TWI882956B - A method of inducing or improving wound healing properties of mesenchymal stem cells - Google Patents

Abstract

The present invention relates to a method of inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising cultivating the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum). The invention also relates to a mesenchymal stem population, wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD105 and lack expression of the following markers: CD34, CD45 and HLA-DR. The invention also relates to a pharmaceutical composition of this mesenchymal stem population.

Description

A method for inducing or improving wound healing properties of mesenchymal stem cells

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

The present invention relates to a method for inducing or improving the wound healing properties of a mesenchymal stem cell population. The present invention also relates to a cell culture medium suitable for inducing or improving the wound healing properties of mesenchymal stem cells and/or suitable for isolating a mesenchymal stem cell population. The present invention also relates to a pharmaceutical composition and the use of an isolated mesenchymal stem cell population. 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. The present invention also relates to a highly homogeneous and well-defined mesenchymal stem cell population such as umbilical cord or placenta.

The method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord was first described in U.S. Patent Application No. 2006/0078993 (leading to U.S. Patents 9,085,755, 9,737,568, and 9,844,571) and the corresponding International Patent Application No. WO2006/019357. Since then, the umbilical cord tissue has attracted attention as a source of multipotent cells; the umbilical cord, especially the stem cells isolated from the amniotic membrane of the umbilical cord (also known as "umbilical cord lining stem cells"), has been regarded as an excellent cell replacement source for regenerative medicine due to its wide availability. 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.

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

Although amniotic mesenchymal stem cells can be easily obtained using the methods disclosed in U.S. Patent Application No. 2006/0078993 and International Patent Application No. WO2006/019357, it would be advantageous for clinical trials using those umbilical cord lining MSCs to allow the isolation of a highly homogeneous population of umbilical cord lining MSCs, which can then be used in clinical trials. In addition, methods of inducing or improving the wound healing properties of mesenchymal stem cell populations are generally advantageous.

Accordingly, the purpose of the present invention is to provide a method for inducing or improving the wound healing properties of a mesenchymal stem cell population. Another purpose is to isolate a mesenchymal stem cell population from the umbilical cord amniotic membrane that meets the requirements. Therefore, another purpose of the present invention is to provide a highly homogeneous mesenchymal stem cell population.

Using methods, mesenchymal stem cell populations, individual pharmaceutical compositions, and cell culture media with independent characteristics to achieve the purpose.

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 a mesenchymal stem cell population in a culture medium containing DMEM (Double Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). The mesenchymal stem cell population may be a mesenchymal stem cell population of the umbilical cord, a mesenchymal stem cell population of the placenta, a mesenchymal stem cell population of umbilical cord blood, a mesenchymal stem cell population of the bone marrow, or a mesenchymal stem cell population derived from adipose tissue.

In a second aspect, the present invention provides an isolated mesenchymal stem cell population, wherein at least about 90% or more 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 a specific embodiment of this second aspect, the isolated mesenchymal stem cell population has 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 expressing each of CD73, CD90, and CD105. In addition, in the specific 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, or about 99% or more of the isolated mesenchymal stem cell population preferably lacks expression markers CD34, CD45, and HLA-DR. The mesenchymal stem cell population can be obtained by using 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 for isolating a mesenchymal stem cell population.

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

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

i. 250ml of DMEM

ii. 118ml M171

iii. 118ml of DMEM/F12

iv. 12.5 ml of fetal bovine serum (FBS) to achieve a final concentration of 2.5% (v/v).

In a fifth aspect, the present invention provides a cell culture medium obtained using the method of the fourth aspect.

In a sixth aspect, the present invention provides a method for isolating a mesenchymal stem cell population, which comprises culturing the mesenchymal stem cell population in a culture medium prepared by the method of the fourth aspect.

In a seventh aspect, the present invention provides a cell culture medium comprising: - DMEM with a final concentration of about 55 to 65% (v/v), - F12 with a final concentration of about 5 to 15% (v/v), - M171 with a final concentration of about 15 to 30% (v/v), and - FBS with a final concentration of about 1 to 8% (v/v).

In an eighth aspect, the present invention provides the use of the cell culture medium of the seventh aspect for inducing or improving the wound healing properties of mesenchymal stem cell populations or isolating mesenchymal stem cell populations.

The present invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting embodiments and drawings, wherein:

Figures 1A-C show the results of flow cytometry experiments in which mesenchymal stem cells isolated from the umbilical cord were analyzed for the expression of mesenchymal stem cell markers CD73, CD90, and CD105. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different culture media, and then the mesenchymal stem cells were subcultured in the respective culture media. The following three media were used in these experiments: a) 90% (v/v/DMEM supplemented with 10% FBS (v/v), b) the medium PTT-4 disclosed in U.S. Patent Application No. US 2008/0248005 and corresponding International Patent Application No. WO2007/046775, which consists of 90% (v/v) CMRL1066 and 10% (v/v) FBS (see paragraph [0183] of WO2007/046775, and c) the medium PTT-6 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 for each of the three media used. The results are shown in Figures 1A to 1C .

In more detail, Figure 1A shows that the umbilical cord tissue was isolated and cultured in DMEM/10% Figure 1B shows the percentage of isolated interstitial umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and cultured in PTT-4, and Figure 1C shows the percentage of isolated interstitial umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after isolation from umbilical cord tissue and cultured in PTT-6.

Figures 2A-B show 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) of mesenchymal stem cells isolated from the umbilical cord was analyzed, which was used to define the suitability of multipotent human mesenchymal stem cells for cell therapy and compared with the expression of those markers of 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 culture medium of the present invention, PTT-6, and bone marrow mesenchymal stem cells were isolated from human bone marrow using standard procedures.

FIG. 2A shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 and lacking CD34, CD45, and HLA-DR after isolation from umbilical cord tissue and culture in PTT-6 medium, while FIG. 2B shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90, and CD105 and lacking CD34, CD45, and HLA-DR.

Figure 3 shows an experimental design, where dark grey wells are standards reconstituted with PTT-4 medium and corresponding samples of MSCs cultured in PTT-4; light grey wells are standards reconstituted with PTT-6 medium and corresponding samples of MSCs cultured in PTT-6. The samples in italics are control group supernatants, which are tested as part of the stored samples for repeated testing.

