cleaned at the end of each shift. b. Prepare / clean the biosafety cabinet. c. Perform biological particle counting while working inside the biosafety cabinet. d. Gather all necessary items into the biosafety cabinet, checking for packaging damage and expiration dates. When handling syringes, serum pipettes, sterile forceps, surgical scalpels, tissue plates, and needles, never touch any surfaces that may come into contact with sterile products. Only the outside of syringes, tubes, plunger tips, and / or needle caps or cases may be handled safely. Discard any items if any surfaces are touched or if any surface comes into contact with a non-sterile surface. e. Record the lot number and expiration date (if applicable) of all reagents and supplies used. f. Receive the thawed vials by cleaning them with a lint-free wipe moistened with 70% alcohol and then moving them into a biosafety cabinet. g. Using an aspiration needle attached to a syringe, remove as much liquid as possible from the vial. Avoid aspirating tissue. h. Using sterile forceps, transfer the tissue to a sterile 100 mm Petri dish. i. Add a fixed fraction of 5 ml of rinse medium to the tissue fragment. j. Swirl the contents for 15-30 seconds, then remove the rinse medium using a pipette or syringe with a suction needle. Repeat this rinsing process twice. k. Add 2 mL of rinse medium to the tissue to prevent it from drying out.

[0093] 2.4. Initiation of MSC proliferation from tissue: a. Label the bottom of the 6-well plate with the MSC lot number or umbilical cord tissue ID and the start date of proliferation, along with the label "Proliferation 1". If using a 60 mm tissue culture dish, draw a grid on the bottom of the dish to divide the plate into four sections. b. Using sterile disposable forceps, place one 3×3 mm to 5×5 mm tissue sample into each well. If using a 60 mm tissue culture dish, place the tissues in the center of each section, keeping them separated (more than 1 cm apart). c. Fill each well with 3 ml of PTT-6. d. Using a suction needle attached to a 30 ml syringe, remove just enough culture medium to barely cover the tissue. Do not tilt the plate. Do not touch the bottom of the wells with the suction needle. e. Observe cell proliferation 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. Replace the culture medium daily. Always allow the medium to equilibrate to room temperature before use. i. Remove the culture medium by aspirating it. ii. Add 3 ml of PTT-6. iii. Aspirate until the tissue is barely immersed in the culture medium. g. Once cell proliferation is observed in the tissue, transfer the tissue to a new 6-well plate using the same procedure as in 4.a-4.e above, except that the plate is labeled "Proliferation 2". Maintain cell proliferation in the "Proliferation 1" plate by adding 2 ml of PTT-6 to each well. Observe the concentration density daily. Replace the culture medium every 2-3 days (always equilibrate the medium to room temperature before use). h. Repeat steps 4a-4e, except that when cell proliferation is observed in the "Proliferation 2" plate, label the plate "Proliferation 3". Maintain cell proliferation in the "Proliferation 2" plate by adding 2 ml of PTT-6 to each well. Observe the concentration density daily. Replace the culture medium every 2-3 days (always equilibrate the medium to room temperature before use). i. Discard the tissue when proliferation is observed in the "Proliferation 3" plate. If the tissue is very small and does not hinder cell proliferation, discard the tissue during subculturing. j. Once the cells reach a density of 40-50%, monitor them daily to prevent excessive growth. k. When the cells reach a density of 70-80%, subculture the cells. Do not allow the cells to grow beyond a density of 80%.

[0094] When the size of the tissue explant is about 1 - 3 mm and the culture of the tissue explant / cells is carried out in a 175 mm square culture dish, the average number of mesenchymal stem cells recovered from the explant is typically about 4,000 - 6,000 cells / explant. Thus, when mesenchymal stem cells are simultaneously proliferated from 48 explants, about 300,000 cells can be obtained at the time of recovery. These 300,000 mesenchymal stem cells collected from the explants can then be used for subculture by seeding such 300,000 cells into a 175 cm 2 cell culture flask (which can be referred to as passage 1). Then, using the mesenchymal stem cells obtained from this passage 1, as described in Example 2.5 below, again seed into a 175 cm 2 flask (passage 2) to increase the cells. The cells obtained from both passage 1 and passage 2 can be "banked" by cryopreservation, and the mesenchymal stem cells obtained after passage 2 are regarded as representing the master cell bank, which is for further increasing the mesenchymal stem cells, for example, in a bioreactor, as described in Example 2.7 below.

