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

Abstract

The present invention relates to a method of inducing or improving wound healing properties of a population of mesenchymal stem cells, the method comprising culturing the population of mesenchymal stem cells in a culture medium comprising DMEM (Dulbecco Modified Eagle Medium), F12 (Hamm F12 Medium), M171 (Medium 171) and FBS (fetal calf serum). The present invention also relates to a population of mesenchymal stem cells wherein at least about 90% or more of the cells of the population of stem cells express each of the following markers: CD73, CD90 and CD105 and lack the expression of the following markers: CD34, CD45 and HLA-DR. The invention also relates to a pharmaceutical composition of the mesenchymal stem cell population.

Description

[0001] Cross-references to Related Patent Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 656,531, filed April 12, 2018, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] 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 for isolating a mesenchymal stem cell population. The present invention also relates to pharmaceutical compositions and uses of such isolated mesenchymal stem cell populations. The present invention also relates to a method for treating a disease or disorder, comprising administering a mesenchymal stem cell population of the present invention or a pharmaceutical composition containing such a mesenchymal stem cell population to a subject in need thereof. The present invention also relates to a highly homogenous and well-defined mesenchymal stem cell population, such as from the umbilical cord or placenta. Background Art

[0004] Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord were first reported in U.S. Patent Application 2006 / 0078993 (granted U.S. Patents 9,085,755, 9,737,568, and 9,844,571) and the corresponding International Patent Application WO 2006 / 019357. Since then, umbilical cord tissue has attracted much attention as a source of multipotent cells. Due to the wide availability of the umbilical cord, stem cells isolated from the umbilical cord, particularly from the amniotic membrane of the umbilical cord (also known as "cord lining stem cells"), have been 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.

[0005] A subsequent study compared the phenotype, proliferation rate, migration, immunogenicity, and immunomodulatory capacity of human mesenchymal stem cells (MSCs) derived from the amniotic membrane of the umbilical cord (umbilical cord lining (CL-MSC), umbilical cord blood (CB-MSC), placenta (P-MSC), and Wharton's jelly (WJ-MSC)) (Stubbendorf et al., Immunological Properties of Extraembryonic Human Mesenchymal Stromal Cells Derived from Gestational Tissue, STEM CELLS AND DEVELOPMENT Volume 22, Number 19, 2013, 2619-2629). Stubbendorf et al. concluded that MSC populations derived from extraembryonic gestational tissues showed different potential to escape immune responses and exert immunomodulatory effects. The authors also found that CL-MSCs showed the most promising potential for cell-based therapies because they showed low immunogenicity, but they also showed enhanced proliferation and migration potential, so future studies should focus on the best disease models in which CL-MSCs can be administered.

[0006] Although amniotic mesenchymal stem cells can be readily obtained using protocols such as those described in U.S. Patent Application 2006 / 0078993 and International Patent Application WO 2006 / 019357, for clinical trials using these umbilical cord lining MSCs, it would be advantageous to have a method that allows the isolation of a population of these umbilical cord lining MSCs that is highly homogeneous and thus amenable to clinical trials. Furthermore, it would be advantageous to have a method for inducing or improving the wound healing properties of a general population of mesenchymal stem cells.

[0007] Therefore, one 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 mesenchymal stem cell population that meets this need from umbilical cord amniotic membrane. Therefore, another object of the present invention is to provide a highly homogenous mesenchymal stem cell population. Summary of the Invention

[0008] The objects of the present invention are achieved by a method, a population of mesenchymal stem cells, a corresponding pharmaceutical composition and a cell culture medium having the features stated in the independent claims.

[0009] In a first aspect, the present invention provides a method for inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum). The mesenchymal stem cell population can be an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, an umbilical cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population, or an adipose tissue-derived mesenchymal stem cell population.

[0010] 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 expression of the following markers: CD34, CD45, and HLA-DR. In embodiments of this second aspect, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105. In addition, in these embodiments of the second aspect, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells in the isolated mesenchymal stem cell population lack expression of the markers CD34, CD45, and HLA-DR. The mesenchymal stem cell population can be obtained by the method of inducing or improving wound healing properties of the first aspect. Therefore, the method of the first aspect can also be a method of isolating a mesenchymal stem cell population.

[0011] In a third aspect, the present invention provides a pharmaceutical composition comprising the mammalian cell of the present invention (the second aspect).

[0012] In a fourth aspect, the present invention provides a method for preparing a culture medium for inducing or improving wound healing properties of a mesenchymal stem cell population or for isolating a mesenchymal stem cell population, the method comprising mixing the following to obtain a final volume of 500 ml of culture medium:

[0013] i. 250 ml of DMEM

[0014] ii.118ml M171

[0015] iii. 118ml DMEM / F12

[0016] iv. 12.5 ml fetal bovine serum (FBS) to give a final concentration of 2.5% (v / v).

[0017] In a fifth aspect, the present invention provides a cell culture medium obtainable by the method described in the fourth aspect.

[0018] 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 described in the fourth aspect.

[0019] In a seventh aspect, the present invention provides a cell culture medium comprising:

[0020] - DMEM at a final concentration of about 55 to 65% (v / v),

[0021] - F12 at a final concentration of about 5 to 15% (v / v),

[0022] - Final concentration of about 15 to 30% (v / v) M171 and

[0023] - FBS at a final concentration of approximately 1 to 8% (v / v).

[0024] In an eighth aspect, the present invention provides use of the cell culture medium according to the seventh aspect for inducing or improving wound healing properties of a mesenchymal stem cell population or for isolating a mesenchymal stem cell population. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be better understood by referring to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings, in which:

[0026] Figure 1 A technical information sheet for Lonza's Dulbecco's modified eagle medium is shown, which includes the catalog number of DMEM used to prepare an exemplary example of the culture medium of the present invention (PTT-6) in the experimental section;

[0027] Figure 2 A technical information sheet of Lonza's Ham's F12 medium is shown;

[0028] Figure 3 A technical information sheet of Lonza's DMEM:F12 (1:1) medium is shown, which includes the product catalog number of the DMEM:F12 (1:1) medium used to prepare an exemplary example (PTT-6) of the culture medium of the present invention in the experimental section;

[0029] Figure 4A technical information sheet of M171 medium from Life Technologies Corporation is shown, which includes the product catalog number of the M171 medium used to prepare an exemplary example of the culture medium of the present invention (PTT-6) in the experimental section;

[0030] Figure 5 Shown is a list of ingredients including commercial suppliers and catalog numbers of each ingredient used to prepare PTT-6 medium in the experimental section;

[0031] Figure 6A -C shows the results of flow cytometry experiments in which the expression of mesenchymal stem cell markers CD73, CD90, and CD105 in mesenchymal stem cells isolated from umbilical cord was analyzed. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the tissue in three different culture media and then subcultured in the corresponding culture media. In these experiments, the following three culture media were used: a) 90% (v / v) DMEM supplemented with 10% FBS (v / v), b) PTT-4 medium described in U.S. Patent Application No. US2008 / 0248005 and the corresponding International Patent Application WO2007 / 046775, which consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS (see paragraph

[0183] of WO2007 / 046775), and c) PTT-6 medium of the present invention, the composition of which is described herein. In this flow cytometric analysis, two different samples of the umbilical cord lining mesenchymal stem cell (CLMC) population were analyzed in each of the three culture media used. The results are shown in Figures 6A to 6C .

[0032] In more detail, Figure 6A Shown is the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in DMEM / 10% FBS. Figure 6B Shown are the percentages of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90, and CD105 following isolation from umbilical cord tissue and culture in PTT-4, and Figure 6C Shown are the percentages of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90, and CD105 following isolation from umbilical cord tissue and culture in PTT-6.

[0033] Figure 7A-B shows the results of flow cytometry experiments in which the expression of stem cell markers (CD73, CD90 and CD105, CD34, CD45 and HLA-DR (human leukocyte antigen-antigen D related) used to define the suitability of multipotent human mesenchymal stem cells for cell therapy) of mesenchymal stem cells isolated from the umbilical cord was analyzed and compared with the expression of these markers in bone marrow mesenchymal stem cells. For this experiment, umbilical cord amniotic mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in the PTT-6 medium of the present invention, while the bone marrow mesenchymal stem cells were isolated from human bone marrow using standard procedures.

[0034] Figure 7A Shown is the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 and lacking expression of CD34, CD45 and HLA-DR following isolation from umbilical cord tissue and culture in PPT-6 medium. Figure 7B Shown are the percentages of isolated bone marrow mesenchymal stem cells that express CD73, CD90, and CD105 and lack expression of CD34, CD45, and HLA-DR.

[0035] Figure 8 The experimental setup is shown, with dark grey wells, standards reconstituted with PTT-4 medium and corresponding samples from MSCs cultured in PTT-4; light grey wells, standards reconstituted with PTT-6 medium and corresponding samples from MSCs cultured in PTT-6. Samples in italics are control supernatants that were assayed as part of repeated assays of stored samples.

[0036] Figure 9 Single-plex measurements of TGFβ1 are shown. As can be seen, CL-MSC and WJ-MSC cultures produced more TGFβ1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced approximately equal amounts of TGFβ1 when grown in either PTT-6 or PTT-4. All error bars represent the standard deviation of three measurements.

[0037] Figure 10A Multiple measurements of PDGF-AA are shown. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC produced more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of three measurements.

[0038] Figure 10BMultiple measurements of VEGF are shown. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC produced more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three measurements.

[0039] Figure 10C Multiple measurements of Ang-1 are shown. As can be seen, CL-MSC and WJ-MSC cultures produced significantly more Ang-1 when grown in PTT-6 compared to PTT-4. AT-MSC and BM-MSC cultures produced virtually no Ang-1. All error bars represent the standard deviation of three measurements.

[0040] Figure 11 Multiple measurements of HGF are shown. As can be seen, cultures CL-MSC and WJ-MSC produced significantly more HGF when grown in PTT-6 compared to cultures grown in PTT-4. Cultures AT-MSC and BM-MSC produced essentially no HGF. All error bars represent the standard deviation of three measurements.

[0041] Figure 12 Multiple measurements of PDGF-AA are shown. 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 equivalent amounts of PDGF-AA in both media. All error bars represent the standard deviation of three measurements.

[0042] Figure 13A Multiple measurements of VEGF are shown. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC produced more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three measurements.

[0043] Figure 13B Multiple measurements of Ang-1 are shown. As can be seen, CL-MSC and WJ-MSC cultures produced significantly more Ang-1 when grown in PTT-6 compared to PTT-4. AT-MSC and BM-MSC cultures produced virtually no Ang-1. All error bars represent the standard deviation of three measurements.

[0044] Figure 13CMultiple measurements of HGF are shown. As can be seen, cultures CL-MSC and WJ-MSC produced significantly more HGF when grown in PTT-6 compared to cultures grown in PTT-4. Cultures AT-MSC and BM-MSC produced essentially no HGF. All error bars represent the standard deviation of three measurements.

[0045] Figure 14 Multiple measurements of bFGF are shown. It can be seen that cultures of CL-MSC and WJ-MSC produced more bFGF when grown in PTT-6 than when grown in PTT-4. Cultures of AT-MSC and BM-MSC produced equivalent amounts of bFGF when grown in PTT-4 and PTT-6. All error bars are the standard deviation of three measurements.

[0046] Figure 15 Summarize the TGFβ1 measurement of 5 different experiments (170328, 170804, 170814, 180105, 180226). The mean fluorescence intensity (MFI) of the TGFβ standard curve measured in each experiment is depicted in the small figure on the left below. The MFI of the TGFβ standard curve obtained in PTT-4 and PTT-6 culture media is shown in the small figure above. The small figure on the right below depicts that culture CL-MSC and WJ-MSC produce more TGFβ1 when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produce equal amounts of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from different measurements for experiments 170328, 170804, 170814, 180105, 180226.

[0047] Figure 16 Ang-1 measurements from six different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the Ang-1 standard curve measured for each experiment is depicted in the lower left panel. The MFI of the Ang-1 standard curves obtained in PTT-4 and PTT-6 culture media is shown in the upper panel. The lower right panel depicts that cultures CL-MSC and WJ-MSC produce more Ang-1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produce essentially equivalent amounts of Ang-1 when grown in PTT-6 or PTT-4. All error bars represent the standard deviation of the different measurements for experiments 170602, 170511, 170414, 170224, 180105, and 180226.

[0048] Figure 17The PDGF-BB measurements of 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) have been summarized. The mean fluorescence intensity (MFI) of the PDGF-BB standard curve measured in each experiment is depicted in the left side panel below. The MFI of the PDGF-BB standard curve obtained in PTT-4 and PTT-6 culture media is shown in the top panel. It should be noted that PDGF-BB was not detected in all experiments.

[0049] Figure 18 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized for PDGF-AA measurement. The mean fluorescence intensity (MFI) of the PDGF-AA standard curve measured in each experiment is depicted in the left side panel below. The MFI of the PDGF-AA standard curve obtained in PTT-4 and PTT-6 culture media is shown in the top panel. The right side panel below depicts that culture CL-MSC, AT-MSC and BM-MSC and WJ-MSC cultures produce slightly more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are the standard deviations of the measured values ​​from experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0050] Figure 19 The IL-10 measurements of 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the IL-10 standard curve measured in each experiment is depicted in the small figure on the left below. The MFI of the IL-10 standard curve obtained in PTT-4 and PTT-6 culture media is shown in the small figure above. It is worth noting that IL-10 was not detected in all experiments.

[0051] Figure 20VEGF measurements from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the VEGF standard curve measured in each experiment is depicted in the small figure on the left below. The MFI of the VEGF standard curve obtained in PTT-4 and PTT-6 culture media is shown in the small figure above. The small figure on the right below depicts 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 of the different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0052] Figure 21 HGF measurements from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the HGF standard curve measured in each experiment is depicted in the small figure on the left below. The MFI of the HGF standard curve obtained in PTT-4 and PTT-6 culture media is shown in the small figure above. The small figure on the right below depicts that cultures CL-MSC and WJ-MSC produce more HGF when grown in PTT-6 than when grown in PTT-4. On the other hand, cultures AT-MSC and BM-MSC do not produce as much HGF as other cultures. All error bars are standard deviations of the different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0053] Figure 22 : Single-plex measurement of TGFβ1. The mean fluorescence intensity (MFI) of the TGFβ1 standard curve measured in each experiment is depicted in the left panel. As can be seen from the right panel, all CL-MSCs, WJ-MSCs and placental MSCs grew in PTT-6 compared to those grown in PTT-4 or DMEM / F12 (in Figure 22 When grown in DMEM (referred to simply as DMEM), more TGFβ1 is produced.

[0054] Figure 23 The measurement of PDGF-BB in the supernatant of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM / F12 is summarized. The mean fluorescence intensity (MFI) of the PDGF-BB standard curve measured in each experiment is depicted in the small figure on the left. It is worth noting that PDGF-BB was not detected in all experiments.

[0055] Figure 24The measurement of IL-10 in the supernatant of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM / F12 analyzed is summarized. The mean fluorescence intensity (MFI) of the VEGF standard curve measured in each experiment is depicted in the left panel. S6 represents the lowest standard used in the assay. Any sample below this is considered to be below the detection limit. As can be seen from the right panel, when grown in PTT-6, all CL-MSC, WJ-MSC and placental MSC produced detectable levels of IL-10, while when MSCs were grown in PTT-4 or DMEM / F12, little or no IL-10 was detected.