Figure 4 shows a single assay of TGFβ1. It can be seen that when CL-MSCs and WJ-MSCs were cultured, more TGFβ1 was produced when grown in PTT-6 than when grown in PTT-4. Only AT-MSCs and BM-MSCs culture medium produced more or less equal amounts of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations of three assays.

Figure 5A shows multiplex assays of PDGF-AA. It can be seen that when CL-MSC, WJ-MSC, AT-MSC, and BM-MSC were cultured in PTT-4, more PDGF-AA was produced than when they were grown in PTT-6. All error bars are standard deviations of three determinations.

Figure 5B shows multiplex assays of VEGF. It can be seen that when CL-MSC, WJ-MSC, AT-MSC, and BM-MSC were cultured in PTT-6, more VEGF was produced than when they were grown in PTT-4. All error bars are standard deviations of three determinations.

Figure 5C shows multiplex assays of Ang-1. It can be seen that when CL-MSCs and WJ-MSCs were cultured in PTT-6, more Ang-1 was produced than when they were grown in PTT-4. AT-MSCs and BM-MSCs did not produce virtually any Ang-1. All error bars are standard deviations of three determinations.

Figure 6 shows multiplex assays of HGF. It can be seen that when CL-MSCs and WJ-MSCs were cultured in PTT-6, more HGF was produced than when they were grown in PTT-4. AT-MSCs and BM-MSCs did not produce virtually any HGF. All error bars are standard deviations of three determinations.

Figure 7 shows multiplexed measurements of PDGF-AA. It can be seen that when CL-MSCs and WJ-MSCs were cultured in PTT-4, more PDGF-AA was produced than when they were grown in PTT-6. AT-MSCs and BM-MSCs cultured in both media produced the same amount of PDGF-AA. All error bars are standard deviations of three measurements.

Figure 8A shows multiplexed assays of VEGF. It can be seen that when CL-MSC, WJ-MSC, AT-MSC, and BM-MSC were cultured in PTT-6, more VEGF was produced than when they were grown in PTT-4. All error bars are standard deviations of three determinations.

Figure 8B shows multiplexed assays of Ang-1. It can be seen that when CL-MSCs and WJ-MSCs were cultured in PTT-6, more Ang-1 was produced than when they were grown in PTT-4. AT-MSCs and BM-MSCs did not produce virtually any Ang-1. All error bars are standard deviations of three determinations.

Figure 8C shows multiplex assays of HGF. It can be seen that when CL-MSCs and WJ-MSCs were cultured in PTT-6, more HGF was produced than when they were grown in PTT-4. AT-MSCs and BM-MSCs did not produce virtually any HGF. All error bars are standard deviations of three determinations.

Figure 9 shows multiplexed measurements of bFGF. It can be seen that when CL-MSCs and WJ-MSCs were cultured in PTT-6, they produced more bFGF than when they were grown in PTT-4. AT-MSCs and BM-MSCs cultured in PTT-6 and PTT-4 produced equal amounts of bFGF. All error bars are standard deviations of three measurements.

Figure 10 summarizes the results of TGFβ1 assays from 5 different experiments (170328, 170804, 170814, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the TGFβ standard curve in the experiment. The upper graph shows the MFI of the TGFβ standard curve taken from PTT-4 and PTT-6 medium. The lower right graph shows that when CL-MSC and WJ-MSC are cultured, more TGFβ1 is produced when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC culture media produce equivalent amounts of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations of different measurements from experiments 170328, 170804, 170814, 180105, and 180226.

Figure 11 summarizes the results of Ang-1 assays from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the Ang-1 standard curve in the experiment. The upper graph shows the MFI of the Ang-1 standard curve taken from PTT-4 and PTT-6 culture media. The lower right graph shows that when CL-MSC and WJ-MSC are cultured, more Ang-1 is produced when grown in PTT-6 than when grown in PTT-4. When grown in PTT-6 or PTT-4, only AT-MSC and BM-MSC culture media produce essentially the same amount of Ang-1. All error bars are standard deviations of different measured values from experiments 170602, 170511, 170414, 170224, 180105, and 180226.

Figure 12 summarizes the results of PDGF-BB assays from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the PDGF-BB standard curve in the experiment. The upper graph shows the MFI of the PDGF-BB standard curve taken from PTT-4 and PTT-6 culture media. It is worth noting that PDGF-BB was not detected in any experiment.

Figure 13 summarizes the results of PDGF-AA assays from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the PDGF-AA standard curve in the experiment. The upper graph shows the MFI of the PDGF-AA standard curve taken from PTT-4 and PTT-6 medium. The lower right graph shows that when CL-MSC, AT-MSC, BM-MSC, and WJ-MSC were cultured in medium, slightly more PDGF-AA was produced when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of different measured values from experiments 170602, 170511, 170414, 170224, 180105, and 180226.

Figure 14 summarizes the results of IL-10 assays from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the IL-10 standard curve in the experiment. The upper graph shows the MFI of the IL-10 standard curve taken from PTT-4 and PTT-6 culture media. It is worth noting that IL-10 was not detected in any experiment.

Figure 15 summarizes the results of VEGF measurement from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left side of the figure depicts the mean fluorescence intensity (MFI) measured by the VEGF standard curve in the experiment. The upper figure shows the MFI of the VEGF standard curve taken from PTT-4 and PTT-6 culture media. The lower right side of the figure shows that when CL-MSC, AT-MSC, BM-MSC, and WJ-MSC were cultured in PTT-6, more VEGF was produced than when grown in PTT-4. All error bars are standard deviations of different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

Figure 16 summarizes the results of HGF measurement from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the HGF standard curve in the experiment. The upper graph shows the MFI of the HGF standard curve taken from PTT-4 and PTT-6 medium. The lower right graph shows that when CL-MSC and WJ-MSC are cultured, more HGF is produced when grown in PTT-6 than when grown in PTT-4. On the other hand, AT-MSC and BM-MSC cultured do not produce as much HGF as in the other media. All error bars are standard deviations of different measured values from experiments 170602, 170511, 170414, 170224, 180105, and 180226.

Figure 17 : Single measurement of TGFβ1. The left side of the figure depicts the mean fluorescence intensity (MFI) measured by the TGFβ1 standard curve in the experiment. It can be seen in the right figure that CL-MSC, WJ-MSC, and placental MSC grown in PTT-6 produce more TGFβ1 than those grown in PTT-4 or DMEM/F12 (only DMEM in Figure 17).