[0095] 2.5. Subculture of MSCs in a cell culture dish a. During work in the biosafety cabinet, perform biological particle counting. Equilibrate all media to room temperature before use. b. When the cell growth reaches a confluence density of about 70 - 80%, subculture the cells. i. Remove PTT - 6 from the Petri dish. ii. Rinse with calcium - and magnesium - free HBSS. iii. Add 0.2 ml of 1×TrypLE - EDTA and swirl for 1 - 2 minutes. iv. Tilt the dish at 30 - 45° so that the cells can move downward by gravity flow. Gently tap the side of the plate to promote detachment. Add 1 ml of v. PTT-6. Gently pipette up and down, then transfer the cells to a 15 ml centrifuge tube. Use a clean pipette tip for each well. Pool the cells from all 6 wells into a single 15 ml tube. vi. Centrifuge at 1200 rpm for 10 minutes. vii. Remove the supernatant and resuspend the cells in 5 ml of PTT-6. c. Subculture the MSCs. i. Take 50 μl of cell suspension and assay for TNC and viability using a trypan blue exclusion assay. ii. Count the cells using a hemocytometer. Predict counting 20-100 cells per compartment. If the number is greater than 100, dilute the original sample 1:5 and repeat the trypan blue test using a hemocytometer. iii. Count live cells / ml and total live cells: 1. Live cells / ml = Number of live cells × Dilution factor × 10 4 2. Total viable cells = Number of viable cells × Dilution factor × Total volume × 10 4 iv. Count the % survival rate: 1. % viability = number of live cells x 100 / (number of live cells + number of dead cells) v. Prepare a cell suspension in 1.0 × 10⁻⁶ units. 6 Dilute to cells / ml: 1. “X” Volume = Total Viable Cells / 10 6 cells / ml 2. For example, if the total number of viable cells is 1.0 × 10⁻⁶ 7 If there is only one; 3. "X" = 10 7 / 10 6 The cell volume is 10 ml, i.e., 10 ml, and therefore, by adding 5 ml to the cell suspension (5 ml), the total cell volume is increased to 10 ml. vi. Cell suspension is 10 6 If the amount is less than 1 / ml, use each 150 mm Petri dish or 175 cm 2 2 × 10 cells in a flask 6Determine the capacity required to sow each individual seed. 1. Cells 2×10 6 Capacity per cell = 2 × 10⁻¹⁰ cells 6 cells ÷ live cells / ml 2. For example, if the number of living cells / ml is 8 × 10 5 If the value is cells / ml, then 2 × 10⁶ cells 6 pieces ÷ 8×10 5 Cells / ml, or 2.5 ml, is required. vii. Set aside 0.5 ml for MSC marker analysis. viii. 2×10 cells 6 Place each 30 ml of PTT-6 in a 150 mm Petri dish or 175 cm² container. 2 Sow the seeds in a flask. ix. Observe adhesion, colony formation, and densification every three days. Once the cells reach 40-50% densification, observe them daily or every two days to prevent excessive growth. Do not allow the cells to grow beyond 80% densification. A real-time cell culture monitoring system can be used instead of an optical microscope. x. Replace the culture medium every 2-3 days.

[0096] 2.6 Cryopreservation of MSC cells a. Perform biological particle counting while working inside the biosafety cabinet. b. When the cells reach 70-80% density, use each 150 mm petri dish or 175 cm 2 Cells are detached from the flask using 2 ml of 1×TrypLE-EDTA. i. Remove the PTT-6 from the Petri dish. ii. Wash with 5 ml of HBSS or PBS that does not contain calcium and magnesium. iii. Add 2 ml of 1×TrypLE-EDTA and swirl for 1-2 minutes. iv. Tilt the dish 30-45° to allow the cells to move downwards due to gravity. Gently tap the sides of the petri dish to help facilitate detachment. Add 10 ml of v. PTT-6 to inactivate TrypLE. Mix thoroughly to dissociate the 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 culture medium and resuspend it in 10 ml of PTT-6. ix. Dispense 50 μl and determine the total viable cell count and % viability as described above. Cell counting should be performed within 15 minutes, as cells may begin to aggregate. c. Prepare cells for cryopreservation. i. Prepare cell suspension media and cryopreservation media, and freeze the cells.

[0097] 2.7. Subculturing (enlargement) of MSCs in a Quantum Bioreactor (Terumo BTC, Inc.) It is also possible to increase the number of MSCs using a Quantum bioreactor. The starting cell count for growth in a Quantum bioreactor should be 20 to 30 million cells per run. A typical yield per run is 300 to 700 million MSCs at harvest. The bioreactor is operated according to the manufacturer's protocol. The mesenchymal stem cells thus obtained are typically cryopreserved (see below) and become a working cell bank.

[0098] Materials / Reagents: 1. Quantum Enlargement Set 2. Quantum waste liquid bag 3. Quantum culture medium bag 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 Combo Tip

[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. Sterilized tube connectors 7. M4 Continuous Pipette 8. RF Sealer

[0100] procedure: 1. Preparation of the Quantum Bioreactor a) Pre-preparation of the Quantum bioreactor b) Bioreactor coating: 1) Prepare the fibronectin solution in a biosafety cabinet. 1) Acclimatize the freeze-dried fibronectin to room temperature (at room temperature for ≥ 15 minutes). 2) Add 5 ml of sterile distilled water; do not swirl or stir. 3) Allow the fibronectin to dissolve over 30 minutes. 4) Using a 10 ml syringe fitted with an 18 g needle, transfer the fibronectin solution to 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 "cleaning" as the inlet supply source). 4) Open or set the bioreactor coating program (Figure 1, steps 3-5). 5) Run the program. 6) While the program is running and coating the bioreactor, prepare a 4 L medium bag of PTT-6 medium. 7) Connect the culture medium bag to the IC culture medium line using a sterile tube connector. 8) Once the bioreactor coating stage is complete, remove the cell inlet bags used with the fibronectin solution using an RF sealer. c) Washing away excess fibronectin d) Acclimatization of the bioreactor with culture medium 2. Cell culture in a Quantum bioreactor a) Cell loading and adhesion using a homogeneous suspension: b) Nutritional support and culture of cells 1) Select the culture medium flow rate to supply nutrients to the cells. 2) Sample lactic acid and glucose daily. 3) Adjust the flow rate of the culture medium as the lactate level rises. The actual maximum acceptable lactate concentration is determined by the flask culture from which the cells originated. Ensure that there is sufficient PTT-6 medium in the medium bag. Replace the PTT-6 medium bag with a new one if necessary. 4) Once the flow rate reaches the desired value, measure the lactate level every 8-12 hours. If the lactate level does not decrease or continues to rise, collect the cells. 3. Cell recovery from the Quantum bioreactor a) Once the lactate concentration has not decreased, collect the cells after the final sampling for lactate and glucose. b) Cell harvesting: 1) Using a sterile tube connector, connect the cell inlet bag filled with TrypLE 100 ml to the "reagent" line. 2) Ensure there is enough PBS in the PBS bag. If not, use a sterile tube connector to connect a new bag containing at least 1.7 liters of PBS to the "wash" line. 3) Execute the recovery program. 4. Cryopreservation of cells 1) Once the cells have been collected, transfer them to a 50 ml centrifuge tube to pellet them. 2) Resuspend the cells in 25 ml of cold cell suspension solution. Count the cells using a Sysmex or Biorad cell counter. Attach the cell count report to each Quantum processing batch record. 3) Cell concentration 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 serial pipette, add 1 ml of the cell suspension in the cryopreservation agent to each 1.8 ml vial. Cryopreserve using a controlled-speed freezer with the CRF program as described in SOP D6.100 CB cryopreservation. 6) Store the vials in the designated liquid nitrogen storage space. 7) Attach the CRF execution report to the form for each MSC P3-Quantum processing batch.