[0056] Figure 25 The VEGF measurements in the supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT-6, PTT-4, or DMEM / F12 are summarized. The mean fluorescence intensity (MFI) of the VEGF standard curves measured for each experiment is depicted in the left panel. S1 represents the highest standard used in the assay. Any sample above this is considered speculative (too concentrated). As can be seen from the right panel, all CL-MSCs, WJ-MSCs, and placental MSCs produced much higher levels of VEGF when grown in PTT-6 than when grown in PTT-4 or DMEM / F12.

[0057] Figure 26 Multiple measurements of bFGF are summarized. The mean fluorescence intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the left panel. As can be seen in the right panel, cultured CL-MSC and WJ-MSC produced more bFGF when grown in PTT-6 than when grown in PTT-4. As can be seen, all CL-MSC, WJ-MSC, and placental MSC produced much lower levels of bFGF when grown in PTT-6 compared to when grown in PTT-4 or DMEM / F12.

[0058] Figure 27 The measurement of PDGF-AA is summarized. The mean fluorescence intensity (MFI) of the PDGF-AA standard curve measured in each experiment is depicted in the small figure on the left. S6 represents the lowest standard used in the assay. Any sample below this is considered to be below the detection limit. It can be seen that all CL-MSCs, WJ-MSCs, and placental MSCs produce higher levels of PDGF-AS when grown in PTT-6 compared to when MSCs are grown in PTT-4 or DMEM / F12.

[0059] Figure 28Ang-1 measurements are summarized. The mean fluorescence intensity (MFI) of the Ang-1 standard curve measured for each experiment is depicted in the left panel. S1 represents the highest standard used in the assay. Any sample above this is considered speculative (too concentrated). The right panel depicts that all CL-MSCs, WJ-MSCs, and placental MSCs produced much higher levels of Ang-1 when grown in PTT-6 compared to when MSCs were grown in PTT-4 or DMEM / F12.

[0060] Figure 29 HGF measurements are summarized. The mean fluorescence intensity (MFI) of the HGF standard curve measured for each experiment is depicted in the left panel. The right panel depicts that all CL-MSCs, WJ-MSCs, and placental MSCs produced significantly higher levels of Ang-1 when grown in PTT-6 compared to when MSCs were grown in PTT-4 or DMEM / F12. Detailed Description of the Invention

[0061] As described above, in a first aspect, the present invention relates to a method for inducing or improving the wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum). Surprisingly, it has been found in the present application that the use of such a culture medium 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 / compartment of the mesenchymal stem cell population. Without wishing to be bound by theory, it is believed that the induction or improvement of the wound healing properties of the mesenchymal stem cell population is caused by the ability of the culture medium of the present invention to increase the expression and / or secretion of at least one, two, three or all four of angiopoietin 1 (Ang-1), TGF-β1, VEGF and HGF. See the experimental section, which shows that the umbilical cord amniotic membrane mesenchymal stem cell population cultured in the PTT-6 medium of the present invention expresses and / or secretes increased angiopoietin 1 (Ang-1), TGF-β1, VEGF and HGF relative to the culture of such mesenchymal stem cell population in the culture medium (PTT-4), which has been used in U.S. patent application US2008 / 0248005 and the corresponding international patent application WO2007 / 046775 for isolating the umbilical cord amniotic membrane mesenchymal stem cell population shown to have excellent wound healing properties in U.S. patent application US2008 / 0248005 and the international patent application WO2007 / 046775 (see WO 2008 / 0248005). Examples 23-26 of 2007 / 046775 show that such umbilical cord amniotic membrane mesenchymal stem cell populations (UCMCs) alleviate full-thickness burns (Example 23), partial-thickness cortical wounds (Example 24), non-healing radiation wounds (Example 25), as well as non-healing diabetic wounds and non-healing diabetic foot wounds (Example 26). As shown in the experimental section herein, culturing in a medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum) increases the amount of angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF not only in the umbilical cord amniotic membrane mesenchymal stem cell population, but also in mesenchymal stem cell populations of other compartments of the umbilical cord (such as Wharton's jelly) or (adjacent) compartments (such as the placenta). Therefore, the present application is considered to provide a general teaching for inducing or improving the wound healing properties of a given mesenchymal stem cell population by culturing the mesenchymal stem cell population in the culture medium of the present invention (such as medium PTT-6).

[0062] In this context, the present invention relates to the discovery that a combined increase in the amount of Ang-1, TGF-β1, VEGF and / or HGF produced by a mesenchymal stem cell population will improve or would improve the wound healing properties of such stem cell populations, and that the mimicry of the wound healing properties of stem cell populations is also enabled by compositions / solutions containing three or four of Ang-1, TGF-β1, VEGF or HGF as the only wound healing proteins.

[0063] In this context, it should be noted that the involvement of angiopoietin-1 (Ang-1), TGF-β1, VEGF, and HGF proteins 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 the study by Li et al., the angiopoietin-1 gene was inserted into bone marrow mesenchymal stem cells, and the results showed that “Ang1-MSCs significantly improved wound healing compared to MSCs, Ad-Ang1, or sham treatment, with increased epidermal and dermal regeneration and enhanced angiogenesis.” Notably, the authors Li et al. noted that mesenchymal stem cells (MSCs) alone do not produce sufficient Ang-1, and for this reason, the authors inserted the Ang1 gene into MSCs to generate genetically modified cells. In contrast to Li's study, the present application surprisingly discovered that culturing "natural" mesenchymal stem cells in a culture medium such as PTT-6 can provide conditions under which, for example, umbilical cord tissue mesenchymal stem cells (i.e., a population of mesenchymal stem cells cultured in PTT-6) produce increased levels of Ang-1, thereby making the mesenchymal stem cells suitable for wound healing or further improving their wound healing properties. This means that the present invention provides the advantage that, instead of genetically modifying naturally occurring mesenchymal stem cells to induce wound healing properties in mesenchymal stem cells (which is not only laborious but also not a preferred option for therapeutic applications due to the inherent risks of gene therapy), the wound healing properties of naturally occurring mesenchymal stem cells can be 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.

[0064] For other proteins such as hepatocyte growth factor (HGF) involved in wound healing, in particular the healing of chronic / non-healing wounds, see, for example, Yoshida et al., "Neutralization of Hepatocyte Growth Factor Leads to Retarded Cutaneous Wound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation" J. Invest. Dermatol. 120: 335-343, 2003; Li, Jin-Feng et al., "HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through β1-Integrin / ILK Pathway." BioMed Research International 2013 (2013): 470418 or Conway et al., "Hepatocyte growth factor regulation: An integral part of why wounds become chronic". Wound Rep Reg (2007) 15 683–692.

[0065] For the involvement of vascular endothelial growth factor (VEGF) in wound healing, particularly the healing of chronic / non-healing wounds, see, for example, Froget et al., Eur. Cytokine Netw., Vol. 14, March 2003, 60-64 or Bao et al., “The Role of Vascular Endothelial Growth Factor in Wound Healing” J Surg Res. 2009 May 15; 153(2): 347-358.

[0066] For the involvement of transforming growth factor β (including TGF-β1, TGF-β2 and TGF-β3) in wound healing, in particular 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.

[0067] In this context, it should also be noted that a further surprising advantage of the present invention is that culturing in the culture medium of the present invention provides for the isolation of a population of mesenchymal stem cells (such as a population of umbilical cord amniotic membrane mesenchymal stem cells) in which greater than 90% or even 99% or more of the cells are positive for the three mesenchymal stem cell markers CD73, CD90, and at the same time these stem cells lack expression of CD34, CD45 and HLA-DR (see Experimental Section), meaning that 99% or even more of the cells in the population express the stem cell markers CD73, CD90 and CD105 but do not express the markers CD34, CD45 and HLA-DR. Such highly homogeneous and well-defined cell populations are ideal candidates for clinical trials and cell-based therapies because they fully meet the generally accepted criteria for human mesenchymal stem cells for 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 mesenchymalstromal / 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. In addition, using a bioreactor such as the Quantum Cell Expansion System, large quantities of mesenchymal stem cells can be obtained, such as 300 million to 700 million mesenchymal stem cells per run (see also the Experimental Section). Thus, the present invention provides the further advantage of providing the number of stem cells required for therapeutic applications (e.g., for use in wound healing) in a cost-effective manner. Furthermore, all components used to prepare the culture medium of the present invention are commercially available in GMP quality. Thus, the present invention paves the way for the GMP production of a highly homogenous population of mesenchymal stem cells, such as a population of mesenchymal stem cells from placental tissue or umbilical cord tissue, such as a population of mesenchymal stem cells from the umbilical cord amniotic membrane or a population of mesenchymal stem cells from Wharton's jelly.

[0068] Mesenchymal stem cell populations suitable for wound healing (by inducing wound healing properties in a population that did not have wound healing properties before the culture process of the present invention or by improving wound healing properties) can be any suitable mesenchymal stem cell known in the art, for example, an adult stem cell population or a neonatal stem cell. Mesenchymal stem cell populations can be derived from any mammalian tissue or compartment / body part known to contain mesenchymal stem cells. In an illustrative example, the mesenchymal stem cell population can be an umbilical cord mesenchymal stem cell population (these are examples of neonatal stem cells), a placental mesenchymal stem cell population (another example of neonatal stem cells), a mesenchymal stem cell population at the umbilical cord-placenta junction (another example of a neonatal stem cell population), an umbilical cord blood mesenchymal stem cell population (another example of a neonatal stem cell population), a bone marrow mesenchymal stem cell population (which can be an adult stem cell population), or a mesenchymal stem cell population derived from adipose-tissue (another example of an adult stem cell population).

[0069] Umbilical cord mesenchymal stem cell populations may be derived from (or originate from) any compartment of the umbilical cord tissue containing mesenchymal stem cells. Mesenchymal stem cell populations may be amniotic membrane (AM) mesenchymal stem cell populations, perivascular (PV) mesenchymal stem cell populations, Wharton's jelly (WJ) mesenchymal stem cell populations, umbilical cord amniotic membrane mesenchymal stem cell populations, and umbilical cord mixed (MC) mesenchymal stem cell populations, which refer to a mesenchymal stem cell population comprising stem cells from two or more of these compartments. Mesenchymal stem cells from these compartments and their isolation are known to those skilled in the art and are described, for example, by 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): e0127992, 2015 and references cited therein, and 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 umbilical cord mesenchymal stem cell population can be obtained, for example, by removing the arteries and veins of the umbilical cord tissue, cutting the remaining tissue and Wharton's jelly into fragments, and culturing the umbilical cord tissue (tissue explant) in a culture medium according to the present invention.Umbilical cord mixed mesenchymal stem cell populations can also be obtained by culturing whole umbilical cord tissue with intact umbilical cord blood vessels as tissue fragments under conditions such as those described in Schugar et al., "High harvest yield, high expansion, and phenotypestability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue. Journal of biomedicine & biotechnology. 2009; 2009: 789526" (cultured in serum-supplemented DMEM with 10% fetal bovine serum, 10% horse serum and 1% penicillin / streptomycin). In this context, it should be noted that mesenchymal stem cell populations at the umbilical cord-placental junction can be isolated 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.

[0070] Based on the above, it should be noted that the mesenchymal stem cell populations cultured in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum) in the present invention to induce or improve their wound healing properties can be isolated from their natural environment prior to culturing in the culture medium of the present invention. Such methods are particularly useful for mesenchymal stem cell populations that are not easily isolated by tissue separation blocks, such as umbilical cord blood mesenchymal stem cell populations or bone marrow mesenchymal stem cell populations. However, this method can also be used for umbilical cord mesenchymal stem cell populations, placental mesenchymal stem cell populations, or mesenchymal stem cell populations derived from adipose tissue. Such stem cell populations, such as Wharton's jelly mesenchymal stem cell populations, can first be isolated as described in Subramanian et al., 2015, PLoS ONE (supra) or International Patent Application WO 2004 / 072273 "Progenitor Cells From Wharton's Jelly Of Human Umbilical Cord", and then the isolated mesenchymal stem cell populations can be cultured in a culture medium of the present invention comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Hamm's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum). Placental mesenchymal stem cell populations can also be isolated from the placenta and subsequently cultured in the culture medium of the present invention, for example as described in European patent application EP1 288 293, Talwadekar et al., “Cultivation and Cryopreservation of Cord Tissue MSCs with Cord Blood AB Plasma” Biomed Res J 2014; 1(2): 126-136, Talwadekar et al., “Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-apaired 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-derived mesenchymal stem cells from liposuction and resected fat can be isolated as described in Schneider et al., “Adipose-derived mesenchymal stem cells from liposuction and resected fat are feasible sources for regenerative medicine” Eur J Med Res. 2017; 22: 17, which is a reference cited therein, and then cultured in the culture medium of the present invention (see also the experimental section). As another illustrative example, a mesenchymal stem cell population from the umbilical cord-placenta junction can also be first isolated 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, and then cultured in the culture medium of the present invention.

[0071] Alternatively, and particularly for mesenchymal stem cells that can be isolated using tissue separation blocks, mesenchymal stem cell populations can be directly isolated from their natural tissue environment by culturing the native tissue in the cell culture medium of the present invention. Such methodology is particularly suitable for culturing mesenchymal stem cell populations from umbilical cord tissue, placental tissue (placental tissue can, for example, include or be placental amniotic membrane), or the umbilical cord-placenta junction.

[0072] In this context, it should be noted that the culture medium of the present invention thus also allows the isolation of a population of mesenchymal stem cells (also referred to herein as "mesenchymal stem cells") from their natural environment. Thus, the culture medium of the present invention also allows the isolation of a population of mesenchymal stem cells under conditions that allow the proliferation of mesenchymal stem cells / progenitor cells without differentiation of said mesenchymal stem cells / progenitor cells.

[0073] In one embodiment, the culture medium of the present invention allows for the isolation of a population of mesenchymal stem cells from amniotic membrane under conditions that allow the proliferation of mesenchymal stem cells / progenitor cells without differentiation of the mesenchymal stem cells / progenitor cells. Thus, after isolating the mesenchymal stem cells from amniotic membrane as described herein, the isolated mesenchymal stem cells / progenitor cell population has the ability to differentiate into a variety of cell types, such as described in 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, umbilical cord amniotic membrane mesenchymal stem cells have a spindle shape, express the following genes: POU5f1, Bmi-1, leukemia inhibitory factor (LIF), and secrete activin A and follistatin. The mesenchymal stem cells separated in the present invention can for example be differentiated into any type of mesenchymal cells, such as, but not limited to skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes, cells producing mucin, cells derived from endocrine glands such as cells (for example, β-islet cells) or neuroectodermal cells producing insulin. The stem cells separated in the present invention can be differentiated in vitro, so that the differentiated cells are used for medical purposes subsequently. An illustrative example of this method is that mesenchymal stem cells are differentiated into the β-islet cells producing insulin, which can then be applied to the patient (this aspect is also referring to WO2007 / 046775) suffering from insulin deficiency (such as diabetes) by implantation. Alternatively, the mesenchymal stem cells of the present invention can be used for cell-based therapy with its undifferentiated state, for example, for wound healing purposes, such as for treating burns or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells of the present invention can promote wound healing by interacting with surrounding diseased tissues, and can also differentiate into corresponding skin cells (see again, for example, WO 2007 / 046775).