Figure 18 shows an excerpt of PDGF-BB assays of supernatants from CL-MSC, WJ-MSC, and placental MSC cultured in PTT-6, PTT-4, or DMEM/F12. The left side of the figure depicts the mean fluorescence intensity (MFI) measured by the PDGF-BB standard curve in the experiments. It is noteworthy that PDGF-BB was not detected in any of the experiments.

Figure 19 : IL-10 assay of supernatants extracted from CL-MSC, WJ-MSC, and placental MSC cultured in PTT-6, PTT-4, or DMEM/F12. The left panel depicts the mean fluorescence intensity (MFI) measured by the VEGF standard curve in the experiment. S6 represents the lowest standard used in the experiment. Any sample falling below is considered below the detection range. It can be seen in the right panel that all CL-MSC, WJ-MSC, and placental MSC produced detectable levels of IL-10 when grown in PTT-6, while little or no IL-10 was detected when MSCs were grown in PTT-4 or DMEM/F12.

Figure 20 : VEGF assay of supernatants extracted from CL-MSC, WJ-MSC, and placental MSC cultured in PTT-6, PTT-4, or DMEM/F12. The left panel depicts the mean fluorescence intensity (MFI) measured by the VEGF standard curve in the experiment. S1 represents the highest standard used in the experiment. Any samples falling above it are considered extrapolated (too concentrated). It can be seen in the right panel that all CL-MSC, WJ-MSC, and placental MSC produce higher levels of VEGF when grown in PTT-6 compared to when the MSC are grown in PTT-4 or DMEM/F12.

Figure 21 : Multiplexed measurement of bFGF. The left panel depicts the mean fluorescence intensity (MFI) measured by the PDGF-AA standard curve in the experiment. It can be seen in the right panel that CL-MSC and WJ-MSC cultured when grown in PTT-6 produced more bFGF than when grown in PTT-4. It can be seen that all CL-MSC, WJ-MSC, and placental MSC produced lower levels of bFGF when grown in PTT-6 compared to when MSC were grown in PTT-4 or DMEM/F12.

Figure 22 : Excerpt of PDGF-AA assay. The left panel depicts the mean fluorescence intensity (MFI) measured by the PDGF-AA standard curve in the experiment. S6 represents the lowest standard used in the experiment. Any sample falling below is considered below the detection range. 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.

Figure 23 : Excerpt of Ang-1 measurement. The left panel depicts the mean fluorescence intensity (MFI) measured by the Ang-1 standard curve in the experiment. S1 represents the highest standard used in the experiment. Any sample falling above it is considered extrapolated (too concentrated). In the right panel, all CL-MSCs, WJ-MSCs, and placental MSCs produced higher levels of Ang-1 when grown in PTT-6 compared to MSCs grown in PTT-4 or DMEM/F12.

Figure 24 : Extracted HGF measurements. The left panel depicts the mean fluorescence intensity (MFI) measured by the HGF standard curve in the experiment. In the right panel, all CL-MSCs, WJ-MSCs, and placental MSCs produced higher levels of Ang-1 when grown in PTT-6 compared to MSCs grown in PTT-4 or DMEM/F12.

As explained above, the first aspect of 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 containing DMEM (Double Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum). It was surprisingly found that in the present application, the use of this culture medium has the effect of inducing or improving the wound healing properties of a wide range of mesenchymal stem cell populations, which is independent 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 wound healing properties of mesenchymal stem cell populations 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 by mesenchymal stem cell populations. The experimental part shows that the expression/secretion of angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF of the umbilical cord amniotic mesenchymal stem cell population cultured in the culture medium PTT-6 of the present invention is increased, compared with culturing the mesenchymal stem cell population in the culture medium (PTT-4), which is used in U.S. Patent Application No. US 2008/0248005 and the corresponding International Patent Application No. WO2007/046775 to isolate the umbilical cord amniotic mesenchymal stem cell population, which is shown in U.S. Patent Application No. US 2008/0248005 and the International Patent Application No. WO2007/046775 to have excellent wound healing properties (see WO Examples 23-26 of 2007/046775 show that the umbilical cord amniotic mesenchymal stem cell population (UCMC) can alleviate third-degree burns (Example 23), second-degree burns (Example 24), non-healing radiation wounds (Example 25), and non-healing diabetic wounds and non-healing diabetic foot injuries (Example 26). As shown in the experimental section of this article, culturing in a medium containing DMEM (Double's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum) can increase the amount of angiopoietin 1 (Ang-1), TGF-β1, VEGF, and/or HGF, not only in the umbilical cord amniotic mesenchymal stem cell population, but also in the mesenchymal stem cell population of other compartments of the umbilical cord, such as Valdenza or (adjacent) compartments, such as the placenta. Therefore, it is believed that the present application provides a generally applicable teaching to induce or improve 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 the culture medium PTT-6.

In this case, the present invention has discovered that the combined increase in the amount of Ang-1, TGF-β1, VEGF, and/or HGF produced by a mesenchymal stem cell population can improve the wound healing properties of this stem cell population, and that the use of a composition/solution containing three or four of Ang-1, TGF-β1, VEGF, or HGF as the sole wound healing protein can also activate the wound healing properties of the simulated stem cell population.

In this context, it should be noted that the involvement of proteins such as angiopoietin-1 (Ang-1), TGF-β1, VEGF, and HGF in the wound healing process is known to those skilled in the art. For the involvement of angiopoietin-1 in wound healing, see, for example, Li et al. Stem Cell Research & Therapy 2013, 4: 113 "Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing" or Bitto et al, "Angiopoietin-1 gene transfer improves the impaired wound healing of the genetically diabetic mice without increasing VEGF expression", Clinical Science May 14, 2008, 114 (12) 707-718. In 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, accompanied by increased epidermal and dermal regeneration, and enhanced angiogenesis compared to MSCs, Ad-Ang1, or pseudotherapy." It is worth noting that the authors Li et al. mentioned that mesenchymal stem cells (MSCs) alone cannot produce enough Ang-1, and for this reason, the authors inserted the Ang1 gene into MSCs to produce a genetically modified cell. In contrast to Li's study, the present application surprisingly found that culturing "natural" mesenchymal stem cells in a culture medium (such as PTT-6) can provide conditions for umbilical cord tissue mesenchymal stem cells to increase Ang-1 levels (i.e., culturing the mesenchymal stem cell population in PTT-6), thereby making the mesenchymal stem cells suitable for wound healing or further improving their wound healing properties. This means that the advantage provided by the present invention is that, instead of genetically modifying naturally occurring mesenchymal stem cells to induce the wound healing properties of mesenchymal stem cells (which is not only laborious but also not a good option for therapeutic applications due to the inherent risks of gene therapy), the mesenchymal stem cell population is "simply" cultured using the culture medium of the present invention to induce or enhance the wound healing properties of naturally occurring mesenchymal stem cells. This method is simpler, safer, and more cost-effective.