[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 the expression of mesenchymal stem cells isolated from the umbilical cord for the 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, the following three culture media were used: a) 90% (v / v / DMEM) supplemented with 10% FBS (v / v), b) culture medium PTT-4 as described in U.S. Patent Application No. 2008 / 0248005 and the 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, whose composition is described herein. In this flow cytometry analysis, two different samples of umbilical cord-lined mesenchymal stem cell (CLMC) populations were analyzed for each of the three culture media used.

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

[0105] material and method TIFF0007876213000006.tif195152

[0106] procedure a) Isolation and culture of cells from the umbilical cord lining membrane 1. As described in Example 2, the explant tissue samples were incubated in cell culture plates, immersed in each medium, and then maintained in a CO2 incubator at 37°C. 2. The culture medium was changed every 3 days. 3. Cell proliferation from tissue culture explants was monitored under a light microscope. 4. At approximately 70% compaction, the cells were separated from the dish by trypsin treatment (0.0125% trypsin / 0.05% EDTA) and used for flow cytometry experiments. b) Trypsin treatment of experimental cells 1. Remove the culture medium from the cell culture plate. 2. Since FBS interferes with the enzymatic action of trypsin, gently rinse with sterile 1x PBS to remove any trace amounts of FBS. 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 have been removed. Neutralize the trypsin by adding complete medium containing FBS (DMEM containing 10% FBS). 5. Using a pipette, break up cell clumps by pipetting the cells against the plate wall in the culture medium. Collect the cell suspension and transfer it to a 50 ml centrifuge tube. 6. Add sterile 1×PBS to the plate, rinse it, and collect the cell suspension in the same centrifuge tube. 7. Centrifuge this at 1800 rpm for 10 minutes. 8. Discard the supernatant and resuspend the cell pellet in PBA medium. c) Cell counting 1. Preferably, the hemocytometer and its coverslip should be washed with 70% ethanol, dried, and then wiped with Kimwipes (lint-free paper) to ensure they are clean and dry. 2. Transfer a small amount of the suspended cells into a microcentrifuge tube and remove it from the BSC hood. 3. Stain the suspended cells 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. Trypan blue is toxic and can lead to an increase in non-viable cells and the production of pseudocell counts; therefore, cells should not be exposed to trypan blue for longer than 30 minutes. 5. Add 20 μl of the cell suspension mixture to each chamber of the hemocytometer and observe under a light microscope. a. For a total of eight compartments in the upper and lower chambers, count the number of viable cells (bright cells; non-viable cells readily absorb trypan blue and are therefore darker in color) in each compartment of the hemocytometer. The total cell count is (average cell count / compartment) × 10 4 It is given as cells / ml. d) Cell staining i. Preparation before staining cells Each cell suspension, containing 50,000 cells, is divided into three tubes (CD73, CD90, CD105) in pairs and two tubes (negative control). ii. Staining with primary antibody (Ab) Add 1 μl [0.5 mg / ml Ab] of primary antibody to 100 μl of cell suspension and incubate at 4°C for 45 minutes. • Adjust to 1 ml with PBA. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. Add 1 ml of PBA and resuspend the pellet. Centrifuge at 8000 rpm for 5 minutes at 4°C. Remove the supernatant. Resuspend in 100 µl of PBA. iii. Staining with secondary Ab - Under the dark Add 1 µl [0.5 mg / ml ab] of secondary antibody to 100 µl 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 µl of PBA. Transfer the cells to a FACS tube for reading using BD FACS CANDO flow cytometry.

[0107] The results of flow cytometry analysis are shown in Figures 6a to 6c. Figure 6a shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in DMEM / 10% FBS; Figure 6b shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT-4; and Figure 6c shows the percentage of isolated mesenchymal umbilical cord-lining stem cells expressing the stem cell markers CD73, CD90, and CD105 after isolation and culture from umbilical cord tissue in PTT-6. As can be seen from Figure 6a, the population isolated using DMEM / 10% FBS as the culture medium had approximately 75% CD73+ cells, 78% CD90+ cells, and 80% CD105+ cells (average of two experiments), whereas the number of CD73-positive, CD90-positive, and CD105-positive mesenchymal stem cells after isolating / culturing umbilical cord tissue using PTT-4 culture medium (see Figure 6b) was approximately 87% (CD73+ cells), 93% (CD90+ cells), and 86% (CD105+ cells) on average of two experiments. The purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention was at least 99.0% for all three markers (CD73, CD90, CD105), which means that the purity of this cell population is significantly higher than that of culture using PTT-4 medium or DMEM / 10% FBS. Furthermore, and more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is essentially a 100% pure and distinct stem cell population. This makes the stem cell population of the present invention an ideal candidate for stem cell-based therapies. Thus, this population of mesenchymal umbilical cord-lining stem cells can serve as the optimal 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 expressed the stem cell markers CD73, CD90, and CD105, and lacked the expression of CD34, CD45, and HLA-DR, after isolation and culture from umbilical cord tissue in PTT-6 medium. As shown in Figure 7a, the mesenchymal stem cell population contained 97.5% viable cells, 100% of which expressed CD73, CD90, and CD105 respectively (see the "CD73+CD90+" and "CD73+CD105+" columns), 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 the "CD34-CD45-" and "CD34-HLA-DR-" columns). Therefore, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is essentially a 100% pure and distinct stem cell population that meets the criteria for enabling mesenchymal stem cells to be 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," see above). It is noted herein that the amniotic mesenchymal stem cells of the present invention are adherent to plastic under standard culture conditions, differentiate into osteoblasts, adipocytes, and chondrocytes in vitro, and meet generally accepted standards for the use of mesenchymal stem cells in cell therapy, see U.S. Patent No. 9,085,755, U.S. Patent No. 8,287,854, or WO2007 / 046775.