[0074] In light of the above disclosure, it should be noted 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) as long as the umbilical cord tissue contains the amniotic membrane (also known as the "cord lining"). Thus, as described in the experimental section of this application, the mesenchymal stem cell populations can be isolated from (a portion of) the entire umbilical cord. Thus, in addition to the amniotic membrane, the umbilical cord tissue can comprise any other tissue / component of the umbilical cord. For example, as described in U.S. Patent Application No. 2006 / 0078993 or International Patent Application No. WO2006 / 019357 Figure 16As shown, the amniotic membrane of the umbilical cord is the outermost part of the umbilical cord, covering it. Furthermore, the umbilical cord contains a vein (which delivers oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-depleted blood away from the fetus). For protection and mechanical support, these three blood vessels are embedded in Wharton's jelly, a jelly-like substance primarily composed of mucopolysaccharides. Therefore, the umbilical cord tissue used in the present invention may also include this vein, two arteries, and Wharton's jelly. Using this entire (intact) portion of the umbilical cord has the advantage that the amniotic membrane does not need to be separated from the other components of the umbilical cord. This reduces the number of separation steps and, therefore, makes the present method simpler, faster, less error-prone, and more economical—all important aspects of GMP production required for therapeutic applications of mesenchymal stem cells. Therefore, the isolation of mesenchymal stem cells can begin with tissue explants, and if larger quantities of mesenchymal stem cells are required, such as for clinical trials, the isolated mesenchymal stem cells can then be subcultured (cultivated). Alternatively, the amniotic membrane may be first separated from other components of the umbilical cord, and then mesenchymal umbilical cord lining stem cells may be isolated from the amniotic membrane by culturing the amniotic membrane in the culture medium of the present invention. The culturing may also be performed by a tissue dissociation block method, optionally followed by subculturing the isolated mesenchymal stem cells.

[0075] In this context, the term "tissue separation block (method)" or "tissue separation block method" is used with its conventional meaning in the art and refers to a method in which, once tissue (e.g., placental tissue or umbilical cord tissue) is harvested, the tissue or a portion of the tissue is placed in a cell culture dish containing a culture (growth) medium, and over time, stem cells migrate out of the tissue to the surface of the culture dish. These primary stem cells can then be further expanded and transferred to a fresh culture dish by micropropagation (passage culture) as described herein. In this context, it should be noted that in the first step of isolating / obtaining a mesenchymal stem cell population of the present invention (e.g., umbilical cord mesenchymal stem cells such as amniotic membrane or Wharton's jelly mesenchymal stem cells), a master cell bank of isolated mesenchymal stem cells is obtained, while a working cell bank can be obtained in subsequent passage culture. If the mesenchymal stem cell population of the present invention (particularly a mesenchymal stem cell population in which at least about 97% or more, 98% or more, or 99% or more of the cells express each of the markers CD73, CD90, and CD105 and lack expression of each of the following markers: CD34, CD45, and HLA-DR) is used for clinical trials or as an approved therapeutic agent, the cell population of the working cell bank is generally used for this purpose. Both the stem cell population of the isolation step (which can constitute the master cell bank) and the stem cell population of the subculture step (which can constitute the working cell bank) can be stored, for example, in a cryopreserved form.

[0076] As described above, the method of inducing or improving the wound healing properties of a mesenchymal stem cell population of the present invention (and optionally, a method of isolating mesenchymal stem cells from a medium such as Wharton's jelly or umbilical cord amniotic membrane at the same time) has the following advantages: all components used in the culture medium of the present invention are available in GMP quality, thus providing the possibility of isolating mesenchymal stem cells under GMP conditions for subsequent therapeutic administration.

[0077] As used herein, "inducing or improving wound healing properties of a mesenchymal stem cell population" refers to 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 the mesenchymal stem cell population. As previously mentioned, all four of these proteins are known to be involved in wound healing. "Inducing or improving wound healing properties" is assessed relative to culturing the mesenchymal stem cell population in a reference (culture) medium, such as the culture medium PTT-4 (composed of 90% (v / v) CMRL1066 and 10% (v / v) FBS) used in U.S. Patent Application No. US2008 / 0248005 and the corresponding International Patent Application No. WO2007 / 046775 for the isolation and culture of umbilical cord amniotic membrane mesenchymal stem cell populations, which were shown in U.S. Patent Application No. US2008 / 0248005 and the corresponding International Patent Application No. WO2007 / 046775 to have excellent wound healing properties. When the mesenchymal stem cell population is cultured in the culture medium of the present invention, the mesenchymal stem cell population secretes at least one of the four marker proteins Ang-1, TGF-β1, VEGF, and HGF into the supernatant / culture medium in greater amounts (corresponding to higher secretion levels or higher concentrations) compared to when the mesenchymal stem cell population is cultured in a reference culture medium, thereby increasing the wound healing properties of the mesenchymal stem cell population. If, during culture in the reference culture medium, the mesenchymal stem cell population is observed to not (detectably) secrete any of the four marker proteins, but detectable secretion of at least one of the four marker proteins is observed during or after culture of the mesenchymal stem cell population in the culture medium of the present invention, wound healing properties of the stem cell population are induced. Furthermore, when the expression or secretion of at least two, at least three, or all four of the four marker proteins Ang-1, TGF-β1, VEGF, and HGF is increased relative to when the stem cell population is cultured in the reference culture medium, the wound healing properties of the mesenchymal stem cell population are improved. The secretion of the four marker proteins into the culture medium (and thus 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 commercially available antibodies / immunoassays (see Experimental Section). Such measurements can be performed in an automated manner, for example, using a system such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).

[0078] "DMEM" refers to Dulbecco's Modified Eagle's Medium, a modification of Basal Eagle's Medium (BME) developed in 1969 (see Figure 1, which shows a data sheet for DMEM available from Lonza). The original DMEM formulation contained 1000 mg / L glucose and was first reported for culturing embryonic mouse cells. Since then, DMEM has become the standard culture medium for cell culture and is commercially available from a variety of sources, such as ThermoFisher Scientific (Cat. No. 11965-084), Sigma Aldrich (Cat. No. D5546), or Lonza, to name just a few. Therefore, any commercially available DMEM can be used in the present invention. In a preferred embodiment, the DMEM used herein is the DMEM culture medium available from Lonza as Catalog No. 12-604F. This culture medium is DMEM supplemented with 4.5 g / L glucose and L-glutamine. In another preferred embodiment, the DMEM used herein is the DMEM culture medium from Sigma Aldrich as Catalog No. D5546, which contains 1000 mg / L glucose and sodium bicarbonate, but does not contain L-glutamine.

[0079] "F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium, a nutrient mixture originally designed for culturing a variety of mammalian and hybridoma cells when used with serum and in combination with hormones and transferrin (see Figure 2 , which shows a data sheet for Ham's F12 medium from Lonza. Any commercially available Ham's F12 medium (e.g., from ThermoFisher Scientific (Cat. No. 11765-054), Sigma Aldrich (Cat. No. N4888), or Lonza, to name a few suppliers) can be used in the present invention. In a preferred embodiment, Ham's F12 medium from Lonza is used.

[0080] "DMEM / F12" or "DMEM:F12" refers to a 1:1 mixture of DMEM and Ham's F12 medium (see Figure 3, which shows a data sheet for DMEM:F12 (1:1) from Lonza. DMEM / F12 (1:1) medium is also a widely used basal medium for supporting the growth of many different mammalian cells and is commercially available from multiple suppliers such as ThermoFisher Scientific (Cat. No. 11330057), Sigma Aldrich (Cat. No. D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM / F12 (1:1) medium available from Lonza as Cat. No. 12-719F (which is DMEM:F12 with L-glutamine, 15 mM HEPES, and 3.151 g / L glucose).

[0081] "M171" refers to Medium 171, which has been developed as a basal medium for growing normal human mammary epithelial cells (see Figure 4 , which shows a data sheet for M171 medium from Life Technologies Corporation). This basal medium is also widely used and can be commercially obtained from, for example, ThermoFisher Scientific or Life Technologies Corporation (catalog number M171500). Any commercially available M171 medium can be used in the present invention. In a preferred embodiment, the M171 medium used herein is the M171 medium available from Life Technologies Corporation under catalog number M171500.

[0082] " FBS " means fetal bovine serum (also referred to as " fetal bovine (calf) serum "), i.e., the portion of blood retained after the blood naturally coagulates and is then centrifuged to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement in eukaryotic cell in vitro cell culture because it has very low levels of antibodies and contains more growth factors, making it versatile in many different cell culture applications. Preferably, FBS is obtained from members of the International Serum Industry Association (ISIA), which focuses on ensuring the safety and safe use of serum and animal-derived products by proper traceability of origin, authenticity of labels, and proper standardization and supervision. Suppliers of FBS as ISIA members include AbattoirBasics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, to name a few. In a currently preferred embodiment, FBS is obtained from GE Healthcare with catalog number A15-151.

[0083] Now let's talk about the culture medium of the present invention. This culture medium can contain DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and PBS at a final concentration of about 1 to 8% (v / v) for inducing or improving the wound healing properties of mesenchymal stem cells, or for isolating or culturing mesenchymal stem cells. As used herein, the values ​​of "% (v / v)" refer to the volume of a single component relative to the final volume of the culture medium. This means, for example, that if DMEM is present in the culture medium at a final concentration of about 55 to 65% (v / v), then 1 liter of culture medium contains about 550 to 650 ml of DMEM.

[0084] In other embodiments, the culture medium may comprise DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v). In further embodiments, the culture medium may comprise DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0085] In addition to the above components, the culture medium may contain supplements that are beneficial for culturing the mesenchymal umbilical cord lining stem cells. For example, the culture medium of the present invention may contain 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 such embodiments, the culture medium may contain EGF at a final concentration of about 10 ng / ml.

[0086] The culture medium of the present invention may also contain insulin. If present, insulin may be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some such embodiments, the culture medium may contain insulin at a final concentration of about 5 μg / ml.

[0087] The culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may comprise all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may comprise adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0088] In one embodiment of the method of the present invention, tissue such as umbilical cord tissue or placenta can be cultured until a suitable number of (primary) mesenchymal stem cells (such as umbilical cord lining stem cells, Wharton's jelly or placental stem cells) grow out of the tissue. In a typical embodiment, umbilical cord tissue is cultured until the cell outgrowth of the mesenchymal stem cells of the corresponding tissue reaches about 70% to about 80% confluence. It should be noted here that the term "confluency" is used in its conventional sense in the field of cell culture and is intended as an estimate / indicator of the number of adherent cells in a culture dish or culture flask, referring to the proportion of the surface covered by cells. For example, 50% confluence means that about half of the surface is covered and there is still room for cells to grow. 100% confluence means that the surface is completely covered with cells and there is no longer room for cell monolayer growth.

[0089] Once the primary cells (mesenchymal stem cells) of appropriate quantity have been obtained from the corresponding tissue by the tissue separation block method, the mesenchymal stem cells are removed from the culture vessel for cultivating. By doing this, it is possible to obtain the master cell bank of the mesenchymal stem cells of (primary generation) separation containing, for example, umbilical cord or placenta. Conventionally, because this type of mesenchymal stem cell is an adherent cell, the cells are harvested using standard enzyme treatment. For example, the enzyme treatment can include trypsinization as described in International U.S. Patent Application 2006 / 0078993, International Patent Application WO2006 / 019357 or International Patent Application WO2007 / 046775, meaning that the cells grown outward can be harvested for further amplification by trypsinization (0.125% trypsin / 0.05% EDTA). If the mesenchymal stem cells gathered in the crops are for example used to produce a master cell bank, then the cells can also be cryopreserved and stored for further use, as described below.

[0090] Once harvested, the mesenchymal stem cells can be transferred to a culture vessel for subculture. If a mesenchymal stem cell population that has been previously isolated from its natural environment is used (as described above, such isolated stem cells used in the method of the present invention can be from umbilical cord blood, bone marrow or adipose tissue, or from umbilical cord tissue or placental tissue), subculture or culture (these two terms are used interchangeably hereinafter) is also performed. The subculture can also be started from frozen primary cells, i.e., from the master cell bank. For subculture, any suitable number of cells can be seeded in a culture vessel such as a cell culture plate. For this purpose, the mesenchymal cells can be plated at, for example, about 0.5 x 10 6 cells / ml to approximately 5.0 x 10 6 The cells are suspended in a medium suitable for subculture (most conveniently, the medium of the present invention) at a concentration of about 1.0 x 10 cells / ml. 6The cells are suspended at a concentration of 10 cells / ml for subculture. The subculture can be carried out either in a simple culture bottle or in a multi-layer system that can be stacked in an incubator, such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (part of Nunc, Thermo Fisher Scientific Inc., Waltham, MA, USA). Alternatively, the subculture can also be carried out in a closed, self-contained system (such as a bioreactor). Bioreactors of different designs are known to those skilled in the art, for example, parallel plates, hollow fibers or microfluidic bioreactors. See, for example, Sensebe et al., "Production of mesenchymalstromal / stem cells according to good manufacturing practices: a review", supra. Illustrative examples of commercially available hollow fiber bioreactors are Cell Expansion System (Terumo BCT, Inc), which has been used, for example, to expand bone marrow mesenchymal stem cells for clinical trials (see Hanley et al., Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048–1058). Another example of a commercially available bioreactor that can be used to subculture the mesenchymal stem cell population of the present invention is the Xuri Cell Expansion System available from GE Healthcare. If a working cell bank for therapeutic applications is to be produced under GMP conditions and a large number of cells are required, then, for example, It is particularly beneficial to culture mesenchymal stem cell populations in an automated system such as the Cell Expansion System.

[0091] Subculture of the mesenchymal stem cells of the present invention occurs in the culture medium of the present invention. Thus, the culture medium of the present invention can be used to isolate a population of mesenchymal stem cells, for example, from the placental amniotic membrane, or from the amniotic membrane, or from Wharton's jelly of the umbilical cord, and subsequently culture the isolated primary cells by subculture. Similarly, for subculture, the mesenchymal stem cells can be cultured until a suitable number of cells are grown. In an exemplary embodiment, the mesenchymal stem cells are subcultured until the mesenchymal stem cells reach about 70% to about 80% confluency.

[0092] The isolation / culture of the mesenchymal stem cell population can be carried out under standard conditions for culturing mammalian cells. Typically, the method of isolating a mesenchymal stem cell population of the present invention is typically carried out under the following conditions (temperature, atmosphere): the conditions are generally used to culture cells of the species from which the cells are derived. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are typically cultured at 37° C. in a normal atmosphere containing 5% CO 2 , respectively. In this context, it is noted that in the present invention, the mesenchymal cell population can be derived from any mammalian species, such as mouse, rat, guinea pig, pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, wherein in one embodiment, human-derived mesenchymal stem cells are preferred.

[0093] Once the mesenchymal stem cells required / appropriate amount are obtained from culture or passage culture, the mesenchymal stem cells are harvested by removing them from the culture container for passage culture. Usually, the mesenchymal stem cells are harvested again by enzyme treatment, including trypsin digestion of cells. The separated mesenchymal stem cells are subsequently collected and used directly or preserved for further use. Usually, they are preserved by cryopreservation. The term "cryopreservation" is used in this article with its conventional meaning to describe the process of preserving mesenchymal stem cells by cooling to a low subzero temperature (such as (usually) -80°C or -196°C (boiling point of liquid nitrogen)). Cryopreservation can be performed as known to those skilled in the art, and may include the use of cryoprotectants such as dimethyl sulfoxide (DMSO) or glycerol, which slow down the formation of ice crystals in umbilical cord cells.

[0094] The isolated mesenchymal stem cell populations obtained by the culture and / or separation methods of the present invention are highly defined and homogenous. In typical embodiments of the method, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD105. In addition, in these embodiments, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45, and HLA-DR. In certain embodiments, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cell population express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.