For other proteins such as hepatocyte growth factor (HGF) involved in wound healing, especially 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 β 1-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.

Regarding the involvement of vascular endothelial growth factor (VEGF) in wound healing, especially 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.

Regarding the involvement of transforming growth factor beta (including TGF-β1, TGF-β2, and TGF-β3) in wound healing, especially 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.

In this context, it should also be noted that a further surprising advantage of the present invention is that culture in the culture medium of the present invention can provide an isolated mesenchymal stem cell population, such as an umbilical cord amniotic mesenchymal stem cell population, in which greater than 90%, or even 99% or more of the cells are positive for the three mesenchymal stem cell markers CD73 and CD90, while at the same time these stem cells lack the expression of CD34, CD45, and HLA-DR (see the Experimental section), meaning that 99% or more of this population of cells express the stem cell markers CD73, CD90, and CD105, but do not express the markers CD34, CD45, and HLA-DR. This extremely homogeneous and well-defined cell population is an ideal candidate for clinical trials and cell-based therapies because it fully meets the generally accepted criteria for human mesenchymal stem cells for use in defined cell therapies, such as Dominici et al, "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement", Cytotherapy (2006) Vol. 8, No. 4, 315-317; Sensebe et al,. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a, review", Stem Cell Research & Therapy 2013, 4: 66; Vonk et al., Stem Cell Research & Therapy (2015) 6: 94; or Kundrotas Acta Medica Lituanica.2012.Vol.19.No.2.P.75-79. At the same time, using a bioreactor, such as the Quantum cell expansion system, high quantities of mesenchymal stem cells can be obtained, such as 300 to 700 million mesenchymal stem cells per time (see also the Experimental section). Therefore, the present invention provides a further advantage of providing the amount of stem cells required for therapeutic applications, such as wound healing, in a cost-effective manner. In addition, all components used to make the culture medium of the present invention are of commercially available GMP quality. Accordingly, the present invention opens up a GMP production route for highly homogeneous mesenchymal stem cell populations, for example, placental tissue or umbilical cord tissue, such as umbilical cord amniotic membrane mesenchymal stem cell populations or Valdenza jelly mesenchymal stem cell populations.

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

The mesenchymal stem cell population of the umbilical cord may come from (or originate from) any compartment of the umbilical cord tissue containing mesenchymal stem cells. The mesenchymal stem cell population may be an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Walton's jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and a mixed umbilical cord (MC) mesenchymal stem cell population, meaning a mesenchymal stem cell population including stem cells of two or more of those compartments. Mesenchymal stem cells of these compartments and their isolation are known to those skilled in the art and are described, for example, in Subramanian et al. "Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton's Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells", PLoS ONE 10(6): e0127992, 2015 and references cited therein, Van Pham et al. "Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications", Cell Tissue Bank (2016) 17: 289-302, 2016. The 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 into pieces with Valdenza's jelly, and culturing the umbilical cord tissue (tissue explant) in the culture medium of the present invention. Mixed umbilical cord mesenchymal stem cell populations can also be obtained by culturing whole umbilical cord tissue using intact umbilical cord vessels as tissue explants under one condition (cultured in serum-supplemented DMEM containing 10% fetal bovine serum, 10% horse serum, and 1% penicillin/streptomycin), as described in Schugar et al. "High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue. Journal of biomedicine & biotechnology. 2009; 2009: 789526". In this context, it should be noted that a population of mesenchymal stem cells from 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.

Based on the above, it should be noted that the mesenchymal stem cell populations cultured in the present invention with the medium DMEM (Duel of Eagle's Modified Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum) to induce or improve their wound healing properties can be separated from their natural environment before being cultured in the medium of the present invention. This method is particularly useful for mesenchymal stem cell populations that are difficult to separate from tissue explants, such as mesenchymal stem cell populations of cord blood or mesenchymal stem cell populations of bone marrow. However, this method can also be used for umbilical cord mesenchymal stem cell populations, placental mesenchymal stem cell populations, or adipose tissue-derived mesenchymal stem cell populations. Such a stem cell population, such as a Wharton's jelly mesenchymal stem cell population, can be first isolated in the manner described above, such as Subramanian et al, 2015, PLoS ONE, supra or International Patent Application No. WO 2004/072273 "Progenitor Cells From Wharton's Jelly Of Human Umbilical Cord", and then the isolated mesenchymal stem cell population can be cultured in the culture medium of the present invention comprising DMEM (Double's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum). Placental mesenchymal stem cell populations can also be isolated from the placenta, as described, for example, in European Patent Application No. EP1 288 293; Talwadekar et al, "Cultivation and Cryopreservation of Cord Tissue MSCs with Cord Blood AB Plasma" Biomed Res J 2014; 1(2): 126-136; Talwadekar et al, "Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts-a paired sample study" Scientific Reports 5: 15784 (2015); or Beeravolu et al. "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp.2017;(122):55224, and subsequently cultured in the culture medium of the present invention. Similarly, adipose tissue-derived mesenchymal stem cell populations can be isolated as described in Schneider et al, "Adipose-derived mesenchymal stem cells from liposuction and resected fat are feasible sources for regenerative medicine" Eur J Med Res.2017;22:17, and references cited therein, and subsequently cultured in the culture medium of the present invention (see also the Experimental section). In a further illustrative example, the mesenchymal stem cell population at the umbilical cord-placental junction may first 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, and then cultured in the culture medium of the present invention.