[0109] Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90, and CD105, and lacking expression of CD34, CD45, and HLA-DR. As shown in Figure 7b, the bone marrow mesenchymal stem cell population contained 94.3% viable cells, of which 100% expressed CD73, CD90, and CD105 respectively (see the "CD73+CD90+" and "CD73+CD105+" columns), while only 62.8% of the bone marrow stem cell population lacked CD45 expression, and 99.9% of the stem cell population lacked CD34 and HLA-DR expression (see the "CD34-CD45-" and "CD34-HLA-DR-" columns). Therefore, bone marrow mesenchymal stem cells, considered to be the optimal standard for mesenchymal stem cells, exhibit far less uniformity / purity with respect to stem cell markers than the mesenchymal stem cell population (from the umbilical cord amniotic membrane) of this application. This finding also indicates that the stem cell population of the present invention could be an ideal candidate for stem cell-based therapies and could serve as the optimal 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 remarkable result that culturing in PTT-6 yields essentially 100% pure and defined mesenchymal stem cell populations, various isolated mesenchymal stem cell populations were cultured in PTT-6 and analyzed for wound healing marker protein secretion compared to culture in PTT-4 medium (which served as a reference medium).

[0111] More specifically, the following isolated mesenchymal stem cell populations were analyzed. - Mesenchymal stem cells from the amniotic membrane of the umbilical cord (umbilical cord-lining MSCs / CL-MSCs). This population of CL-MSCs was isolated from tissue explants of human umbilical cord-lining membrane as described in Example 2 of WO2007 / 046775 (cultured in DMEM supplemented with 10% fetal bovine serum, and DMEM / 10% FBS). - Wharton's gelatinous mesenchymal stem cells (WJ-MSCs). This population of WJ-MSCs was isolated by tissue explants of Wharton's gelatinous material 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. - Mesenchymal stem cells derived from adipose tissue (AT-MSCs). This population of AT-MSCs was isolated from adipose tissue of skin tissue donated after abdominal wall reconstruction using tissue explants (cultured 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 donated 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 5 million MSCs from each source were plated in DMEM / F12 / 10%FCS in 100 mm tissue culture dishes and left for 24 hours. The culture medium was discarded, and PTT-6 / PTT-4 was added to the culture for 24 hours. The culture medium was discarded, and the cells were washed with PBS. 10 ml of DMEM was added to the culture medium for 24 hours. The culture medium was discarded, and 5 ml of DMEM was added to the culture. After 24 hours of incubation, the conditioned medium was collected, centrifuged to remove cell fragments, and the supernatant was collected in tubes for storage at -80°C and subsequent analysis of marker protein secretion by cytokine assay.

[0113] Cytokine assay in PTT-6 vs. PTT-4 medium supernatant from CL-MSCs, WJ-MSCs, bone marrow MSCs, and adipose-derived MSCs. Cytokine detection was performed in the MSC supernatant. Measurement and analysis were performed using Luminex 200 and Xponent software.

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

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

[0116] The purpose of this experiment was to create the cytokine profiles of MSCs cultured either in PTT-4 or PTT-6, and to compare the profiles of MSCs from different tissue origins (umbilical cord lining vs Wharton's jelly vs adipose tissue vs bone marrow). This profile will reveal which stem cell population grown in which medium secretes more of the cytokines of interest for promoting wound healing.

[0117] The plate layout for the entire plate is described in Figure 8. Hereinafter, the following abbreviations are used: MSC, mesenchymal stem cell; CL, umbilical cord lining; WJ, Wharton's jelly; 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 and 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 the A chain, and PDGF-BB consists of the B homodimer. R&D states that the PDGF-BB antibody also detects the PDGF-AB heterodimer similarly) · PDGF-BB HGF, hepatocyte growth factor IL-10, Interleukin-10

[0119] TGFβ1 Singleplex Information: R&D Systems / Bio-techne) • Basic kit, catalog number LTGM00, lot number P156217, received February 27, 2018, expiration date August 30, 2018. TGFβ1 component, catalog number LTGM100, lot number P161760, received February 27, 2018, expiration date November 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 January 31, 2020. bFGF2 component, catalog number EPX01A-12074-901, lot number 169751102, expiration date December 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 January 31, 2020. bFGF2 component, catalog number EPX01A-12074-901, lot number 166916102, expiration date December 31, 2019.

[0122] Multiplex information: R&D Systems / Bio-techne catalog number LXSAHM. This kit is lot number L123999, expired on 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 supernatant was performed according to detailed protocol information. As part of this experiment, there is one modification to the protocol: standard substance 8 in the multiplex kit is no longer used. The reason for discontinuing standard substance 8 is that the R&D Systems protocol itself uses only standards substances 1-6. Furthermore, standard substance 8 was validated in ClinImmune for only two of the six analytes constituting 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 intermediate region of the standard curve. Since the standards are reconstituted using growth medium, the standard curves were constructed using both PTT-6 and PTT-4. Test samples grown in either PTT-6 or PTT-4 were estimated from their respective standard curves.

[0125] The results were estimated using Luminex software from analyte-specific standard curves created by the same software: the analysis algorithm was set to Logistic 5P Weighted, using a weighted analysis with a weighting of 1 / y².

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

[0127] Summary of experimental results TGFβ1 singleplex assay · Figure 9 shows one of the three equal aliquots used. All error bars are the standard deviation from triplicate measurements.