[0095] Therefore, in accordance with the above disclosure, the present invention also relates to a population of mesenchymal stem cells, such as a placental mesenchymal stem cell population or an umbilical cord mesenchymal stem cell population (e.g., isolated from Wharton's jelly or the amniotic membrane of the umbilical cord), wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90, and CD 105. In preferred embodiments, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the cells of the isolated mesenchymal stem cell population are CD73+, CD90+, and CD105+, meaning that this percentage of the isolated cell population expresses each of CD73, CD90, and CD105 (see the Experimental section of this application). In addition, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45 and HLA-DR. In a specific embodiment, about 97% or more, about 98% or more or about 99% or more of the isolated mesenchymal stem cells express CD73, CD90 and CD105, while lacking expression of CD34, CD45 and HLA-DR. This paper reports for the first time such a highly homogeneous population of mesenchymal stem cells derived from the umbilical cord amniotic membrane, and the cell population meets the criteria for mesenchymal stem cells for cell therapy (see also the experimental section and, for example, Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra). In this context, it should be noted that the mesenchymal stem cell population can be obtained by the isolation method of the present invention, but can also be obtained by a different method such as cell sorting if necessary. In one embodiment of such an umbilical cord mesenchymal stem cell population of the present invention, at least about 91% or more of the cells of the stem cell population express CD73, CD90, and CD105, and lack expression of CD34, CD45, and HLA-DR, excluding mesenchymal stem cell populations isolated from umbilical cord amniotic membrane.

[0096] As described above, the present invention also relates to a pharmaceutical composition comprising a mesenchymal stem cell population as described herein, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90, and CD105, and optionally lack expression of CD34, CD45, and HLA-DR. The pharmaceutical composition may comprise any pharmaceutically acceptable excipient and may be formulated for any desired pharmaceutical route of administration. The pharmaceutical composition may, for example, be suitable for systemic or topical application. In a related aspect, the present invention also provides a pharmaceutical composition comprising three or four of Ang-1, TGF-β1, VEGF, or HGF as the sole wound healing protein. Such pharmaceutical compositions may be formulated as a liquid or as a lyophilisate / freeze-dried formulation, for example, using a pharmaceutically suitable buffer, such as 0.9% saline, Ringer's solution, or phosphate-buffered saline (PBS).

[0097] In another 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, wherein the method comprises mixing the following to obtain a final volume of 500 ml of culture medium:

[0098] i. 250 ml of DMEM

[0099] ii.118ml M171

[0100] iii. 118ml DMEM / F12

[0101] iv. 12.5 ml fetal bovine serum (FBS) to give a final concentration of 2.5% (v / v).

[0102] As mentioned above, DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Therefore, 118 ml of DMEM / F12 medium contains 59 ml of DMEM and 59 ml of F12. Therefore, when using this method of preparing the medium, the final concentrations (v / v) in a total volume of 500 ml are as follows:

[0103] DMEM: 250 ml + 59 ml = 309 ml, corresponding to 309 / 500 = 61.8% (v / v)

[0104] M171: 118 ml, corresponding to 118 / 500 = 23.6% (v / v)

[0105] F12: 59 ml, corresponding to 59 / 500=11.8% (v / v).

[0106] This embodiment of the method of preparing a culture medium further comprises adding:

[0107] v. 1 ml of EGF stock solution (5 μg / ml) to give a final EGF concentration of 10 ng / ml, and

[0108] vi. Insulin 0.175 ml stock solution (14.28 mg / ml) to give a final insulin concentration of 5 μg / ml.

[0109] It should be noted here that, in these embodiments, the volume mixing of above-mentioned these components i to vi obtains the substratum that final volume is 499.675ml.If no longer add other components in substratum, then remaining 0.325ml (volume being added to 500ml) can be for example arbitrarily among component i to iv, and this means can be DMEM, M171, DMEM / F12 or FBS.Alternatively, certainly can regulate the concentration of EGF or insulin storing solution so that the cumulative volume of substratum is 500ml.In addition, also should be noted that component i to iv need not add according to the order that they are listed, but certainly can use any order to make these components mix to obtain substratum of the present invention. This means, for example, that M171 and DMEM / F12 can be mixed together and then combined with DMEM and FBS to achieve final concentrations as described herein, i.e., a final concentration of about 55 to 65% (v / v) of DMEM, a final concentration of about 5 to 15% (v / v) of F12, a final concentration of about 15 to 30% (v / v) of M171, and a final concentration of about 1 to 8% (v / v) of FBS.

[0110] In other embodiments, the method further comprises adding one or more of the following supplements to the DMEM: adenine, hydrocortisone, 3,3',5-triiodo-L-thyronine sodium salt (T3) in a volume of 0.325 ml, thereby obtaining a total volume of 500 ml of culture medium. In this embodiment, the final concentrations of these supplements in the DMEM may be as follows:

[0111] about 0.05 to 0.1 μg / ml adenine, for example about 0.025 μg / ml adenine,

[0112] About 1 to 10 μg / ml hydrocortisone,

[0113] About 0.5 to 5 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0114] In light of the above disclosure, the present invention also relates to a cell culture medium obtainable or obtained by the method for preparing a culture medium as described herein.

[0115] Furthermore, the present invention also relates to a method for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, wherein the method comprises culturing the amniotic membrane tissue in a culture medium prepared by the method described herein.

[0116] Therefore, the present invention also relates to a cell culture medium comprising:

[0117] - DMEM at a final concentration of about 55 to 65% (v / v),

[0118] - F12 at a final concentration of about 5 to 15% (v / v),

[0119] - Final concentration of about 15 to 30% (v / v) M171 and

[0120] - FBS at a final concentration of approximately 1 to 8% (v / v).

[0121] In some embodiments of the culture medium described herein, the culture medium comprises DMEM at a final concentration of about 57.7 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v). In other embodiments, the culture medium may comprise DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

[0122] In addition, the culture medium may further comprise epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml. In some embodiments, the culture medium comprises EGF at a final concentration of about 10 ng / ml. The culture medium described herein may further comprise insulin at a final concentration of about 1 μg / ml to about 10 μg / ml. In such embodiments, the culture medium may comprise insulin at a final concentration of about 5 μg / ml.

[0123] The cell culture medium of the present invention may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In some embodiments, the culture medium comprises all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). If present, the culture medium may comprise adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine or about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml hydrocortisone or about 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0124] In an embodiment of the cell culture medium, 500 ml of the cell culture medium of the present invention comprises:

[0125] i. 250 ml of DMEM

[0126] ii.118ml M171

[0127] iii. 118ml DMEM / F12

[0128] iv. 12.5 ml fetal bovine serum (FBS) (final concentration 2.5%).

[0129] In further embodiments, the cell culture medium may further comprise:

[0130] v. EGF at a final concentration of 10 ng / ml, and

[0131] vi. Insulin at a final concentration of 5 μg / ml.

[0132] Insulin and EGF can be added to the culture medium using selected stock solutions so that the total volume of the culture medium does not exceed 500 ml.

[0133] In a specific embodiment, components i to vi of the culture medium of the present invention are Figure 5 The components shown in Figure 5 The catalog numbers shown are obtained from the respective manufacturers. Figure 5 The culture medium obtained by mixing the components i to vi shown is also referred to herein as "PTT-6". It should also be noted in this context that components i to vi and any other components (such as antibiotics from any other commercial supplier) can be used to prepare the culture medium of the present invention.

[0134] In addition, the cell culture medium of the present invention can comprise adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine or about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml, about 0.5 to about 10 μg / ml, or about 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.1 to about 5 ng / ml or about 0.5 to about 5 ng / ml.

[0135] Finally, the present invention also provides a method for treating a non-human mammal (such as a cat, dog, horse, to name a few) or a human patient suffering from a disease or condition, the method comprising administering to the non-human mammal or human patient a mesenchymal stem cell population disclosed herein or a pharmaceutical composition containing the stem cell population disclosed herein. The disease can be any disease or condition, particularly any disease or condition in which wound healing is desired or required. The subject (patient or non-human mammal) suffers from a wound caused by a burn, bite, trauma, surgery, or a disease such as a skin disease or metabolic disorder. As an example of such a metabolic disorder, the patient may, for example, suffer from type I or type II diabetes, as well as a chronic foot ulcer. To treat the subject, the mesenchymal stem cell population of the present invention can be administered in any suitable manner, including, but not limited to, topical administration, by implantation, or by injection. In principle, any topical administration method is intended herein. Administration of the mesenchymal stem cell population can be carried out by means of a syringe. However, before applying the mesenchymal stem cells to the subject, the mesenchymal stem cells can also be contacted in a cream, ointment, gel, suspension, or any other suitable substance. The stem cell population can then be placed, for example, directly on a wound such as a burn or diabetic wound (see International Patent Application WO 2007 / 046775). After its application to the subject, the stem cell population can be placed on a wound such as a burn or diabetic wound (see International Patent Application WO 2007 / 046775). Dressing) and covering The crepe bandage of the dressing can hold the mesenchymal stem cell population in place. Alternatively, the stem cell population can also be implanted subcutaneously, for example, directly under the skin, in body fat, or in the peritoneum.

[0136] The present invention also relates to a unit dose comprising about 20 million cells, about 15 million cells, about 10 million cells, about 5 million cells, about 4 million cells, about 3 million cells, about 2 million cells, about 1 million cells, about 0.5 million cells, about 0.25 million cells, or less than 0.25 million cells of the mesenchymal stem cell populations described herein.

[0137] It is also contemplated that the unit dose comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, about 0.5, about 0.25, or about 0.1 million cells. Preferably, the unit dose comprises about 10 million cells. It is further contemplated that the unit dose comprises about 1000 cells to about 5 million cells. The unit dose can be applied at a dose of about 100,000 cells, 300,000 cells, or 500,000 cells. As described herein, the unit dose can be applied topically, particularly if used for wound healing. For example, the unit dose can be applied topically per square centimeter.

[0138] If desired, the unit dose can be applied once, twice, three times, or more times per week. For example, the unit dose can be applied for one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more weeks. A unit dose containing about 100,000 cells, about 300,000 cells, or about 500,000 cells can be applied twice a week for eight weeks, preferably at a 1 cm 2 On application.

[0139] 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 cases, for example, 0.1 ml of the vial can be applied to the subject, preferably per square centimeter. The unit dose can optionally be contained in a syringe.

[0140] In the unit dose of the present invention, the cells may be contacted with a pharmaceutically acceptable carrier (e.g., a liquid carrier). The carrier may be any known carrier, such as HypoThermosol TM 、Hypothermosol TM -FRS or PlasmaLyte. The culture medium of the present invention can also be used as a carrier for the mesenchymal stem cell population (unit dose) of the present invention. In this case, the mesenchymal stem cells can be separated from the carrier before administration. For example, the cells can be centrifuged and separated before administration to the subject.

[0141] The treatment methods and unit doses of the present invention may include the use of living cells. The viability of the mesenchymal stem cell population is determined using known methods, such as Tryphan Blue staining as described in the experimental section.

[0142] The present invention is further illustrated by the following non-limiting experimental examples.

[0143] The present invention is further illustrated by the following non-limiting experimental examples.

[0144] The sequences used herein are depicted in Table 1 below.

[0145] Table 1. Sequences of proteins used in this article

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] Experimental Examples

[0157] 1. Cryopreservation of umbilical cord tissue before mesenchymal stem cell isolation

[0158] As described below, umbilical cord tissue (donated with the mother's informed consent) was processed to allow for the subsequent isolation of mesenchymal stem cells from the amniotic membrane of the umbilical cord.

[0159] 1.1 Washing of umbilical cord tissue samples:

[0160] a. Remove the scalpel from the protective cover.

[0161] b. Clamp the umbilical cord with forceps and cut it into 10 cm long sections with a scalpel. Place the unusable umbilical cord back into the original tissue cup.

[0162] c. Transfer the 10 cm long umbilical cord segment to a new 150 mm culture dish. A 150 mm culture dish can be used instead of the cup.

[0163] d. Use the lid of a 150 mm culture dish as a place to store forceps and a scalpel.

[0164] e. Use a 30 ml syringe to remove 25 ml of Plasmalyte A (Baxter, catalog # 2B2543Q). Hold the syringe at a 45° angle with one hand and dispense Plasmalyte A directly onto the umbilical cord tissue.

[0165] f. Tilt the culture dish slightly and remove Plasmalyte A using a 30 ml syringe and blunt needle.

[0166] g. Collect the used Plasmalyte A in a 300 ml transfer bag which serves as a waste container and dispose of it in a biohazard bin.

[0167] h. Repeat the washing procedure, using a new dish for each wash if necessary. Ensure that all blood clots on the surface have been removed. If washing the tissue is necessary, use more Plasmalyte A.

[0168] i. Place the tissue into a new labeled tissue culture dish to continue cutting the tissue. Add 20 ml of Plasmalyte A to the dish so that the tissue does not dry out while cutting.

[0169] j. Cut the umbilical cord into 10 equal-length pieces of approximately 1 cm each.

[0170] k. Then cut each 1 cm piece into smaller segments, each piece is about 0.3 cm x 0.3 cm to 0.5 cm x 0.5 cm.

[0171] l. Remove any Plasmalyte A from the culture dish.

[0172] m. Use a 30 ml syringe to remove 25 ml of Plasmalyte A from the original Plasmalyte A bag and dispense directly onto the umbilical cord tissue piece.

[0173] n. Hold the dish at an angle to collect all the Plasmalyte A used to wash the tissue on one side and remove it with a syringe and blunt needle.

[0174] Repeat the wash cycle again. No clots should remain.

[0175] NOTE: If the umbilical cord is not frozen immediately, store the cord tissue in Plasmalyte A until ready to freeze.

[0176] 1.2 Cryopreservation of umbilical cord tissue:

[0177] a. Prepare cryopreservative solution:

[0178] i. Prepare 50 ml of a frozen solution consisting of 60% Plasmalyte A, 30% of 5% human serum albumin, and 10% dimethyl sulfoxide (DMSO).

[0179] ii. Label a 150 ml transfer bag "Tissue Freeze" and connect the plasma transfer device to the port using aseptic technique.

[0180] 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 Freeze Solution" with the date and time the solution was prepared.

[0181] iv. Use a 20 ml syringe to take 15 ml of 5% human serum albumin and transfer it to the labeled transfer bag.

[0182] v. Add 5 ml of DMSO to the transfer bag.

[0183] vi. Mix well and record the mixing status of the freezing liquid.

[0184] b. Remove Plasmalyte A from tissue before adding freezing solution.

[0185] c. Using a 60 ml syringe, draw all 50 ml of the cryovial into the syringe and add approximately 30 ml of the cryovial into the 150 mm cell culture dish containing the umbilical cord tissue. Place a blunt needle on the syringe to maintain sterility.

[0186] d. Spin the culture dish containing the tissue and freezing solution at 4°C / min for 10 minutes.

[0187] e. Use forceps to select 8 randomly selected pieces and place them into each of four 4 ml cryovials. Select 4 randomly selected pieces and place them into one 1.8 ml cryovial. These pieces should be free of blood clots.

[0188] f. Add the remaining cryovial to each cryovial containing umbilical cord tissue, filling the 4 ml tube to the 3.6 ml line and the 1.8 ml Nunc vial to the 1.8 ml line.

[0189] g. Label one bottle of Bactec Lytic / 10-Anaerobic / F and one bottle of Bactec PlusAerobic / F with your tissue ID.

[0190] h. Use a syringe and blunt needle to remove 20 ml of frozen solution from the culture dish. After wiping the Bactec vial with an alcohol cotton swab, convert the blunt needle into an 18G needle and inoculate the aerobic and anaerobic Bactec bottles, 10 ml per bottle.

[0191] i. Start the controlled rate freezer.

[0192] j. After controlled-rate freezing is complete, place the device in a liquid nitrogen freezer with continuous temperature monitoring until further use.