Alternatively, and particularly for mesenchymal stem cells isolated from tissue explants, mesenchymal stem cell populations may be isolated directly from their natural tissue environment by culturing the natural tissue in the cell culture medium of the present invention. This methodology is particularly suitable for culturing mesenchymal stem cell populations from umbilical cord tissue, placental tissue (placental tissue may, for example, include or be placental amnion), or the umbilical cord-placental junction.

In this context, it should be noted that the culture medium of the present invention thereby also allows the isolation of a population of mesenchymal stem cells (also referred to herein as "mesenchymal stem cells") from their natural environment. Accordingly, the culture medium of the present invention can also isolate a population of mesenchymal stem cells under conditions that allow cell proliferation of mesenchymal stem cells/progenitor cells without differentiating the mesenchymal stem cells/progenitor cells.

In one embodiment, the culture medium of the present invention allows the isolation of a population of mesenchymal stem cells from the amniotic membrane under conditions that allow proliferation of the mesenchymal stem cells/pioneer cells without differentiating the mesenchymal stem cells/pioneer cells. Thus, after the mesenchymal stem cells are isolated from the amniotic membrane as described herein, the isolated population of mesenchymal stem cells/pioneer cells has the ability to differentiate into multiple cell types, as described, for example, 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. As described in U.S. Patent Application No. 2006/0078993, for example, umbilical cord amniotic mesenchymal stem cells have a spindle shape and express the following genes: POU5f1, Bmi-1, leukemia inhibitory factor (LIF), secrete Activin A, and Follistatin. The mesenchymal stem cells isolated in the present invention can, for example, differentiate into any type of mesenchymal cells, such as, but not limited to, skin fibroblasts, chondrocytes, osteoblasts, tendon cells, ligament fibroblasts, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, adipocytes, mucin-producing cells, cells from endocrine glands, such as insulin-producing cells (e.g., β-islet cells), or neuroectoderm cells. The stem cells isolated in the present invention can be differentiated in vitro to subsequently use the differentiated cells for medical purposes. An illustrative example of this method is to differentiate mesenchymal stem cells into insulin-producing β-islet cells, which can then be administered by implantation to patients suffering from insulin deficiency, such as diabetes (see also WO2007/046775). Alternatively, the mesenchymal stem cells of the present invention can be used in their undifferentiated state for cell-based therapies, for example, for wound healing purposes, such as treating burns or chronic diabetic wounds. In such therapeutic applications, the mesenchymal stem cells of the present invention can promote wound healing by interaction with surrounding diseased tissue, or can also differentiate into corresponding skin cells (again, see, for example, WO2007/046775).

According to the foregoing disclosure, it should be noted that this mesenchymal stem cell population described herein can be separated and cultured (i.e., derived) from any umbilical cord tissue, as long as the umbilical cord tissue contains amniotic membrane (also referred to as "umbilical cord lining"). Accordingly, the mesenchymal stem cell population can be separated from the entire umbilical cord (fragment thereof), as described in the experimental part of this application. This umbilical cord tissue can thus contain, in addition to the amniotic membrane, any other tissue/component of the umbilical cord. As shown in Figure 16 of U.S. Patent Application No. 2006/0078993 or International Patent Application No. WO2006/019357, the umbilical cord amniotic membrane is the outermost part of the umbilical cord, which covers the umbilical cord. In addition, the umbilical cord contains a vein (which transports 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 Valden's jelly, a gel-like substance made primarily of mucopolysaccharides. Accordingly, the umbilical cord tissue used in the present invention may also include this vein, two arteries, and Valden'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 isolation steps, making the method of the present invention simpler, faster, less prone to error, and more economical - all of which are important aspects of GMP production required for therapeutic applications of mesenchymal stem cells. The isolation of mesenchymal stem cells can therefore begin with tissue explants, and if larger quantities of mesenchymal stem cells are required, for example, for clinical trials, the isolated mesenchymal stem cells can then be cultured (cultivated). Alternatively, the amniotic membrane can first be separated from the other components of the umbilical cord, and the amniotic membrane can be cultured in the culture medium of the present invention to separate the mesenchymal umbilical cord lining stem cells from the amniotic membrane. The culture can also be performed using tissue explants, optionally followed by subsequent culture of isolated mesenchymal stem cells.

In this context, the terms "tissue explant" or "tissue explant method" are used in their ordinary sense in the art to refer to a method in which a tissue (such as placental tissue or umbilical cord tissue), once harvested, or a piece of tissue is placed in a cell culture dish containing a (growth) medium, over time, stem cells migrate from the tissue to the surface of the culture dish. Those primary stem cells can then be further expanded and transferred to a fresh culture dish by micropropagation (subculture), also as described herein. In this context, it should be noted that, with respect to cell production for therapeutic purposes, in the first step of isolating/obtaining a population of mesenchymal stem cells according to the invention, for example, umbilical cord mesenchymal stem cells, such as amnion or Feldmann-Straussl mesenchymal stem cells, a master cell bank of isolated mesenchymal stem cells is obtained, whereas in the subsequent subculture, a working cell bank can be obtained. 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 each of the expression markers CD34, CD45, and HLA-DR) is used in clinical trials or as an approved therapeutic agent, a cell population of a working cell bank is usually used for this purpose. Both the stem cell population of the separation step (which can constitute the master cell bank) and the stem cell population of the secondary culture step (which can constitute the working cell bank) are stored, for example, by cryopreservation.

As described above, the present method of inducing or improving the wound healing properties of a mesenchymal cell population (and optionally isolating mesenchymal stem cells from tissues (such as Valden's jelly or umbilical cord amnion) at the same time) has the advantage that the components used in the culture medium of the present invention are of GMP quality, thereby providing the possibility of isolating mesenchymal stem cells under GMP conditions for subsequent therapeutic administration.