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

[0129] First multiplex assay · One of the three equal aliquots 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] Figure 10: Figure 10A Multiplex measurement of PDGF-AA. As can be seen, 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 the triple measurement. Figure 10B Multiplex measurement of VEGF. As can be seen, 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 the triple measurement. Figure 10C Multiplex measurement of Ang-1. As can be seen, CL-MSC and WJ-MSC cultures produce significantly more Ang-1 when grown in PTT-6 than when grown in PTT-4. The AT-MSC and BM-MSC cultures essentially produced no Ang-1. All error bars represent the standard deviation from the triplicate measurements.

[0132] Figure 11: Multiplex measurement of HGF. As can be seen, CL-MSC and WJ-MSC cultures produce significantly more HGF when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produced virtually no HGF. All error bars are standard deviations from the triplet measurements.

[0133] Multiplex assay (including bFGF) • We used a fixed division of 3 out of the three options. The data is shown in Figures 12-14.

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

[0135] Figure 13: Figure 13A. Multiplex measurement of VEGF. As can be seen, 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 the triple measurements. Figure 13B. Multiplex measurement of Ang-1 multiplex assay. As can be seen, CL-MSC and WJ-MSC cultures produce significantly more Ang-1 when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produced virtually no Ang-1. All error bars are standard deviations from the triple measurements. Figure 13C. Multiplex measurement of HGF. As can be seen, CL-MSC and WJ-MSC cultures produce significantly more HGF when grown in PTT-6 than when grown in PTT-4. The AT-MSC and BM-MSC cultures essentially produced no HGF. All error bars represent the standard deviation from the triplicate measurements.

[0136] Figure 14: Multiplex measurement of bFGF. As can be seen, CL-MSC and WJ-MSC cultures produced more bFGF when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produced the same amount of bFGF when grown in PTT-4 and PTT-6. All error bars are the standard deviation from the triplicate measurements. It should be noted that the bFGF sample is present in very small amounts, at or near the lower limit of the detection limit.

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

[0138] Figure 15: Summarizes TGFβ1 measurements across 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 left graph. The MFI for the TGFβ standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. The lower right graph shows that CL-MSC and WJ-MSC cultures 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, and 180226.

[0139] Figure 16: Summarizes Ang-1 measurements across 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 left graph. The MFI for the Ang-1 standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. The lower right graph shows that CL-MSC and WJ-MSC cultures 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 represent the standard deviation from different measurements in experiments 170602, 170511, 170414, 170224, 180105, and 180226.

[0140] Figure 17 summarizes the measurements of PDGF-BB across 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 lower left graph. The MFI for the PDGF-BB standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. Notably, PDGF-BB was not detected in any of the experiments.

[0141] Figure 18: Summarizes PDGF-AA measurements across 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 left graph. The MFI for the PDGF-AA standard curve obtained in PTT-4 medium and PTT-6 medium is shown in the upper graph. The lower right graph shows that cultures CL-MSC, AT-MSC, BM-MSC, and WJ-MSC produce slightly more PDGF-AA when grown in PTT-4 medium than when grown in PTT-6 medium. All error bars are standard deviations from the measurements in experiments 170602, 170511, 170414, 170224, 180105, and 180226.

[0142] Figure 19 summarizes the measurements of IL-10 across six different experiments (170602, 170511, 170414, 170224, 180105, 180226). The mean fluorescence intensity (MFI) measured for the IL-10 standard curve throughout the experiments is shown in the lower left graph. The MFI for the IL-10 standard curve obtained in PTT-4 and PTT-6 media is shown in the upper graph. Notably, IL-10 was not detected in any of the experiments.

[0143] Figure 20: Summarizes VEGF measurements across 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 left graph. The MFI for the VEGF standard curve obtained in PTT-4 medium and PTT-6 medium is shown in the upper graph. The lower right graph shows that cultures CL-MSC, AT-MSC, 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 different measurements in experiments 170602, 170511, 170414, 170224, 180105, and 180226.

[0144] Figure 21: Summarizes HGF measurements across 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 lower left graph. The MFI for the HGF standard curve obtained in PTT-4 medium and PTT-6 medium is shown in the upper graph. The lower right graph 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 the standard deviation from different measurements in experiments 170602, 170511, 170414, 170224, 180105, and 180226.

[0145] Cytokine assays in PTT-6 vs. PTT-4 medium or DMEM / F12 supernatant from CL-MSCs, WJ-MSCs, and placental MSC-derived MSCs. Cytokine detection was performed in the MSC supernatant. Measurement and analysis were carried out as described above.

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

[0147] Each sample was tested in triplets, with the exception of the placental supernatant sample. The objective of this experiment was to create cytokine profiles 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 gelatinous tissue vs. placental MSCs). Cytokine measurements were performed as described above. These profiles will reveal which stem cell populations, grown in which medium, 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 from the graph on the right, all CL-MSCs, WJ-MSCs, and placental MSCs 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 summarizes the measurement of PDGF-BB in the 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 PDGF-BB standard curve throughout the experiments is shown in the graph on the left. Notably, PDGF-BB was not detected in any of the experiments.

[0150] Figure 24 summarizes the measurement of IL-10 in the 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 left graph. S6 indicates the lowest standard used in the assay. Any sample below this is considered subdetectable. As can be seen from the right graph, all CL-MSCs, WJ-MSCs, and placental MSCs produced detectable levels of IL-10 when grown in PTT-6, while IL-10 was little to no detectable when MSCs were grown in PTT-4 or DMEM / F12.

[0151] Figure 25 summarizes the measurement of VEGF in the 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 left graph. S1 indicates the highest standard substance used in the assay. Any sample exceeding this is considered estimated (too high concentration). As can be seen from the right graph, all CL-MSCs, WJ-MSCs, and placental MSCs produce significantly higher levels of VEGF when grown in PTT-6 compared to when the MSCs are grown in PTT-4 or DMEM / F12.

[0152] Figure 26: Summarizes the 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 the MSCs are grown in PTT-4 or DMEM / F12.