[0193] 2. Isolation of Mesenchymal Umbilical Cord Lining Stem Cells from Umbilical Cord Tissue

[0194] 2.1 Preparation of culture medium for treating MSCs from umbilical cord tissue:

[0195] a. Prepare 500 ml of PTT-6 (culture / growth medium) by mixing the following in the order listed:

[0196] i.DMEM, 250ml

[0197] ii.M171 118ml

[0198] iii. DMEM F12 118ml

[0199] iv. FBS 12.5ml (final concentration 2.5%)

[0200] v. EGF 1ml (final concentration 10ng / ml)

[0201] vi. Insulin 0.175 ml (final concentration 5 μg / ml)

[0202] The volume of above-mentioned components i to vi obtains the culture medium that final volume is 499.675ml.If no longer add other components in culture medium, then remaining 0.325ml (volume added to 500ml) can be any one among for example component i to iv, and this means can be DMEM, M171, DMEM / F12 or FBS.Alternatively, certainly can regulate the concentration of EGF or insulin storing solution so that the cumulative volume of culture medium is 500ml.Alternatively, can add the storing solution of antibiotic (such as penicillin-streptomycin-amphotericin) to obtain final volume is 500ml.Can also add one or more in the following fill-in of 0.325ml volume in described culture medium: adenine, hydrocortisone, 3,3 ', 5-triiodo-L-thyronine sodium salt (T3), obtain thus the culture medium that cumulative volume is 500ml.

[0203] vii. Label the bottle with "PTT-6," the date the medium was prepared, the operator's initials, and the expiration date followed by the phrase "Expiration." The expiration date is the earliest expiration date of any component or one month from the preparation date, whichever comes first.

[0204] b. In a 50 ml centrifuge tube, add 2.5 ml FBS to 47.5 ml HBSS to prepare rinse medium (Hank's buffered saline solution (HBSS) with 5% FBS without calcium or magnesium). Label the tube "Rinse Medium," the operator's initials, and the date the medium was prepared.

[0205] c. All culture media were sterile tested using Bactec Lytic / 10–Anaerobic / F (Becton Dickinson & Company) and Bactec Pluc+Aerobic / F (Becton Dickinson & Company). 20 ml of the prepared culture medium was injected into each bottle.

[0206] 2.2 Thawing of umbilical cord tissue for MSC harvesting:

[0207] a. When the operator is ready to process the samples in the cleanroom, begin thawing. Do not thaw more than one vial at a time unless the vials are from the same donor.

[0208] b. Wipe the water bath with disinfectant, then with 70% isopropyl alcohol, and fill the water bath with 1 L of sterile water. Heat the water bath to 36-38°C.

[0209] c. Prepare 10 mL of rinse medium consisting of 70% to 90% PlasmaLyte A in a cleanroom under a biosafety cabinet. Sterile filter the solution using a 0.2-μm syringe filter attached to a 10 ml syringe and keep the solution refrigerated until use.

[0210] d. Place treatment labels on 50 ml conical tubes.

[0211] e. Confirm that the water bath temperature is 36-38°C.

[0212] f. Remove tissue vials from liquid nitrogen storage and quickly thaw in a 37°C water bath filled with 1 L of sterile water. The vial holder for the Mr. Frosty Nalgene Cryo 1°C freezing container floats with the vial and can be used as a floating rack while thawing samples.

[0213] g. Remove the vial from the water bath and spray it with 70% isopropyl alcohol solution. A good time to remove the vial from the water bath is when you can see small ice floating in the vial - it is recommended that the internal temperature of the vial be below 37°C.

[0214] h. Place the vial into the transfer channel and alert the cleanroom processing technician.

[0215] 2.3 Preparation of tissue processing:

[0216] a. Umbilical cord tissue processing should be performed in an environmentally monitored (EM) cleanroom. The entire room and safety hood should be cleaned at the end of each shift.

[0217] b. Prepare / clean the biosafety cabinet.

[0218] c. Perform viable particle counts while working in a biosafety cabinet.

[0219] d. Assemble all necessary supplies in a biosafety cabinet, inspecting each supply for damage to packaging and expiration dates. When handling syringes, serological pipettes, sterile forceps, scalpels, tissue plates, and needles, ensure that no surfaces will come into contact with the sterile product. Only the outside of the syringe barrel, tubing, plunger tip, and / or needle cap or sheath are safe to handle. Discard any surface that has been touched or comes into contact with a non-sterile surface.

[0220] e. Record the lot numbers and expiration dates (if applicable) of all reagents and supplies to be used.

[0221] f. Receive the thawed vials by cleaning them with a lint-free cloth soaked in 70% alcohol and then transferring to a biosafety cabinet.

[0222] g. Using the aspiration needle attached to the syringe, remove as much liquid as possible from the vial. Avoid aspirating the tissue.

[0223] h. Using sterile forceps, transfer the tissue to a sterile 100 mm culture dish.

[0224] i. Add a 5 ml aliquot of rinse medium to the tissue fragments.

[0225] j. Spin the contents for 15-30 seconds, then remove the rinse medium using a pipette or syringe with a needle. Repeat this rinse process two more times.

[0226] k. Add 2 mL of rinse medium to the tissue to prevent it from drying out.

[0227] 2.4 Start of MSC self-organized outgrowth:

[0228] a. Label the bottom of a 6-well plate with "Outgrowth 1," the MSC lot number or umbilical cord tissue ID, and the date the outgrowth was initiated. If using a 60 mm tissue culture dish, divide the plate into four quadrants by drawing a grid on the bottom of the dish.

[0229] b. Using sterile disposable forceps, place a 3 x 3 mm to 5 x 5 mm piece of tissue into each well. If using a 60 mm tissue culture dish, place the tissue in the middle of each quadrant to keep the tissues separate (more than 1 cm apart).

[0230] c. Add 3 ml of PTT-6 to each well.

[0231] d. Using a pipette needle attached to a 30 ml syringe, draw up enough medium to barely cover the tissue. Do not tilt the plate. Do not touch the bottom of the well with the pipette needle.

[0232] e. Using an inverted light microscope, observe cell growth every day (24 ± 6 hours). A real-time cell culture imaging system can be used instead of a light microscope.

[0233] f. Change the culture medium every day. Ensure that the culture medium is equilibrated to room temperature before use.

[0234] i. Aspirate the culture medium.

[0235] ii. Add 3 ml of PTT-6.

[0236] iii. Aspirate until the tissue is almost submerged in the medium.

[0237] g. When cell outgrowth is observed from the tissue, transplant the tissue into a new 6-well plate using the same procedures as described in 4.a through 4.e, except that the plate is labeled "Outgrowth 2." Maintain the cell outgrowth in the "Outgrowth 1" plate by adding 2 ml of PTT6 to each well. Observe confluence daily. Change the culture medium every 2-3 days (ensure that the medium is equilibrated to room temperature before use).

[0238] h. When an outgrowth is observed in the "Outgrowth 2" plate, repeat steps 4.a through 4.e, labeling the plate "Outgrowth 3." Maintain the outgrowth in the "Outgrowth 2" plate by adding 2 ml of PTT-6 to each well. Observe confluency daily. Change the culture medium every 2-3 days (ensure it is equilibrated to room temperature before use).

[0239] i. When outgrowth is observed in the "Outgrowth 3" plate, discard the tissue. If the tissue is very small and does not appear to interfere with cell growth, dispose of it at the time of subculture.

[0240] j. When cells reach 40-50% confluency, observe cells daily to prevent overexpansion.

[0241] k. Subculture cells when they reach 70-80% confluency. Do not allow cells to expand beyond 80% confluency.

[0242] When the size of the tissue fragments is about 1-3 mm and the tissue fragment method / cell culture is performed in a 175 square millimeter culture dish, the average number of mesenchymal stem cells harvested from the fragments is generally about 4,000-6,000 cells / fragment. Thus, when mesenchymal stem cells grow from 48 fragments simultaneously, about 300,000 cells can be obtained at the time of harvest. These 300,000 cells are then seeded in a 175 cm2 dish as described in Example 2.5 below. 2The 300,000 mesenchymal stem cells collected from the separation block were used for subculture in a cell culture flask (this can be called the first generation). The mesenchymal stem cells obtained from this first generation can then be seeded again in a 175 cm 2 The cells are cultured in a cell culture flask (passage 2) and expanded as described in Example 2.5 below. The cells obtained from passages 1 and 2 can be cryopreserved as a "bank", wherein the mesenchymal stem cells obtained after passage 2 are considered to represent the master cell bank, which will be used for further expansion of the mesenchymal stem cells, for example, in a bioreactor as described in Example 2.7 below.

[0243] 2.5. Subculturing MSCs in cell culture dishes

[0244] a. Perform particle counts while working in a biosafety cabinet. Before use, equilibrate all culture media to room temperature.

[0245] b. When the cell outgrowth reaches approximately 70-80% confluence, subculture the cells.

[0246] i. Remove PTT-6 from the culture dish.

[0247] ii. Rinse with HBSS without calcium or magnesium.

[0248] iii. Add 0.2 ml 1X TrypLE-EDTA and rotate for 1-2 minutes.

[0249] iv. Tilt the culture dish 30-45° to allow the cells to move downward by gravity flow. Gently tap the side of the plate to accelerate detachment.

[0250] v. Add 1 ml of 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. Cells from all 6 wells can be combined into a single 15 ml tube.

[0251] vi. Centrifuge at 1200 rpm for 10 minutes.

[0252] vii. Remove the supernatant and resuspend the cells in 5 ml of PTT-6.

[0253] c. Subculture MSCs.

[0254] i. Aliquot 50 μl of the cell suspension and analyze TNC and viability by Trypan Blue Exclusion Assay.

[0255] ii. Count the cells using a hemocytometer. Expect a count of 20-100 cells / square. If the count is higher than 100, dilute the original sample 1:5 and repeat the trypan blue method using a hemocytometer.

[0256] iii. Calculate viable cells / ml and total viable cells:

[0257] 1. Viable cells / ml = viable cell count × dilution factor × 10 4

[0258] 2. Total viable cells = viable cell count × dilution factor × total volume × 10 4

[0259] iv. Calculate % survival:

[0260] 1. % survival rate = viable cell count × 100 / (viable cell count + dead cell count)

[0261] v. Dilute the cell suspension to 1.0×10 6 cells / ml

[0262] 1. Volume of "X" = total viable cells / 10 6 cells / ml

[0263] 2. For example, if the total number of viable cells is 1.0×10 7 ;

[0264] 3. “X” = 10 7 / 10 6 cells / ml or 10 ml, therefore, by adding 5 ml to the cell suspension (i.e. 5 ml), the total cell volume can be brought to 10 ml.

[0265] vi. If the cell suspension is less than 10 6 / ml, then determine the 2 The bottle was inoculated with 2×10 6 The volume required for each cell.

[0266] 1.2×10 6 The volume required for cells = 2×10 6 cells ÷ viable cells / ml

[0267] 2. For example, if the viable cells / ml is 8×10 5 cells / ml, 2×10 6 cells÷8×10 5 cells / ml or 2.5ml.

[0268] vii. Set aside 0.5 ml for MSC marker analysis.

[0269] viii. In each 150 mm dish or 175 cm dish with 30 ml of PTT-6 2 The bottle was inoculated with 2×10 6 cells.

[0270] ix. Observe cells every three days for attachment, colony formation, and confluence. When cells reach 40-50% confluence, observe cells every 1-2 days to prevent overexpansion. Do not allow cells to expand beyond 80% confluence. A real-time cell culture monitoring system can be used instead of a light microscope.

[0271] x. Change the culture medium every 2-3 days.

[0272] 2.6 Cryopreservation of MSCs

[0273] a. Perform presence particle counts while working in a biosafety cabinet.

[0274] b. When cells reach 70-80% confluence, 2 The flask was treated with 2 ml of 1X TrypLE-EDTA to detach the cells.

[0275] i. Remove PTT-6 from the culture dish.

[0276] ii. Wash with 5 ml of HBSS or PBS without calcium or magnesium.

[0277] iii. Add 2 ml of 1X TrypLE-EDTA and rotate for 1-2 minutes.

[0278] iv. Tilt the dish 30-45° to allow the cells to move downward by gravity. Gently tap the side of the dish to help accelerate separation.

[0279] v. Add 10 ml of PTT-6 to inactivate TrypLE and mix thoroughly to separate cell clumps.

[0280] vi. Use a Pasteur pipette to transfer the cells to a 15 ml centrifuge tube.

[0281] vii. Centrifuge at 1200 rpm for 10 minutes.

[0282] viii. Aspirate the culture medium and resuspend with 10 ml of PTT-6.

[0283] ix. Aliquot 50 μl and determine the total viable cell count and % viability as described above. Since cells may begin to clump, the cell count should be completed within 15 minutes.

[0284] c. Prepare cells for cryopreservation.

[0285] i. Prepare cell suspension medium and cryopreservation medium, and freeze cells

[0286] 2.7. Subculturing (expansion) of MSCs in a Quantum bioreactor (Terumo BTC, Inc.)

[0287] A Quantum bioreactor can also be used to expand MSCs. The starting cell count for expansion in a Quantum bioreactor should be 20 to 30 million cells per run. Harvesting typically yields 300 to 700 million MSCs per run. The bioreactor is operated according to the manufacturer's instructions. The mesenchymal stem cells thus obtained are typically cryopreserved (see below) and used as a working cell bank.

[0288] Materials / Reagents:

[0289] 1. Quantum Amplification Device

[0290] 2. Quantum waste bags

[0291] 3. Quantum culture medium bag

[0292] 4. Quantum enters the bag

[0293] 5. PTT-6

[0294] 6. PBS

[0295] 7. Fibronectin

[0296] 8. TrypLE

[0297] 9. 3ml syringe

[0298] 10. Glucose test strips

[0299] 11. Lactic acid test paper

[0300] 12. 60ml cell culture plate or equivalent

[0301] 13. Medical grade 5% CO2 gas mixture

[0302] 14. 50ml Combi-tip

[0303] equipment:

[0304] 1. Biological safety cabinet

[0305] 2. Blood glucose meter (Bayer Healthcare / Ascensia Contour blood glucose meter)

[0306] 3. Lactate Plus (Nova Biomedical)

[0307] 4. Peristaltic pump with head

[0308] 5. Centrifuge, Eppendorf 5810

[0309] 6. Sterile tube connector

[0310] 7. M4 Repeating Pipette

[0311] 8. RF sealer

[0312] process:

[0313] 1. Prepare the Quantum Bioreactor

[0314] a) Start-up of Quantum Bioreactor

[0315] b) Coating bioreactor:

[0316] 1) Prepare fibronectin solution in a biosafety cabinet.

[0317] 1) Allow the freeze-dried fibronectin to adapt to room temperature (≥15 min at room temperature)

[0318] 2) Add 5 ml of sterile distilled water; do not rotate or stir

[0319] 3) Allow fibronectin to form a solution for 30 minutes.

[0320] 4) Using a 10 ml syringe with an 18 g needle attached, transfer the fibronectin solution into the cell access bag containing 95 ml of PBS.

[0321] 2) Connect the bag to the "reagent" line

[0322] 3) Check for air bubbles (air bubbles can be removed by using "Remove IC Air" or "Remove EC Air" and using "Scrub" as the inlet source.

[0323] 4) Open or set up the coating bioreactor program ( Figure 1 , steps 3-5).

[0324] 5) Run the program

[0325] 6) While the program is running to coat the bioreactor, prepare a media bag with 4 L of PTT-6 media.

[0326] 7) Connect the media bag to the IC media line using a sterile tubing connector.

[0327] 8) When the bioreactor coating step is complete, separate the cells for the fibronectin solution into bags using an RF sealer.

[0328] c) Wash off excess fibronectin

[0329] d) Conditioning the bioreactor with culture medium.