Herein, "inducing or improving the wound healing properties of mesenchymal stem cell populations" means that the culture medium increases or activates (induces) the ability of mesenchymal stem cell populations to express and/or secrete at least one of Ang-1, TGF-β1, VEGF, and HGF proteins. As mentioned above, it is known that all four proteins are involved in wound healing. Evaluation of "inducing or improving wound healing properties", and relative to culturing the mesenchymal stem cell population in a reference (culture) medium, such as the medium PTT-4 (which consists of 90% (v/v) CMRL1066 and 10% (v/v) FBS), which is used in US Patent Application No. US 2008/0248005 and the corresponding International Patent Application No. WO2007/046775 to isolate and culture the mesenchymal stem cell population of the umbilical cord amniotic membrane, and US Patent Application No. US 2008/0248005 and International Patent Application No. WO2007/046775 show excellent wound healing properties. Compared to culturing the mesenchymal stem cell population in a reference medium, when cultured in the medium of the present invention, the mesenchymal stem cell population secretes a greater amount (corresponding to a higher secretion level or a higher concentration) of at least one of the four marker proteins Ang-1, TGF-β1, VEGF, and HGF into the supernatant/medium, subsequently increasing the wound healing properties of the mesenchymal stem cell population. During the culture period in the reference medium, it was observed that the mesenchymal stem cell population did not (detectably) secrete the four marker proteins, while during or after the culture period in the medium of the present invention, it was observed that the mesenchymal stem cell population could detectably secrete at least one of the four marker proteins, and then the wound healing properties of the stem cell population were induced. Compared with culturing the stem cell population in the reference medium, when the expression or secretion of at least two or at least three or all four of the four marker proteins Ang-1, TGF-β1, VEGF, and HGF is increased, the wound healing properties of the mesenchymal stem cell population are also improved. The secretion of the four marker proteins into the culture medium (and thus the production of those 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).

"DMEM" means Dulbecco's Modified Eagle's Medium, which was developed in 1969 and is a modification of Basal Medium Eagle (BME) (see Table 1, which shows a data sheet for DMEM purchased from Lonza). The original DMEM formulation contained 1000 mg/L of glucose and was first documented for use in culturing mouse embryonic cells. DMEM has since become the standard cell culture medium and is commercially available from a variety of sources, such as ThermoFisher Scientific (Catalog No. 11965-084), Sigma Aldrich (Catalog No. D5546), or Lonza, to name just a few suppliers. Thus, any commercially available DMEM may be used in the present invention. In a preferred embodiment, the DMEM used herein is a DMEM medium purchased from Lonza, and its catalog number is 12-604F (this medium is DMEM supplemented with 4.5 g/L glucose and L-glutamine). In another preferred embodiment, the DMEM used herein is a DMEM medium from Sigma Aldrich (catalog number D5546), which contains 1000 mg/L glucose and sodium bicarbonate, but no L-glutamine.

"F12" medium refers to Ham's F12 medium. This medium is also a standard cell culture medium and is a nutrient mixture originally designed for culturing a variety of mammalian and hybrid tumor cells, and is used with serum binding hormone and transferrin (see Table II, which shows a data sheet for Ham's F12 medium purchased from Lonza). Any commercially available Ham's F12 medium can be used in the present invention (e.g., purchased from ThermoFisher Scientific (Catalog No. 11765-054), Sigma Aldrich (Catalog No. N4888), or Lonza, to name a few suppliers). In a preferred embodiment, Ham's F12 medium purchased from Lonza is used.

"DMEM/F12" or "DMEM:F12" means a mixture of DMEM and Ham's F12 medium at a ratio of 1:1 (see Table III, which shows a data sheet of DMEM:F12 (1:1) medium purchased from Lonza). DMEM/F12 (1:1) medium is also widely used as a basic medium to support the growth of many different mammalian cells, and is commercially available from multiple suppliers, such as ThermoFisher Scientific (Catalog No. 11330057), Sigma Aldrich (Catalog No. D6421), or Lonza. Any commercially available DMEM:F12 medium can be used in the present invention. In a preferred embodiment, the DMEM:F12 medium used herein is DMEM/F12 (1:1) medium purchased from Lonza, catalog number 12-719F (which is DMEM:F12, containing L-glutamine, 15mM HEPES, and 3.151g/L glucose).

"M171" means medium 171, which was developed as a basal medium for the growth of normal human mammary epithelial cells (see Table IV, which shows a data sheet of M171 medium purchased from Life Technologies Corporation). This basal medium is also widely used and commercially available from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (Catalog No. M171500). The present invention may use any commercially available M171 medium. In a preferred embodiment, the M171 medium used herein is the M171 medium purchased from Life Technologies Corporation (Catalog No. M171500).

"FBS" means fetal bovine serum (also known as "calf serum"), which is the blood component remaining after the blood has naturally coagulated, followed by centrifugation to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement for in vitro cell culture of eukaryotic cells because it has very low levels of antibodies and contains more growth factors, making it versatile in many different cell culture applications. FBS is preferably purchased from a member of the International Serum Industry Association (ISIA), whose primary concern is the safety and secure use of serum and animal-derived products through proper traceability of origin, authenticity of labeling, and appropriate standardization and oversight. Suppliers of FBS are ISIA members, including Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco of Thermo Fisher Scientific, and Life Science Production, to name a few. In a currently preferred embodiment, FBS is purchased from GE Healthcare (Catalog No. A15-151).

The culture medium of the present invention is now described. For inducing or improving wound healing properties or isolating or culturing mesenchymal stem cells, the culture medium may include DMEM at a final concentration of about 55 to 65% (v/v), F12 at a final concentration of about 5 to 15% (v/v), M171 at a final concentration of about 15 to 30% (v/v), and FBS at a final concentration of about 1 to 8% (v/v). The numerical value "% (v/v)" used herein means the volume of the individual 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.

In other specific embodiments, the culture medium may include 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 a further specific embodiment, the culture medium may include 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).

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

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

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

In one embodiment of the method of the present invention, a tissue (such as umbilical cord tissue or placenta) can be cultured until an appropriate number of (primary) mesenchymal stem cells, such as umbilical cord lining stem cells, Valdenza jelly, or placental stem cells, grow from the tissue. In a typical embodiment, the umbilical cord tissue is cultured until the cell outgrowth of individual tissue mesenchymal stem cells reaches about 70 to about 80% confluence. It should be noted here that the words "full" or "full" are used in their conventional sense in the field of cell culture and refer to an estimate/indicator of the number of adherent cells in a culture dish or culture flask, meaning the proportion of the surface covered by the cells. For example, 50% full means that approximately half of the surface is covered, and there is still room for cells to grow. 100% full means that the cells completely cover the surface and there is no room for cells to grow as a monolayer.