[0153] Figure 27: Summarizes PDGF-AA measurement. The mean fluorescence intensity (MFI) measured for the PDGF-AA standard curve throughout the experiment is shown in the graph on the left. S6 indicates the lowest standard used in the assay. Any sample below this is considered subdetectable. As can be seen, CL-MSCs, WJ-MSCs, and placental MSCs all produce higher levels of PDGF-AA when 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 substance used in the assay. Any sample exceeding this is considered estimated (too high concentration). The graph on the right shows that CL-MSCs, WJ-MSCs, and placental MSCs all produce much higher levels of Ang-1 when grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12.

[0155] Figure 29: Summarizing 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 produce much higher levels of Ang-1 when grown in PTT-6 compared to when MSCs are 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 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 commercially available 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 the secretion of all Ang-1, TGF-β1, VEGF, and HGF (whose involvement in wound healing is known, as discussed herein) in mesenchymal stem cell populations, it is clear 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 originally originated—it should be noted here again that Experiment 4 was performed using cell populations isolated from their native environment before being cultured in PTT-6.

[0158] In addition, culturing mesenchymal stem cells in PTT-6 using tissue explants results in a highly homogeneous mesenchymal stem cell population from the umbilical cord amniotic membrane (containing 97.5% viable cells, of which 100% expressed CD73, CD90, and CD105 respectively, 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 the rows "CD34-CD45-" and "CD34-HLA-DR-"). PTT-6 Since culturing Wharton's colloid 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 its effect on the production of these cytokines in umbilical cord lining stem cells, it can be predicted that culturing Wharton's colloid in PTT-6 will also result in such a highly homogeneous mesenchymal Wharton's colloid stem cell population. Therefore, it can be predicted that explants of tissues from other compartments of the umbilical cord, such as umbilical cord blood vessels, will yield similarly homogeneous perivascular tissue. (PV) It is also predicted that this will result in a mesenchymal stem cell population. Similarly, tissue explants of placental tissue, including the amniotic membrane of the placenta, cultured in PTT-6 may also be predicted to result in a placental mesenchymal stem cell population of similar homogeneity. 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, and about 99% or more cells of the isolated mesenchymal stem cell population express CD73, CD90, and CD105 respectively, and lack the expression of CD34, CD45, and HLA-DR respectively.