[0330] 2. Cultivating cells in a Quantum bioreactor

[0331] a) Loading and attaching cells using a uniform suspension:

[0332] b) Feeding and culturing cells

[0333] 1) Select the medium flow rate to feed the cells.

[0334] 2) Lactate and glucose samples were collected daily.

[0335] 3) As lactate levels increase, adjust the flow rate of the medium. The actual maximum tolerated lactate concentration will be determined by the culture flask culture from which the cells were derived. Determine if there is sufficient PTT-6 medium in the medium bag. If necessary, replace the PTT-6 medium bag with a new one.

[0336] 4) When the flow rate reaches the desired value, measure the lactate level every 8-12 hours. If the lactate level does not decrease or if the lactate level continues to increase, harvest the cells.

[0337] 3. Harvesting cells from the Quantum bioreactor

[0338] a) When lactate concentration has not decreased, cells are harvested after the last lactate and glucose sampling.

[0339] b) Harvesting cells:

[0340] 1) Connect the cell access bag containing 100 ml of TrypLE to the "reagent" line using a sterile tubing connector.

[0341] 2) Confirm that there is enough PBS in the PBS bag. If not, connect a new bag with at least 1.7 liters of PBS to the "Wash" line using a sterile tubing connector.

[0342] 3) Run the Harvest program

[0343] 4. Cryopreservation of cells

[0344] 1) Once the cells are harvested, transfer them to a 50 ml centrifuge tube to pellet the cells.

[0345] 2) Resuspend in 25 ml of cold cell suspension buffer. Count the cells using a Sysmex or Biorad cell counter. Attach the cell count report to the corresponding Quantum processing batch record.

[0346] 3) Adjust the cell concentration to 2×10 7 / ml

[0347] 4) Add an equal volume of cryopreservative solution and mix thoroughly (do not shake or vortex)

[0348] 5) Using a repeating pipette, add 1 ml of the cell suspension in cryopreservative solution to each 1.8 ml vial and cryopreserve using the CRF procedure as described in SOP D6.100 CB Cryopreservation Using Controlled Rate Freezers.

[0349] 6) Store the vials in a designated liquid nitrogen storage space.

[0350] 7) Attach the CRF run report to the corresponding MSC P3-Quantum processing batch record form.

[0351] 3. Using different culture media to analyze the stem cells in the mesenchymal umbilical cord lining stem cell population isolated from umbilical cord tissue Marker expression

[0352] Flow cytometry experiments were performed to analyze the expression of mesenchymal stem cell markers CD73, CD90, and CD105 in mesenchymal stem cells isolated from umbilical cord.

[0353] For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the tissue in three different culture media, and then subcultured in the corresponding culture media as described in Example 2.

[0354] In these experiments, the following three culture media were used: a) 90% (v / v) DMEM supplemented with 10% (v / v) FBS, b) PTT-4 medium described in U.S. Patent Application 2008 / 0248005 and the corresponding International Patent Application WO 2007 / 046775, which consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS (see paragraph

[0183] of WO 2007 / 046775), and c) PPT-6 medium of the present invention, the composition of which is described herein. In this flow cytometric analysis, two different samples of umbilical cord lining mesenchymal stem cell (CLMC) populations were analyzed in each of the three culture media used.

[0355] The flow cytometry analysis employed the following protocol.

[0356] Materials and methods

[0357]

[0358] process:

[0359] a) Isolation and culture of cells from the umbilical cord lining

[0360] 1. The excised tissue samples were incubated in cell culture plates and immersed in the corresponding culture medium, and then maintained in a CO2 incubator at 37°C as described in Example 2.

[0361] 2. Replace the culture medium every 3 days.

[0362] 3. Monitor cell outgrowth from the tissue culture inserts under a light microscope.

[0363] 4. When approximately 70% confluent, cells were detached from the culture dish by trypsinization (0.0125% trypsin / 0.05% EDTA) and used for flow cytometry experiments.

[0364] b) Trypsinization of cells used for experiments

[0365] 1. Remove the culture medium from the cell culture plate.

[0366] 2. Gently rinse with sterile 1X PBS to remove traces of FBS, as FBS will interfere with the enzymatic action of trypsin.

[0367] 3. Add 1X trypsin to the cell culture plate and incubate at 37°C for 3-5 minutes.

[0368] 4. Observe the cells under a microscope and ensure that the cells have detached. Neutralize the trypsin by adding complete medium containing FBS (DMEM containing 10% FBS).

[0369] 5. Use a pipette to break up the cell clumps by drawing the cells in the culture medium against the wall of the culture plate. Collect the cell suspension and transfer it to a 50ml centrifuge tube.

[0370] 6. Add sterile 1X PBS to the plate and rinse. Collect the cell suspension into the same centrifuge tube.

[0371] 7. Centrifuge at 1800 rpm for 10 minutes.

[0372] 8. Discard the supernatant and resuspend the cell pellet in PBA medium.

[0373] c) Count cells

[0374] 1. Ensure that the hemocytometer and its cover glass are clean and dry. Preferably, wash with 70% ethanol and let dry, then wipe with Kim paper towel (dust-free paper).

[0375] 2. Aliquot a small amount of suspended cells into microcentrifuge tubes and remove from the BSC hood.

[0376] 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, to obtain 0.2% trypan blue solution).

[0377] 4. Avoid exposing cells to trypan blue for more than 30 minutes because trypan blue is toxic and can lead to an increase in non-viable cells, thus giving an erroneous cell count.

[0378] 5. Add 20 μl of the cell suspension mixture to each well of the hemocytometer and observe under a light microscope.

[0379] a. Count the number of viable cells (bright cells; non-viable cells readily absorb trypan blue and therefore appear dark) in each quadrant of a hemocytometer, for a total of 8 quadrants in the upper and lower chambers.

[0380] The total cell count was expressed as (average number of cells / quadrant) × 10 4 Cells / ml are given.

[0381] d) Staining cells

[0382] i. Preparation before staining cells

[0383] • The cell suspension was aliquoted in duplicate into 3 tubes (CD73, CD90, CD105) and 2 tubes (negative control), each containing 50,000 cells.

[0384] ii. Staining with primary antibody (Ab)

[0385] Add 1 μl of primary antibody (0.5 mg / ml Ab) to 100 μl of cell suspension and incubate at 4°C for 45 min.

[0386] Use PBA to make up to 1 ml.

[0387] Centrifuge at 8000 rpm for 5 min at 4°C.

[0388] Remove the supernatant.

[0389] • Add 1 ml PBA and resuspend the pellet.

[0390] Centrifuge at 8000 rpm for 5 min at 4°C.

[0391] Remove the supernatant.

[0392] • Resuspend in 100 μl PBA.

[0393] iii. Staining with secondary antibody -In the dark

[0394] Add 1 μl of 0.5 mg / ml ab secondary antibody to 100 μl of cell suspension and incubate at 4°C for 30 min.

[0395] Use PBA to make up to 1 ml.

[0396] Centrifuge at 8000 rpm for 5 min at 4°C.

[0397] Remove the supernatant.

[0398] • Add 1 ml PBA and resuspend the pellet.

[0399] Centrifuge at 8000 rpm for 5 min at 4°C.

[0400] Remove the supernatant.

[0401] • Resuspend in 200-300 μl PBA for flow cytometry analysis.

[0402] • Transfer cells to FACS tubes for reading in a BD FACS CANDO flow cytometer.

[0403] The results of the 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 from umbilical cord tissue and culture 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 from umbilical cord tissue and culture 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 from umbilical cord tissue and culture in PTT-6. As can be seen from Figure 6a, the isolated cell population cultured in DMEM / 10% FBS has approximately 75% CD73+ cells, 78% CD90+ cells, and 80% CD105+ cells (average of two sets of experiments), while after isolation / culture of umbilical cord tissue using PPT-4 medium (see Figure 6b), the number of CD73-positive, CD90-positive, and CD105-positive mesenchymal stem cells is approximately 87% (CD73+ cells), 93% (CD90+ cells), and 86% (CD105+ cells), which is the average of two sets of experiments. With respect to all three markers (CD73, CD90, CD105), the purity of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention is at least 99.0%, meaning that the purity of the cell population is significantly higher than that of the cells cultured using PPT-4 medium or DMEM / 10% FBS. Furthermore, and even more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is essentially a 100% pure and defined stem cell population. This makes the stem cell population of the present invention an ideal candidate for stem cell-based therapies. Therefore, this mesenchymal umbilical cord lining stem cell population may become the gold standard for such stem cell-based therapies.

[0404] The results of flow cytometric analysis shown in Figures 7a and 7b further confirmed the findings shown in Figure 6. Figure 7a shows the percentage of isolated mesenchymal umbilical cord lining stem cells (umbilical cord amniotic mesenchymal stem cells) expressing the stem cell markers CD73, CD90, and CD105 and lacking expression of CD34, CD45, and HLA-DR after isolation from umbilical cord tissue and culture in PPT-6 medium. As shown in Figure 7a, the mesenchymal stem cell population contained 97.5% viable cells, of which 100% expressed each of CD73, CD90, and CD105 (see "CD73+CD90+" and "CD73+CD105+" rows), 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 "CD34-CD45-" and "CD34-HLA-DR-" rows). Thus, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is essentially a 100% pure and defined stem cell population that meets the criteria that mesenchymal stem cells should meet to be used in cell therapy (95% or more of the stem cell population express CD73, CD90, and CD105, while 98% or more of the stem cell population lack expression of CD34, CD45, and HLA-DR, see Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", supra). It should be noted here that the mesenchymal stem cells of the amniotic membrane of the present invention adhere to plastic under standard culture conditions and differentiate into osteoblasts, adipocytes, and chondroblasts in vitro, see U.S. Patent No. 9,085,755, U.S. Patent No. 8,287,854, or WO2007 / 046775, and therefore meet the generally accepted criteria for mesenchymal stem cells for cell therapy.

[0405] Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90 and CD105 and lacking the expression of CD34, CD45 and HLA-DR. As shown in Figure 7b, the bone marrow mesenchymal stem cell population contains 94.3% of viable cells, of which 100% express each of CD73, CD90 and CD105 (see "CD73+CD90+" and "CD73+CD105+" rows), while only 62.8% of the bone marrow stem cell population lack the expression of CD45, and 99.9% of the stem cell population lack the expression of CD34 and HLA-DR (see "CD34-CD45-" and "CD34-HLA-DR-" rows). Therefore, compared to the (umbilical cord amniotic membrane) mesenchymal stem cell population of the present application, the bone marrow mesenchymal stem cells, which are considered to be the gold standard of mesenchymal stem cells, have much lower homogeneity / purity in terms of stem cell markers. This finding also suggests that the stem cell population of the present invention may be an ideal candidate for stem cell-based therapy and that the stem cell population may become the gold standard for stem cell-based treatment methods.

[0406] 4. Secretion of wound healing marker proteins by culturing the mesenchymal stem cell population isolated in the culture medium of the present invention Analysis

[0407] Based on the highly significant results (essentially 100% pure and defined mesenchymal stem cell populations were obtained by culture in PTT-6), various isolated mesenchymal stem cell populations were cultured in PTT-6 and analyzed for secretion of wound healing marker proteins compared to culture in PTT-4 medium (as a reference medium).

[0408] In more detail, the following isolated mesenchymal stem cell populations were analyzed.

[0409] - Umbilical cord amniotic membrane mesenchymal stem cells (umbilical cord lining MSC / CL-MSC): This CL-MSC population was isolated from human umbilical cord lining tissue pieces as described in Example 2 of WO2007 / 046775 (cultured in DMEM supplemented with 10% fetal bovine serum (DMEM / 10% FBS)).

[0410] - Wharton's jelly mesenchymal stem cells (WJ-MSCs). This WJ-MSC population was isolated from Wharton's jelly tissue pieces of human umbilical cord (cultured in DMEM with 4,500 mg / mL glucose and 2 mM L-glutamine supplemented with 10% human serum / FBS and antibiotic solution) as described by Beeravolu et al., "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017;(122):55224.

[0411] Adipose-derived mesenchymal stem cells (AT-MSCs) were isolated from adipose tissue of donated skin tissue after abdominoplasty by the tissue dissociation method (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.

[0412] - Bone marrow mesenchymal stem cells (BM-MSCs). This BM-MSC population was donated by the AO Foundation, Davos, Switzerland.

[0413] Placental mesenchymal stem cells (PT-MSCs). These PT-MSCs were isolated from the placenta as described by Beeravolu et al., “Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.” J Vis Exp. 2017; (122): 55224.

[0414] Culture protocol for culturing isolated MSCs

[0415] • 5 million MSCs of each source were plated in 100 mm tissue culture dishes containing DMEM / F12 / 10% FCS for 24 hours.

[0416] ● Discard the culture medium and add PTT-6 / PTT-4 and culture for 24 hours.

[0417] • Discard the culture medium and wash the cells with PBS.

[0418] • Add 10 ml of DMEM to the culture for 24 hours.

[0419] • Discard the culture medium and add 5 ml of DMEM to the culture.

[0420] After 24 hours of culture, the conditioned medium was harvested, cell debris was removed by centrifugation, and the supernatant was aliquoted into tubes and stored at -80°C before analysis of marker protein secretion by cytokine assay.

[0421] Cytokine analysis of PTT-6 and PTT-4 culture supernatants of CL-MSC, WJ-MSC, bone marrow MSC, and adipose MSC-derived MSCs

[0422] Cytokine detection was performed in MSC supernatants using Luminex 200 and Xponent software for measurement and analysis.

[0423] The purpose of this experiment was to measure the relative levels of multiple (PDGF-AA, PDGF-BB, IL-10, VEGF, Ang-1, and HGF), TGFβ1 alone, and bFGF2 alone in cell culture supernatants (MSC, mesenchymal stem cells; CL, umbilical cord lining; WJ, Wharton's jelly; AT, adipose tissue; BM, bone marrow):

[0424] CL-MSCs cultured in PTT-4

[0425] WJ-MSCs cultured in PTT-4

[0426] AT-MSCs cultured in PTT-4

[0427] BM-MSCs cultured in PTT-4

[0428] CL-MSCs cultured in PTT-6

[0429] WJ-MSCs cultured in PTT-6

[0430] AT-MSCs cultured in PTT-6

[0431] BM-MSCs cultured in PTT-6

[0432] Each sample was tested in triplicate (3 wells) except for the sample provided in PTT-4 which was tested in 6 wells. In addition, samples CR001A, CR001C, CR001D, and CR001G were included as positive controls to validate the cytokine assays (conditioned medium from CR001A, CR001C, CR001D, and CR001G was not prepared by culturing cells in PTT-6 or PTT-4).

[0433] The purpose of this experiment is to generate a cytokine profile of MSCs cultured in PTT-4 or PTT-6 and compare the profiles of MSCs from different tissue sources (umbilical cord lining, Wharton's jelly, adipose tissue, and bone marrow). This profile will clarify which stem cell populations secrete more of the desired cytokines when grown in which medium to promote wound healing.

[0434] Figure 8 Plate setup for all culture plates is described in The abbreviations used are as follows: MSC, mesenchymal stem cell; CL, umbilical cord lining; WJ, Wharton's jelly; AT, adipose tissue; BM, bone marrow.

[0435] Multiplex analysis

[0436] Multiple information:

[0437] R&D Systems / Bio-techne cat.#LXSAHM. The kit is lot#L123680, expired 08 / 28 / 18, and has the following analytes:

[0438] Ang-1, angiopoietin

[0439] VEGF, vascular endothelial growth factor

[0440] PDGF-AA, platelet-derived growth factor (PDGF-AA refers to the disulfide-linked homodimer composed of the A chain, while PDGF-BB consists of the B homodimer. R&D indicates that the PDGF-BB antibody also detects the PDGF-AB heterodimer)

[0441] PDGF-BB

[0442] HGF, hepatocyte growth factor

[0443] IL-10, interleukin-10

[0444] TGFβ1 single compound information: R&D Systems / Bio-techne)

[0445] Basic kit, cat.#LTGM00, lot#P156217, received 02 / 27 / 18, expired 08 / 30 / 18.