Once an appropriate number of primary cells (mesenchymal stem cells) have been obtained from the corresponding tissue of the tissue explant, the mesenchymal stem cells are removed from the culture container for culture. In this way, a master cell bank containing (primary) isolated mesenchymal stem cells from, for example, the umbilical cord or placenta can be obtained. Typically, since these mesenchymal stem cells are adherent cells, the cells must be harvested using standard enzyme treatment methods. For example, the enzyme treatment may include trypsinization, as described in US Patent Application No. 2006/0078993, WO2006/019357, or WO2007/046775, meaning that the growing cells may be harvested by trypsinization (0.125% trypsin/0.05% EDTA) for further expansion. If the harvested mesenchymal stem cells are, for example, used to generate a master cell bank, the cells may also be frozen and stored for further use, as described below.

Once harvested, the mesenchymal stem cells can be moved to a culture container for secondary culture. If a population of mesenchymal stem cells previously isolated from their natural environment is used (as previously described, the isolated stem cells used in the method of the present invention can be derived from umbilical cord blood, bone marrow, or adipose tissue, but can also be derived from umbilical cord tissue or placental tissue), secondary culture or culture (these two terms can be used interchangeably below) will also be performed. Secondary culture can also start with frozen primary cells, that is, from a master cell bank. For secondary culture, any suitable amount of cells can be inoculated in a culture container (such as a cell culture dish). For this purpose, the mesenchymal cells can be suspended in a suitable medium (most conveniently, the medium of the present invention) and subcultured at a concentration, such as about 0.5 x 10 ⁶ cells/ml to about 5.0 x 10 ⁶ cells/ml. In a specific embodiment, the suspended cells are subcultured at a concentration of about 1.0 x 10 ⁶ cells/ml. Subculturing can be performed by culturing in simple culture bottles, but can also be performed, for example, in multilayer systems such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA), which can be stacked in a cell culture incubator. Alternatively, subculturing can also be performed in a closed, stand-alone system, such as a bioreactor. Different bioreactor 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 mesenchymal stromal/stem cells according to good manufacturing practices: a review", supra. One illustrative example of a commercially available hollow fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT, Inc), which is used, for example, for expansion of bone marrow mesenchymal stem cells in 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, and one that can be used for subculture of the mesenchymal stem cell populations of the present invention, is the Xuri Cell Expansion System available from GE Heathcare. Cultivating MSC populations in an automated system, such as the Quantum® Cell Expansion System, is particularly advantageous when large numbers of cells are required to produce working cell banks for therapeutic applications under GMP conditions.

The mesenchymal stem cells of the present invention are cultured in the culture medium of the present invention. Accordingly, the culture medium of the present invention can be used to isolate a population of mesenchymal stem cells, for example, from the placental amnion, or from the amnion, or from the Walton's jelly of the umbilical cord, and then culture the isolated primary cells using a culture method. At the same time, for the culture, the mesenchymal stem cells can be cultured until an appropriate number of cells are grown. In an illustrative embodiment, the mesenchymal stem cells are cultured until the mesenchymal stem cells reach about 70 to about 80% full.

Isolation/culture of mesenchymal stem cell populations can be performed under standard conditions for culturing mammalian cells. Typically, the method of isolating mesenchymal stem cell populations of the present invention is usually performed under cell conditions (temperature, atmosphere) used to culture the cell source species. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are cultured under normal atmospheric conditions of 37°C and 5% _CO2 , respectively. In this case, it should be noted that in the present invention, the mesenchymal cell population can be derived from any mammalian species, such as mice, rats, guinea pigs, pigs, rabbits, goats, horses, dogs, cats, sheep, monkeys, or humans. In a specific embodiment, human-derived mesenchymal stem cells are preferred.

Once the required/appropriate amount of mesenchymal stem cells are obtained from the culture medium or subculture medium, they are removed from the culture container used for subculture to collect the mesenchymal stem cells. The collection of mesenchymal stem cells is typically carried out again by enzyme treatment, including trypsinization of the contained cells. The separated mesenchymal stem cells are then collected and used directly or stored for later use. Typically, preservation is carried out by cryopreservation. The term "cryopreservation" used in this article describes in its conventional meaning the process of preserving mesenchymal stem cells by cooling to subzero temperatures, such as (typically) -80°C or -196°C (the boiling point of liquid nitrogen). Cryopreservation can be performed in a manner known to those skilled in the art and may include the use of a cryoprotectant, such as dimethyl sulfoxide (DMSO) or glycerol, which slows ice crystal formation in zona cells.

The isolated population of mesenchymal stem cells obtained by the culture and/or separation methods of the present invention is highly defined and homogeneous. In typical embodiments of the present methods, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD105. In addition, in those embodiments, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45, and HLA-DR. In specific 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, and lack expression of CD34, CD45, and HLA-DR.

Therefore, according to the above disclosure, the present invention also relates to a mesenchymal stem cell population, such as a placental mesenchymal stem cell population, or an umbilical cord mesenchymal stem cell population (such as isolated from Valdenza jelly or umbilical cord amniotic membrane), wherein at least about 90% or more of the stem cell population express each of the following markers: CD73, CD90, and CD105. 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 in the isolated mesenchymal stem cell population are CD73+, CD90+, and CD105+, meaning the percentage of the isolated cell population expressing 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, or about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45, and HLA-DR. In specific 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, and lack expression of CD34, CD45, and HLA-DR. This highly homogeneous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord is described for the first time and meets the criteria for the use of mesenchymal stem cells in cell therapy (see also the Experimental section and, for example, Sensebe et al. "Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review", supra). It should be noted herein that, if desired, this population of mesenchymal stem cells can be obtained using the isolation method of the present invention or by a different method (e.g., cell sorting). In one embodiment of the umbilical cord mesenchymal stem cell population of the present invention, at least about 91% or more of the stem cell population express each of CD73, CD90, and CD105 and lack expression of CD34, CD45, and HLA-DR, wherein the mesenchymal stem cell population isolated from the umbilical cord amniotic membrane is excluded.

In accordance with the 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 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 formulation and may be formulated for any desired mode of drug administration. The pharmaceutical composition may, for example, be suitable for systemic or local application. In a related aspect, the present invention also provides a pharmaceutical composition containing three or four of Ang-1, TGF-β1, VEGF, or HGF as the sole wound healing protein. Such a pharmaceutical composition can be formulated into a liquid, for example, using a pharmaceutically suitable buffer, such as 0.9% saline, Ringer's solution, or phosphate buffered saline (PBS), or a lyophilized/freeze-dried preparation.