[0159] The present invention is also characterized by the following: 1. A method for inducing or improving the wound healing properties of a mesenchymal stem cell population, comprising the step of culturing the mesenchymal stem cell population in a culture medium containing DMEM (Dulbeccoo's modified Eagle medium), F12 (Ham 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 a group consisting of umbilical cord mesenchymal stem cell population, placental mesenchymal stem cell population, umbilical cord-placental junction mesenchymal stem cell population, umbilical cord blood mesenchymal stem cell population, bone marrow mesenchymal stem cell population, and adipose tissue-derived mesenchymal stem cell population. 3. The method of item 2, wherein the umbilical cord mesenchymal stem cell population is selected from a group consisting of the amnion (AM) mesenchymal stem cell population, the perivascular (PV) mesenchymal stem cell population, the Wharton's gelatinous (WJ) mesenchymal stem cell population, the amnion mesenchymal stem cell population of the umbilical cord, and the mixed mesenchymal stem cell population (MC) of the umbilical cord. 4. One of the methods described in items 1-3, wherein the culture medium contains 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 FBS at a final concentration of approximately 1-8% (v / v). 5. The method of item 4, wherein the culture medium contains 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). 6. The method of item 5, wherein the culture medium contains DMEM at a final concentration of approximately 61.8% (v / v), F12 at a final concentration of approximately 11.8% (v / v), M171 at a final concentration of approximately 23.6% (v / v), and FBS at a final concentration of approximately 2.5% (v / v). 7. One of the methods from items 1 to 6, wherein the culture medium further contains epidermal growth factor (EGF) at a final concentration of approximately 1 ng / ml to approximately 20 ng / ml. 8. The method described in item 7, wherein the culture medium contains EGF at a final concentration of approximately 10 ng / ml. 9. One of the methods from items 1 to 8, wherein the culture medium contains insulin at a final concentration of approximately 1 μg / ml to 10 μg / ml. 10. The method described in item 9, wherein the culture medium contains insulin at a final concentration of approximately 5 μg / ml. 11. Any one of the methods from item 1 to 10, wherein the culture medium further comprises at least one of the supplements adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 12. One of the methods from items 1 to 11, wherein the culture medium contains all three: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 13. The method of item 12 or 13, wherein the culture medium contains adenine at a final concentration of approximately 0.01 to approximately 0.1 μg / ml, hydrocortisone at a final concentration of approximately 0.1 to approximately 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of approximately 0.5 to approximately 5 ng / ml. 14. Any method 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 the 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 compared to a reference culture medium that does not contain any of DMEM (Dulbecc's modified Eagle medium), F12 (Ham F12 medium), M171 (Medium 171), and FBS (fetal bovine serum). 15. Method of item 14, in which the reference medium consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS. 16. Any method of the preceding items, wherein a population of mesenchymal stem cells is isolated from its natural environment before being cultured in a culture medium defined in any one of the preceding items 1 to 13. 17. Any method from items 1 to 15, comprising the step of isolating a population of mesenchymal stem cells from a native tissue environment by culturing native tissue in a culture medium defined in any one of items 1 to 13 above. 18. Method of item 17, where the tissue is umbilical cord tissue. 19. The method of item 18, wherein the umbilical cord tissue is selected from the group consisting of the entire umbilical cord, the tissue containing the amnion of the umbilical cord, the tissue containing Wharton's gelatinous substance, the tissue containing the amnion, the amnion and Wharton's gelatinous substance, isolated umbilical cord vessels, Wharton's gelatinous substance separated from other components of the umbilical cord tissue, and isolated amnion of the umbilical cord. 20. The method of item 17, wherein the tissue includes or is amniotic tissue of the placenta. 21. Any one of the methods described in items 17-20 above, wherein the umbilical cord tissue is a fragment from the entire umbilical cord, a fragment from the amniotic membrane of the umbilical cord, or a fragment from the amniotic membrane of the placenta. 22. Any one of items 19-22, comprising the step of culturing umbilical cord tissue or placental amniotic tissue until the proliferation of the amniotic mesenchymal stem cell population reaches a concentration density of approximately 70-80%. 23. The method of item 22, including the step of removing the mesenchymal stem cell population from the culture vessel used for cultivation. 24. The method described in item 23, in which the step of removing the mesenchymal stem cell population from the culture vessel is performed by enzymatic treatment. 25. The method of item 24, in which the enzymatic treatment includes trypsin treatment. 26. One of the methods described in items 23-25, by which the mesenchymal stem cell population is transferred to a culture vessel for subculturing. 27. One of the methods described in items 1-16, by which the mesenchymal stem cell population is transferred to a culture vessel for subculturing. 28. Mesenchymal cell populations are required for culture or subculturing (1.0 × 10⁶). 6 Suspension at a concentration of cells / ml, according to the method of item 26 or 27. 29. The method of item 28, wherein a population of mesenchymal stem cells is subcultured in a culture medium defined in any one of items 1 to 13. 30. The method described in item 29, in which the mesenchymal stem cell population is subcultured until it reaches a concentration density of approximately 70-80%. 31. One of the methods described in items 26-30, in which culture or subculturing is performed 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. Any one of the above methods, wherein the culture is performed in a CO2 cell culture incubator at a temperature of 37°C. 34. The method of item 33, which includes the step of removing the mesenchymal stem cell population from the culture vessel used for (passaging). 35. The method described in item 34, in which the step of removing the mesenchymal stem cell population from the culture vessel is performed by enzymatic treatment. 36. The method of item 35, wherein the enzymatic treatment includes trypsin treatment. 37. The method of item 36, further comprising the step of collecting an isolated mesenchymal stem cell population. 38. Any one of the above methods 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, or about 99% or more of isolated mesenchymal stem cells express the markers CD73, CD90, and CD105. 39. Any one of the above methods, 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, or 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. Approximately 97% or more, approximately 98% or more, or approximately 99% or more of the isolated mesenchymal stem cells express CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR, in any one of the methods of item 38 or 39. 41. Any one of the above methods, further comprising the step of preserving an isolated stem / progenitor cell population for further use. 42. The method of item 41, wherein the preservation stage is carried out by cryopreservation. 43. An isolated population of mesenchymal stem cells, wherein at least about 90% or more of the cells in the population express the markers CD73, CD90, and CD105, respectively. 44. A mesenchymal stem cell population of item 43 in which at least approximately 90% or more of the cells lack expression of the markers CD34, CD45, and HLA-DR. 45. A mesenchymal stem cell population of item 44 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, or about 99% or more cells of the isolated mesenchymal stem cell population express CD73, CD90, and CD105 respectively, and lack expression of CD34, CD45, and HLA-DR respectively. 46. ​​One mesenchymal stem cell population selected from items 43-45, consisting of the umbilical cord mesenchymal stem cell population, the placental mesenchymal stem cell population, the umbilical cord blood mesenchymal stem cell population, the bone marrow mesenchymal stem cell population, and the adipose tissue-derived mesenchymal stem cell population. 47. A mesenchymal stem cell population selected from any one of items 43-46, where the umbilical cord mesenchymal stem cell population consists of the amniotic membrane (AM) mesenchymal stem cell population, the perivascular (PV) mesenchymal stem cell population, the Wharton's gelatinous (WJ) mesenchymal stem cell population, the amniotic membrane mesenchymal stem cell population of the umbilical cord, and the mixed mesenchymal stem cell population (MC) of the umbilical cord. 48. A population of mesenchymal stem cells from any one of items 43-47, obtainable by a method defined in any one of items 1-42. 49. A population of mesenchymal stem cells obtained by any one of items 43-48, by a method defined in any one of items 1-42. 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 in the stem cell population express the markers CD73, CD90, and CD105, respectively, and lack the expression of the markers CD34, CD45, and HLA-DR, respectively. 51. A pharmaceutical composition of item 50 that is suitable for systemic or topical application. 52. A pharmaceutical composition of item 50 or 51, further comprising pharmaceutically acceptable excipients. 53. A method for preparing a culture medium suitable for inducing or improving the wound healing properties of a mesenchymal stem cell population, wherein a final volume of 500 ml of culture medium is obtained. i. DMEM 250 ml ii. M171 118 ml iii. DMEM / F12 118 ml iv. Fetal bovine serum (FBS) 12.5 ml (final concentration 2.5%) The method comprising the step of mixing the following. 54. v. EGF preservation solution (5 μg / ml) 1 ml to achieve a final concentration of 10 ng / ml vi. 0.175 ml of insulin storage solution (14.28 mg / ml) to achieve a final concentration of 5 μg / ml Method 53, further including the step of adding [a certain substance]. 55. The method of item 53 or 54, further comprising the step of adding one or more of the supplementing substances adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3) to DMEM to make a culture medium of a total volume of 500 ml. 56. The final concentration of the supplement in DMEM is Adenine at approximately 0.05-0.1 μg / ml, for example, adenine at approximately 0.025 μg / ml, Approximately 1-10 μg / ml of hydrocortisone, Approximately 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). This is the method described in item 55. 57. A cell culture medium that can be obtained by any one of the methods listed in items 53-56. 58. A method for inducing or improving the wound-healing properties of mesenchymal stem cells, comprising the step of culturing amniotic tissue in a culture medium prepared by a 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 a group consisting of umbilical cord mesenchymal stem cell population, placental mesenchymal stem cell population, umbilical cord blood mesenchymal stem cell population, 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 a group consisting of the amniotic membrane (AM) mesenchymal stem cell population, the perivascular (PV) mesenchymal stem cell population, the Wharton's gelatinous (WJ) mesenchymal stem cell population, the amniotic membrane mesenchymal stem cell population of the umbilical cord, and the mixed mesenchymal stem cell population (MC) of the umbilical cord. 61. - DMEM with a final concentration of approximately 55-65% (v / v), - F12 with a final concentration of approximately 5-15% (v / v), - M171 with a final concentration of approximately 15-30% (v / v), and - FBS with a final concentration of approximately 1-8% (v / v) A cell culture medium containing the cell culture medium. 62. 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. Cell culture medium of item 62, containing DMEM at a final concentration of approximately 61.8% (v / v), F12 at a final concentration of approximately 11.8% (v / v), M171 at a final concentration of approximately 23.6% (v / v), and FBS at a final concentration of approximately 2.5% (v / v). 64. A cell culture medium from item 61 to 62, further containing epidermal growth factor (EGF) at a final concentration of approximately 1 ng / ml to approximately 20 ng / ml. 65. A cell culture medium from any one of items 61-65, containing EGF at a final concentration of approximately 10 ng / ml. 66. A cell culture medium containing insulin at a final concentration of approximately 1 μg / ml to 10 μg / ml, one of the cells specified in items 61 to 65. 67. Cell culture medium of item 66, containing insulin at a final concentration of approximately 5 μg / ml. 68. A cell culture medium from any one of items 61-67, further comprising at least one of the supplementing substances adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 69. Cell culture medium of item 68, containing all three: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). 70. Cell culture medium of item 68 or 69, containing adenine at a final concentration of approximately 0.05 to 0.1 μg / ml, hydrocortisone at a final concentration of approximately 1 to 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of approximately 0.5 to 5 ng / ml. 71. 500 ml of cell culture medium, i. DMEM 250 ml ii. M171 118 ml iii. DMEM / F12 118 ml iv. Fetal bovine serum (FBS) 12.5 ml (final concentration 2.5%) A cell culture medium containing any one of items 61-70, including the above. 72. v. EGF at a final concentration of 10 ng / ml vi. Insulin with a final concentration of 5 μg / ml vi. Insulin 0.175 ml (final concentration 5 μg / ml) Cell culture media for item 71, including further items. 73. Cell culture medium of item 71 or 72, further comprising adenine at a final concentration of approximately 0.05 to 0.1 μg / ml, hydrocortisone at a final concentration of approximately 1 to 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of approximately 0.5 to 5 ng / ml. 74. Use of a cell culture medium as defined in any one of items 61-73 to induce or improve the wound healing properties of a mesenchymal stem cell population. 75. Use of a cell culture medium as defined in any one of items 61-73 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 umbilical cord mesenchymal stem cell population, placental mesenchymal stem cell population, umbilical cord blood mesenchymal stem cell population, 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 a group consisting of the amniotic membrane (AM) mesenchymal stem cell population, the perivascular (PV) mesenchymal stem cell population, the Wharton's gelatinous (WJ) mesenchymal stem cell population, the amniotic membrane mesenchymal stem cell population of the umbilical cord, and the mixed mesenchymal stem cell population (MC) of the umbilical cord. 78. Use of any one of items 74-77, wherein at least approximately 90% or more of the cells in the mesenchymal stem cell population express each of the markers CD73, CD90, and CD105. 79. Use of item 78, in which at least approximately 90% or more of the cells in the mesenchymal stem cell population lack expression of markers CD34, CD45, and HLA-DR. 80. Use of item 79, 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, or about 99% or more cells of an isolated mesenchymal stem cell population express CD73, CD90, and CD105 respectively, and lack expression of CD34, CD45, and HLA-DR respectively. 81. A pharmaceutical composition containing three or four of the following as the sole wound healing protein: Ang-1, TGF-β1, VEGF, or HGF. 82. A pharmaceutical composition of item 81, formulated as a liquid or as a lyophilized substance / lyophilized preparation.