[0446] TGFβ1 component, cat.#LTGM100, lot#P161760, received 02 / 27 / 18, expired 11 / 27 / 19.

[0447] bFGF2 single-plex information (used on March 19, 2018): eBioscience / Thermo:

[0448] Basic kit, cat.# EPX010-10420-901, lot# 172174000, expires 01 / 31 / 20.

[0449] bFGF2 component, cat.#EPX01A-12074-901, lot#169751102, expires 12 / 31 / 19.

[0450] bFGF2 singleplex information (used on March 22, 2018): eBioscience / Thermo:

[0451] Basic kit, cat.# EPX010-10420-901, lot# 172174000, expires 01 / 31 / 20.

[0452] bFGF2 component, cat.#EPX01A-12074-901, lot#166916102, expires 12 / 31 / 19.

[0453] Multiple information:

[0454] R&D Systems / Bio-techne cat.#LXSAHM. This kit is lot#L123999, expired 09 / 25 / 18, and has the following analytes:

[0455] Ang-1, angiopoietin

[0456] VEGF, vascular endothelial growth factor

[0457] PDGF-AA, platelet-derived growth factor 2

[0458] PDGF-BB

[0459] HGF, hepatocyte growth factor

[0460] IL-10, interleukin-10

[0461] bFGF, basic fibroblast growth factor

[0462] Data Input

[0463] The raw data is exported in PDF and Excel formats. The Excel format data is used for processing data.

[0464] program

[0465] Cytokine detection was performed in MSC supernatants according to the detailed protocol information. As part of this experiment, there was a revision to the protocol: Std.8 in the multiplex kit was no longer used. The reason for discontinuing the use of Std.8 was that the R&D Systems protocol itself only used standards 1 to 6. In addition, Std.8 was validated at ClinImmune for only two of the six analytes that made up the multiplex: PDGF-BB and HGF. In the case of PDGF-BB, the analyte was never detected in the supernatant. In the case of HGF, the analyte fell in the middle region of the standard curve. Since the standards were reconstituted using growth medium, standard curves were constructed using both PTT-6 and PTT-4. Test samples grown in PTT-6 or PTT-4 were extrapolated from the corresponding standard curves.

[0466] Results were extrapolated by Luminex software based on analyte-specific standard curves generated by the same software: the analysis algorithm was set to Logistic 5P Weighted with weighted analysis, with weighting as 1 / y2.

[0467] sample

[0468] 1. PTT-4 and PTT-6 culture media (not exposed to MSCs)

[0469] 2. Supernatant of MSC to be tested

[0470] 3. Optional: Supernatant of CL-MSCs from different donors; CR001A, C, D, and G.

[0471] Overview of experimental results

[0472] TGFβ1 singleplex analysis

[0473] Use aliquot 3 of 1- as Figure 9 All error bars are the standard deviation of three measurements.

[0474] Figure 9Figure 3: Single-plex measurement of TGFβ1. As can be seen, CL-MSC and WJ-MSC cultures produced more TGFβ1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced approximately equal amounts of TGFβ1 when grown in either PTT-6 or PTT-4. All error bars represent the standard deviation of three measurements.

[0475] First multiplex analysis

[0476] Use aliquot 1 of 3.

[0477] PDGF-BB and IL-10 were not detected in any samples.

[0478] The data are depicted in Figures 10 and 11.

[0479] Figure 10: Figure 10A =Multiple measurements of PDGF-AA. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC produced more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of three measurements. Figure 10B =Multiple measurements of VEGF. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC produced more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three measurements. Figure 10C Figure 1 shows multiple measurements of Ang-1. As can be seen, CL-MSC and WJ-MSC cultures produced more Ang-1 when grown in PTT-6 compared to when grown in PTT-4. AT-MSC and BM-MSC cultures produced virtually no Ang-1. All error bars represent the standard deviation of three measurements.

[0480] Figure 11 Figure 2: Multiple measurements of HGF. As can be seen, CL-MSC and WJ-MSC cultures produced significantly more HGF when grown in PTT-6 compared to PTT-4. AT-MSC and BM-MSC cultures produced virtually no HGF. All error bars represent the standard deviation of three measurements.

[0481] Multiplex analysis (including bFGF)

[0482] · Aliquot 3 of 3 was used. Data are shown in Figure 12-14 .

[0483] Figure 12Figure 3: Multiplex measurement of PDGF-AA. As can be seen, CL-MSC and WJ-MSC cultures produced more PDGF-AA when grown in PTT-4 compared to when grown in PTT-6. AT-MSC and BM-MSC cultures produced equivalent amounts of PDGF-AA in both media. All error bars represent the standard deviation of three measurements.

[0484] Figure 13: Figure 13A =Multiple measurements of VEGF. As can be seen, cultures CL-MSC, WJ-MSC, AT-MSC, and BM-MSC produced more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three measurements. Figure 13B Figure 1 shows multiple measurements from a multiplex analysis of Ang-1. As can be seen, CL-MSC and WJ-MSC cultures produced significantly more Ang-1 when grown in PTT-6 compared to when grown in PTT-4. AT-MSC and BM-MSC cultures produced virtually no Ang-1. All error bars represent the standard deviation of three measurements. Figure 13C Figure 2 is a multiplex measurement of HGF. As can be seen, cultures CL-MSC and WJ-MSC produced significantly more HGF when grown in PTT-6 compared to cultures grown in PTT-4. Cultures AT-MSC and BM-MSC produced virtually no HGF. All error bars represent the standard deviation of three measurements.

[0485] Figure 14 Figure 2: Multiple measurements of bFGF. As can be seen, CL-MSC and WJ-MSC cultures produced more bFGF when grown in PTT-6 compared to when grown in PTT-4. AT-MSC and BM-MSC cultures produced equivalent amounts of bFGF when grown in PTT-4 and PTT-6. All error bars represent the standard deviation of three measurements.

[0486] It should be noted that the abundance of bFGF samples was very low and at or near the limit of detection.

[0487] Figures 15 to 21 An overview of the data obtained in the different experiments is described.

[0488] Figure 15: TGFβ1 measurements from 5 different experiments (170328, 170804, 170814, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the TGFβ standard curve measured in each experiment is depicted in the small figure on the left below. The MFI of the TGFβ standard curve obtained in PTT-4 and PTT-6 culture media is shown in the small figure above. The small figure on the right below depicts that cultures CL-MSC and WJ-MSC produce more TGFβ1 when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produce equal amounts of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from the different measurements for experiments 170328, 170804, 170814, 180105, 180226.

[0489] Figure 16 Ang-1 measurements from six different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the Ang-1 standard curve measured for each experiment is depicted in the lower left panel. The upper panel shows the MFI of the Ang-1 standard curves obtained in PTT-4 and PTT-6 culture media. The lower right panel shows that cultures CL-MSC and WJ-MSC produce more Ang-1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produce essentially equivalent amounts of Ang-1 when grown in PTT-6 or PTT-4. All error bars represent the standard deviation of the different measurements for experiments 170602, 170511, 170414, 170224, 180105, and 180226.

[0490] Figure 17 :Summarize the PDGF-BB measurement of 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).The mean fluorescence intensity (MFI) of the PDGF-BB standard curve measured in each experiment is depicted in the small figure on the left below.The MFI of the PDGF-BB standard curve obtained in PTT-4 and PTT-6 culture medium is shown in the small figure above.It is worth noting that PDGF-BB was not detected in all experiments.

[0491] Figure 18: PDGF-AA measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the PDGF-AA standard curve measured in each experiment is depicted in the small figure on the left below. The MFI of the PDGF-AA standard curve obtained in PTT-4 and PTT-6 culture media is shown in the small figure above. The small figure on the right below depicts that culture CL-MSC, AT-MSC and BM-MSC and WJ-MSC cultures produce slightly more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of the measured values ​​from experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0492] Figure 19 :Summarizes the IL-10 measurement of 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).The mean fluorescence intensity (MFI) of the IL-10 standard curve measured in each experiment is depicted in the small figure on the left below.The MFI of the IL-10 standard curve obtained in PTT-4 and PTT-6 culture medium is shown in the small figure above.It is worth noting that IL-10 was not detected in all experiments.

[0493] Figure 20 : VEGF measurements from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the VEGF standard curve measured in each experiment is depicted in the lower left panel. The MFI of the VEGF standard curve obtained in PTT-4 and PTT-6 culture media is shown in the upper panel. The lower right panel depicts 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 of the different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0494] Figure 21: HGF measurements from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) are summarized. The mean fluorescence intensity (MFI) of the HGF standard curve measured in each experiment is depicted in the small figure on the left below. The MFI of the HGF standard curve obtained in PTT-4 and PTT-6 culture media is shown in the small figure above. The small figure on the right below depicts that cultures CL-MSC and WJ-MSC produce more HGF when grown in PTT-6 than when grown in PTT-4. On the other hand, cultures AT-MSC and BM-MSC do not produce as much HGF as the other cultures. All error bars are standard deviations of the different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

[0495] Cytokine analysis of PTT-6 and PTT-4 culture media or DMEM / F12 supernatants of CL-MSC, WJ-MSC, and placental MSC-derived MSCs

[0496] Cytokine detection was performed in MSC supernatants. Measurements and analyses were performed as described above.

[0497] The purpose of this experiment was to measure the relative levels of multiple (PDGF-AA, PDGF-BB, IL-10, VEGF, Ang-1, and HGF), TGFβ1 alone, and bFGF2 alone in cell culture supernatants. The supernatants were obtained from mesenchymal stem cells derived from umbilical cord lining (CL), Wharton's jelly (WJ), and placenta. The mesenchymal stem cells were cultured in PTT-6, PPT-4, or DMEM / F12 medium.

[0498] CL-MSCs cultured in PTT-4

[0499] WJ-MSCs cultured in PTT-4

[0500] Placental MSCs cultured in PTT-4

[0501] CL-MSCs cultured in PTT-6

[0502] WJ-MSCs cultured in PTT-6

[0503] Placental MSCs cultured in PTT-6

[0504] CL-MSCs cultured in DMEM / F12

[0505] WJ-MSCs cultured in DMEM / F12

[0506] All samples, except for the placental supernatant sample, were tested in triplicate. The purpose of this experiment was to generate a cytokine profile for MSCs cultured in PTT-4 or PTT-6 and to compare the profiles of MSCs from different tissue sources (umbilical cord lining vs. Wharton's jelly vs. placental MSCs). Cytokine measurements were performed as described above. This profile will clarify which stem cell populations secrete more of the target cytokines in which culture medium to promote wound healing.

[0507] Figure 22 : Single-plex measurement of TGFβ1. The mean fluorescence intensity (MFI) of the TGFβ1 standard curve measured in each experiment is depicted in the left panel. As can be seen from the right panel, all CL-MSCs, WJ-MSCs and placental MSCs grew in PTT-6 compared to those grown in PTT-4 or DMEM / F12 (in Figure 22 When grown in DMEM (referred to simply as DMEM), more TGFβ1 is produced.

[0508] Figure 23 : The measurement of PDGF-BB in the supernatant of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM / F12 is summarized. The mean fluorescence intensity (MFI) of the PDGF-BB standard curve measured in each experiment is depicted in the small figure on the left. It is worth noting that PDGF-BB was not detected in all experiments.

[0509] Figure 24 : The measurement of IL-10 in the supernatant of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM / F12 analyzed is summarized. The mean fluorescence intensity (MFI) of the VEGF standard curve measured in each experiment is depicted in the left panel. S6 represents the lowest standard used in the assay. Any sample below this is considered to be below the detection limit. As can be seen from the right panel, when grown in PTT-6, all CL-MSC, WJ-MSC and placental MSC produced detectable levels of IL-10, while when MSCs were grown in PTT-4 or DMEM / F12, little or no IL-10 was detected.

[0510] Figure 25: Summary of the measurements of VEGF in the supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT-6, PTT-4, or DMEM / F12 analyzed. The mean fluorescence intensity (MFI) of the VEGF standard curve measured for each experiment is depicted in the left panel. S1 represents the highest standard used in the assay. Any sample above this is considered speculative (too concentrated). As can be seen from the right panel, all CL-MSCs, WJ-MSCs, and placental MSCs produced much higher levels of VEGF when grown in PTT-6 compared to when the MSCs were grown in PTT-4 or DMEM / F12.

[0511] Figure 26 Multiple bFGF measurements are summarized. The mean fluorescence intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the left panel. As can be seen in the right panel, cultured CL-MSCs and WJ-MSCs produced more bFGF when grown in PTT-6 than when grown in PTT-4. As can be seen, all CL-MSCs, WJ-MSCs, and placental MSCs produced significantly lower levels of bFGF when grown in PTT-6 compared to when grown in PTT-4 or DMEM / F12.

[0512] Figure 27 : PDGF-AA measurements are summarized. The mean fluorescence intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the left panel. S6 represents the lowest standard used in the assay. Any sample below this is considered below the limit of detection. As can be seen, all CL-MSCs, WJ-MSCs, and placental MSCs produced higher levels of PDGF-AS when grown in PTT-6 compared to when MSCs were grown in PTT-4 or DMEM / F12.

[0513] Figure 28 : Ang-1 measurements are summarized. The mean fluorescence intensity (MFI) of the Ang-1 standard curve measured for each experiment is depicted in the left panel. S1 represents the highest standard used in the assay. Any sample above this is considered speculative (too concentrated). The right panel depicts that all CL-MSCs, WJ-MSCs, and placental MSCs produced significantly higher levels of Ang-1 when grown in PTT-6 compared to when grown in PTT-4 or DMEM / F12.

[0514] Figure 29: HGF measurements are summarized. The mean fluorescence intensity (MFI) of the HGF standard curve measured for each experiment is depicted in the left panel. The right panel depicts that all CL-MSCs, WJ-MSCs, and placental MSCs produced significantly higher levels of Ang-1 when grown in PTT-6 compared to when grown in PTT-4 or DMEM / F12.

[0515] From the above experiments, the following conclusions can be drawn. When mesenchymal stem cells (particularly mesenchymal stem cells separated from the umbilical cord compartment or from the placenta) are cultured in PTT-6 culture medium, the mesenchymal stem cell group secretes factors angiogenin (Ang-1), TGF-β1, VEGF, and HGF significantly increased compared to their production levels in PTT-4 culture medium or commercially available culture medium (such as DMEM / F12). It is noteworthy that PTT-6 culture medium can increase the production / secretion of these factors, regardless of the natural environment / compartment of the mesenchymal stem cell group.

[0516] Since PTT-6 medium causes the secretion of all of Ang-1, TGF-β1, VEGF, and HGF (which are known to be involved in wound healing as discussed herein) by the mesenchymal stem cell population, it is clear that PTT-6 medium has the effect of inducing or improving the wound healing properties of a broad population of mesenchymal stem cells, regardless of the natural environment / compartment of the mesenchymal stem cell population from which the mesenchymal stem cells were originally derived - it is also noted that Experiment 4 was performed using a cell population that had been isolated from its natural environment prior to culturing in PTT-6.

[0517] Furthermore, culturing mesenchymal stem cells in PTT-6 cells using the tissue dissociation block method provided a highly homogenous population of umbilical cord amniotic membrane mesenchymal stem cells (containing 97.5% viable cells, 100% of which expressed each of CD73, CD90, and CD105, while 99.2% of the stem cell population did not express CD45, and 100% of the stem cell population did not express CD34 and HLA-DR (see "CD34-CD45-" and "CD34-HLA-DR-" rows). Since culturing the Wharton's jelly mesenchymal stem cell population in PTT-6 cells had a positive effect on the production of cytokines Ang-1, TGF-β1, VEGF, and HGF, similar to the positive effects on these cytokines in umbilical cord lining stem cells, it is expected that culturing Wharton's jelly cells in PTT-6 cells would also yield such a highly homogenous population of mesenchymal Wharton's jelly stem cells. Therefore, it is also expected that tissue explants from other compartments of the umbilical cord (such as umbilical cord blood vessels) will yield similarly homogenous populations of perivascular (PV) mesenchymal stem cells. Similarly, tissue explants from placental tissue (including placental amniotic membrane) cultured in PTT-6 can be expected to yield similarly homogenous populations of placental mesenchymal stem cells. Thus, the present invention provides a generally applicable method for obtaining a population of mesenchymal stem cells, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

[0518] The present invention is also characterized by the following items.

[0519] 1. A method for inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a culture medium comprising Dulbecco's Modified Eagle's Medium (DMEM), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum).

[0520] 2. The method according to item 1, 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-placenta 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.

[0521] 3. The method according to item 2, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed (MC) mesenchymal stem cell population.

[0522] 4. The method according to any one of items 1 to 3, wherein the culture medium comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

[0523] 5. The method according to claim 4, wherein the culture medium comprises DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v).

[0524] 6. The method according to claim 5, wherein the culture medium comprises DMEM with a final concentration of about 61.8% (v / v), F12 with a final concentration of about 11.8% (v / v), M171 with a final concentration of about 23.6% (v / v) and FBS with a final concentration of about 2.5% (v / v).

[0525] 7. The method according to any one of items 1 to 6, wherein the culture medium further comprises epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml.

[0526] 8. The method according to item 7, wherein the culture medium contains EGF at a final concentration of about 10 ng / ml.

[0527] 9. The method according to any one of items 1 to 8, wherein the culture medium comprises insulin at a final concentration of about 1 μg / ml to 10 μg / ml.

[0528] 10. The method according to item 9, wherein the culture medium contains insulin at a final concentration of about 5 μg / ml.

[0529] 11. The method according to any one of items 1 to 10, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0530] 12. The method according to any one of items 1 to 11, wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0531] 13. The method according to item 12 or 13, wherein the culture medium comprises adenine at a final concentration of about 0.01 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0532] 14. The method of any one of items 1 to 13, wherein culturing the mesenchymal stem cell population in a culture medium as described in any one of items 1 to 13 results in an increase in at least one of angiopoietin 1 (Ang-1), TGF-β (particularly TGF-β1), VEGF and HGF expressed and / or secreted by the mesenchymal stem cell population relative to a reference culture medium that does not contain all of DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (culture medium 171) and FBS (fetal bovine serum).

[0533] 15. The method according to item 14, wherein the reference culture medium consists of 90% (v / v) CMRL1066 and 10% (v / v) FBS.

[0534] 16. The method according to any one of the preceding items, wherein the population of mesenchymal stem cells has been isolated from its natural environment before being cultured in the culture medium according to any one of the preceding items 1 to 13.

[0535] 17. The method according to any one of items 1 to 15, comprising isolating the mesenchymal stem cell population from a natural tissue environment by culturing the natural tissue in the cell culture medium according to any one of items 1 to 13.

[0536] 18. The method according to item 17, wherein the tissue is umbilical cord tissue.

[0537] 19. The method of claim 18, wherein the umbilical cord tissue is selected from the group consisting of whole umbilical cord tissue, tissue comprising umbilical cord amnion, tissue comprising Wharton's jelly, tissue comprising amnion, amnion and Wharton's jelly, isolated umbilical cord blood vessels, Wharton's jelly separated from other components of umbilical cord tissue, and isolated umbilical cord amnion.

[0538] 20. The method according to item 17, wherein the tissue comprises or is placental amniotic tissue.

[0539] 21. The method according to any one of items 17 to 20, wherein the umbilical cord tissue is a portion of the entire umbilical cord, a portion of the umbilical cord amniotic membrane, or a portion of the placental amniotic membrane.

[0540] 22. The method of any one of items 19 to 22, comprising culturing the umbilical cord tissue or the placental amniotic tissue until the outgrowth of the amniotic mesenchymal stem cell population reaches about 70-80% confluence.

[0541] 23. The method according to item 22, comprising removing the mesenchymal stem cell population from a culture container used for the culture.

[0542] 24. The method according to item 23, wherein the mesenchymal stem cell population is removed from the culture container by enzyme treatment.

[0543] 25. The method according to item 24, wherein the enzymatic treatment comprises trypsin digestion.

[0544] 26. The method according to any one of items 23 to 25, wherein the mesenchymal stem cell population is transferred to a culture container for subculture for subculture.

[0545] 27. The method according to any one of items 1 to 16, wherein the mesenchymal stem cell population is transferred to a culture container for subculture for culture.

[0546] 28. The method according to item 26 or 27, wherein the mesenchymal cell population is 1.0 x 10 6 The cells are suspended at a concentration of 10 cells / ml for culture or subculture.

[0547] 29. The method according to item 28, wherein the population of mesenchymal stem cells is subcultured in the culture medium according to any one of items 1 to 13.

[0548] 30. The method according to item 29, wherein the mesenchymal stem cell population is subcultured until the mesenchymal stem cells reach about 70-80% confluence.

[0549] 31. The method according to any one of items 26 to 30, wherein the cultivation or subcultivation is carried out in a self-contained bioreactor.

[0550] 32. The method according to item 31, wherein the bioreactor is selected from the group consisting of a parallel plate bioreactor, a hollow fiber bioreactor and a microfluidic bioreactor.

[0551] 33. The method according to any one of the preceding items, wherein the culture is carried out at a temperature of 37°C in a CO2 cell culture incubator.

[0552] 34. The method according to item 33, comprising removing the mesenchymal stem cell population from a culture container used for the (sub)culture.

[0553] 35. The method according to item 34, wherein the mesenchymal stem cell population is removed from the culture container by enzyme treatment.

[0554] 36. The method according to item 35, wherein the enzymatic treatment comprises trypsin digestion.

[0555] 37. The method according to item 36, further comprising collecting the isolated mesenchymal stem cell population.

[0556] 38. The method of any of the preceding items, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD105.

[0557] 39. A method according to any of the preceding items, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cells lack expression of the following markers: CD34, CD45, and HLA-DR (human leukocyte antigen-antigen D-related).

[0558] 40. The method of any one of items 38 or 39, wherein about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cells express CD73, CD90, and CD105 and lack expression of CD34, CD45, and HLA-DR.

[0559] 41. The method according to any of the preceding items, further comprising preserving the isolated stem / progenitor cell population for further use.

[0560] 42. The method according to item 41, wherein the preservation is carried out by cryopreservation.

[0561] 43. 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.

[0562] 44. The mesenchymal stem cell population of item 43, wherein at least about 90% or more of the cells of the stem cell population lack expression of the following markers: CD34, CD45, and HLA-DR.

[0563] 45. A population of mesenchymal stem cells according to item 44, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the cells of the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

[0564] 46. ​​The mesenchymal stem cell population according to any one of items 43 to 45, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, an umbilical cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population, and an adipose tissue-derived mesenchymal stem cell population.

[0565] 47. The mesenchymal stem cell population according to any one of items 43 to 46, wherein the umbilical cord mesenchymal stem cell population is selected from an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed (MC) mesenchymal stem cell population.

[0566] 48. The mesenchymal stem cell population according to any one of items 43 to 47, wherein the population is obtainable by the method according to any one of items 1 to 42.

[0567] 49. The mesenchymal stem cell population according to any one of items 43 to 48, wherein the population is obtained by the method according to any one of items 1 to 42.

[0568] 50. A pharmaceutical composition comprising the isolated mesenchymal stem cell population of any one of items 43 to 47, wherein at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90 and CD105, and lack expression of each of the following markers: CD34, CD45 and HLA-DR.

[0569] 51. The pharmaceutical composition according to item 50, wherein the pharmaceutical composition is suitable for systemic or local application.

[0570] 52. The pharmaceutical composition according to item 50 or 51, further comprising a pharmaceutically acceptable excipient.

[0571] 53. A method for preparing a culture medium suitable for inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising mixing the following to obtain a final volume of 500 ml of culture medium:

[0572] i. 250 ml of DMEM

[0573] ii.118ml M171

[0574] iii. 118ml DMEM / F12

[0575] iv. 12.5 ml fetal bovine serum (FBS) (final concentration 2.5%).

[0576] 54. The method according to item 53, further comprising adding:

[0577] v. 1 ml of EGF stock solution (5 μg / ml) to obtain a final concentration of 10 ng / ml,

[0578] vi. Insulin 0.175 ml stock solution (14.28 mg / ml) to give a final concentration of 5 μg / ml.

[0579] 55. The method according to item 53 or 54, further comprising adding one or more of the following supplements to DMEM: adenine, hydrocortisone, 3,3',5-triiodo-L-thyronine sodium salt (T3), thereby obtaining a total volume of 500 ml of culture medium.

[0580] 56. The method according to item 55, wherein the final concentration of the supplement in DMEM is as follows:

[0581] about 0.05 to 0.1 μg / ml adenine, for example about 0.025 μg / ml adenine,

[0582] About 1 to 10 μg / ml hydrocortisone,

[0583] About 0.5 to 5 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3), for example 1.36 ng / ml 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0584] 57. A cell culture medium obtainable by the method of any one of items 53 to 56.

[0585] 58. A method for inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing amniotic tissue in a culture medium prepared by the method of any one of items 53 to 56.

[0586] 59. The method according to item 58, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, an umbilical cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population, and an adipose tissue-derived mesenchymal stem cell population.

[0587] 60. The method according to item 59, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed (MC) mesenchymal stem cell population.

[0588] 61. A cell culture medium comprising:

[0589] - DMEM at a final concentration of about 55 to 65% (v / v),

[0590] - F12 at a final concentration of about 5 to 15% (v / v),

[0591] - Final concentration of about 15 to 30% (v / v) M171 and

[0592] - FBS at a final concentration of approximately 1 to 8% (v / v).

[0593] 62. The cell culture medium of claim 61, wherein the cell culture medium comprises DMEM at a final concentration of about 57.7 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v).

[0594] 63. The cell culture medium according to item 62, wherein the cell culture medium comprises DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v) and FBS at a final concentration of about 2.5% (v / v).

[0595] 64. The cell culture medium according to any one of items 61 to 62, wherein the culture medium further comprises epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml.

[0596] 65. The cell culture medium according to any one of items 61 to 65, wherein the culture medium comprises EGF at a final concentration of about 10 ng / ml.

[0597] 66. The cell culture medium according to any one of items 61 to 65, wherein the culture medium comprises insulin at a final concentration of about 1 μg / ml to 10 μg / ml.

[0598] 67. The cell culture medium according to item 66, wherein the culture medium comprises insulin at a final concentration of about 5 μg / ml.

[0599] 68. The cell culture medium according to any one of items 61 to 67, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0600] 69. The cell culture medium according to item 68, wherein the culture medium comprises all three of adenine, hydrocortisone and 3,3',5-triiodo-L-thyronine sodium salt (T3).

[0601] 70. The cell culture medium of item 68 or 69, wherein the culture medium comprises adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0602] 71. The cell culture medium according to any one of items 61 to 70, wherein 500 ml of the cell culture medium comprises:

[0603] i. 250 ml of DMEM

[0604] ii.118ml M171

[0605] iii. 118ml DMEM / F12

[0606] iv. 12.5 ml fetal bovine serum (FBS) (final concentration 2.5%).

[0607] 72. The cell culture medium according to item 71, further comprising:

[0608] v. EGF at a final concentration of 10 ng / ml

[0609] vi. Insulin at a final concentration of 5 μg / ml

[0610] vi. Insulin 0.175 ml (final concentration 5 μg / ml).

[0611] 73. The cell culture medium according to item 71 or 72, further comprising adenine at a final concentration of about 0.05 to about 0.1 μg / ml adenine, hydrocortisone at a final concentration of about 1 to about 10 μg / ml hydrocortisone, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0612] 74. Use of the cell culture medium according to any one of items 61 to 73 for inducing or improving wound healing properties of a mesenchymal stem cell population.

[0613] 75. Use of the cell culture medium according to any one of items 61 to 73 for isolating a population of mesenchymal stem cells.

[0614] 76. The use according to 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.

[0615] 77. The use according to item 76, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed (MC) mesenchymal stem cell population.

[0616] 78. The use of any one of items 74 to 77, wherein at least about 90% or more of the cells of the mesenchymal stem cell population express each of the following markers: CD73, CD90, and CD105.

[0617] 79. The use according to item 78, wherein at least about 90% or more of the cells of the mesenchymal stem cell population lack expression of each of the following markers: CD34, CD45, and HLA-DR.

[0618] 80. The use of claim 79, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the cells of the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

[0619] 81. A pharmaceutical composition comprising three or four of Ang-1, TGF-β1, VEGF or HGF as the sole wound healing proteins.

[0620] 82. The pharmaceutical composition according to item 81, which is formulated as a liquid or a lyophilized / freeze-dried preparation.

[0621] It will be 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.

[0622] All patents and publications mentioned in this specification represent the levels of ordinary skill in the art to which the invention pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0623] The invention described herein in an illustrative manner can be practiced in the absence of any element or elements, or any limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprise," "include," and "contain" are to be construed broadly and without limitation. Furthermore, the terms and expressions employed herein have been used as words of description, not limitation, and their use is not intended to exclude any equivalents of the features shown and described, or portions thereof, but rather to recognize that various modifications may be made within the scope of the claimed invention. Thus, it should be understood that while the invention has been specifically disclosed with reference to preferred embodiments and optional features, modifications and variations of the invention presented herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This generic description of the invention is encompassed, without precondition or negative limitation, by removing any subject matter from the genus, whether or not the deleted matter is specifically described herein. Furthermore, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Other embodiments of the invention will be apparent from the following claims.

Claims

1. A method for inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171) and FBS (fetal bovine serum).

2. The method of claim 1, wherein the mesenchymal stem cell population is selected from an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, an umbilical cord-placenta junction mesenchymal stem cell population, an umbilical cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population, and an adipose tissue-derived mesenchymal stem cell population.

3. The method of claim 2, wherein the umbilical cord mesenchymal stem cell population is selected from an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed (MC) mesenchymal stem cell population.

4. The method of any one of claims 1 to 3, wherein the culture medium comprises DMEM at a final concentration of about 55 to 65% (v / v), F12 at a final concentration of about 5 to 15% (v / v), M171 at a final concentration of about 15 to 30% (v / v), and FBS at a final concentration of about 1 to 8% (v / v).

5. The method of claim 4, wherein the culture medium comprises DMEM at a final concentration of about 57.5 to 62.5% (v / v), F12 at a final concentration of about 7.5 to 12.5% ​​(v / v), M171 at a final concentration of about 17.5 to 25.0% (v / v), and FBS at a final concentration of about 1.75 to 3.5% (v / v).

6. The method of claim 5, wherein the culture medium comprises DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v).

7. The method according to any one of claims 1 to 6, wherein the culture medium further comprises epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml.

8. The method of claim 7, wherein the culture medium comprises EGF at a final concentration of about 10 ng / ml.

9. The method according to any one of claims 1 to 8, wherein the culture medium comprises insulin at a final concentration of about 1 μg / ml to 10 μg / ml.

10. The method of claim 9, wherein the culture medium comprises insulin at a final concentration of about 5 μg / ml.

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