[0160] It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0161] All patents and publications referenced herein represent the level of skill of those skilled in the art in which the present invention relates. All patents and publications are incorporated herein by reference to the same extent that individual publications are shown to be incorporated by reference specifically and individually.

[0162] The inventions described exemplary herein can be adequately carried out in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted comprehensively and non-restrictively. Furthermore, the terms and expressions used herein are descriptive rather than restrictive, and the use of such terms and expressions is not intended to exclude any equivalent of the exhibited and described features or any part thereof, and it should be recognized that various modifications are possible within the scope of the claimed invention. Thus, while the invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and alterations of the inventions embodied herein are left to those skilled in the art, and that such modifications and alterations are considered to be within the scope of the invention. The invention is described broadly and generically herein. Each of the narrower species and subgenera groups that fall within the scope of the generic disclosure also forms part of the invention. This includes generic descriptions of the invention using conditional or negative limitations that exclude any subject matter from the group, regardless of whether the excluded subject matter is specifically mentioned herein. In addition, where a feature or aspect of the invention is described in terms of a Markush group, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of any member 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 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

[Claim 1] A method for preparing a conditioned medium from isolated mesenchymal stem cells, The isolated mesenchymal stem cells are either mesenchymal stem cells from the amniotic membrane of the umbilical cord or mesenchymal stem cells from Wharton's gelatinous jelly (WJ). The method is - The step of culturing isolated mesenchymal stem cells in a culture medium containing 55–65% (v / v) final concentration of DMEM, 5–15% (v / v) final concentration of F12, 15–30% (v / v) final concentration of M171, and 1–8% (v / v) final concentration of FBS; - The step of discarding the culture medium; - A step of washing the cultured mesenchymal stem cells; - The step of culturing the cultured mesenchymal stem cells in a medium containing DMEM to obtain a conditioned medium; and - The step of collecting the acclimatized culture medium. including, The aforementioned method.

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