In a further embodiment of the present invention, it is related to a method for producing a culture medium for inducing or improving wound healing properties and/or for isolating a mesenchymal stem cell population, wherein the method comprises mixing to obtain a final volume of 500 ml of culture medium:

i. 250ml of DMEM.

ii. 118ml M171.

iii. 118ml of DMEM/F12.

iv. 12.5ml of fetal bovine serum (FBS) to achieve a final concentration of 2.5% (v/v).

As mentioned above, DMEM/F12 medium is a mixture of DMEM and Ham's F12 medium in a 1:1 ratio. Therefore, 118 ml of DMEM/F12 medium contains 59 ml of DMEM and 59 ml of F12. Accordingly, when the medium is prepared in this way, the final concentration (v/v) of a total volume of 500 ml is as follows:

DMEM: 250ml+59ml=309ml, equivalent to 309/500=61.8% (v/v).

M171: 118ml, equivalent to 118/500=23.6% (v/v).

F12: 59ml, equivalent to 59/500=11.8% (v/v).

A specific embodiment of the preparation of the culture medium of the present method further comprises adding: v. 1 ml of EGF reserve solution (5 μg/ml) to achieve a final EGF concentration of 10 ng/ml, and vi. 0.175 ml of insulin reserve solution (14.28 mg/ml) to achieve a final insulin concentration of 5 μg/ml.

It should be noted that in those specific embodiments, when the volumes of the above-mentioned components i. to vi. are mixed, a final volume of 499.675 ml of medium is produced. If no other components are added to the medium, the remaining 0.325 ml (added to a volume of 500 ml) can be, for example, any one of components i. to iv., meaning DMEM, M171, DMEM/F12, or FBS. Alternatively, of course, the concentration of the stock solution of EGF or insulin can be adjusted so that the total volume of the medium is 500 ml. In addition, it should also be noted that components i. to iv. do not need to be added in the order listed, but of course, the components can be mixed in any order to complete the medium of the present invention. This means, for example, that M171 and DMEM/F12 can be mixed together and then combined with DMEM and FBS to achieve the final concentrations described herein, i.e., a final concentration of about 55 to 65% (v/v) for DMEM, about 5 to 15% (v/v) for F12, about 15 to 30% (v/v) for M171, and about 1 to 8% (v/v) for FBS.

In other specific embodiments, the method further comprises adding 0.325 ml volume of one or more of the following supplements: adenine, hydrocortisone, 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3) to DMEM to achieve a total volume of 500 ml of culture medium. In this specific embodiment, the final concentrations of the supplements in DMEM can be as follows: about 0.05 to 0.1 μg/ml of adenine, such as about 0.025 μg/ml of adenine, about 1 to 10 μg/ml of hydrocortisone, about 0.5 to 5 ng/ml of 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3), such as 1.36 ng/ml of 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3).

According to the above disclosure, the present invention also relates to a cell culture medium that can be obtained or a cell culture medium obtained using the method for producing a culture medium as described herein.

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

Therefore, the present invention also relates to a cell culture medium, which comprises: - DMEM with a final concentration of about 55 to 65% (v/v), - F12 with a final concentration of about 5 to 15% (v/v), - M171 with a final concentration of about 15 to 30% (v/v), and - FBS with a final concentration of about 1 to 8% (v/v).

In specific embodiments of the culture medium described herein, 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). In other specific embodiments, the culture medium may include 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).

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 a specific embodiment, the culture medium comprises EGF at a final concentration of about 10 ng/ml. The culture medium described herein may further comprise insulin at a final concentration of about 1 μg/ml to 10 μg/ml. In this embodiment, the culture medium may comprise insulin at a final concentration of about 5 μg/ml.

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 a specific embodiment, the culture medium comprises all three of adenine, hydrocortisone, and 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3). If present, the medium may include adenine at a final concentration of about 0.01 to about 0.1 μg/ml or adenine at about 0.05 to about 0.1 μg/ml, hydrocorticosterone at a final concentration of about 0.1 to about 10 μg/ml or hydrocorticosterone at about 1 to about 10 μg/ml, and/or 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng/ml.

In a specific embodiment of the cell culture medium, 500 ml of the cell culture medium of the present invention comprises:

i. 250ml of DMEM

ii. ii. 118ml M171

iii. 118ml of DMEM/F12

iv. 12.5ml of fetal bovine serum (FBS) (final concentration is 2.5%)

In a further specific embodiment, the cell culture medium may further comprise: v. EGF at a final concentration of 10 ng/ml, and vi. Insulin at a final concentration of 5 μg/ml.

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

In a specific example, components i. to vi. of the culture medium of the present invention are the components indicated in Table V, which means that they are obtained from various manufacturers and use the catalog numbers shown in Table V. The culture medium obtained by mixing components i. to vi. indicated in Table V is also referred to herein as "PTT-6". It should be noted again in this article that components i. to vi., as well as any other ingredients (such as antibiotics from any other commercial supplier) can be used to prepare the culture medium of the present invention.

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

Finally, the present invention also provides a method of treating a non-human mammal (e.g., cat, dog, horse, to name a few) or a human patient suffering from a disease or a disorder, the method comprising administering to the non-human mammal or human patient a population of mesenchymal stem cells or a pharmaceutical composition containing the population of stem cells disclosed herein. The disease may be any disease or disorder, particularly any disease or disorder in which wound healing is desired/needed. The subject (patient or non-human mammal) may have a wound resulting from a burn, bite, trauma, surgery, or a disease such as a skin disease, or a metabolic disorder. An example of a metabolic disorder is, for example, that the patient may suffer from type I or type II diabetes, and from chronic foot ulcers. To treat a subject, the mesenchymal stem cell population of the present invention may be administered in any suitable manner, for example, including but not limited to, local administration, implantation or injection. In principle, any local administration method is referred to herein. Administration of the mesenchymal stem cell population may be performed using a syringe. However, before applying the mesenchymal stem cells to the subject, the mesenchymal stem cells may also be contacted in a cream, ointment, gel, suspension, or any other suitable substance. The stem cell population may be placed directly on a wound (such as a burn or diabetic wound) (see International Patent Application No. WO2007/046775). After it is applied to the subject, the mesenchymal stem cell population can be held in place using a dressing such as a Tegaderm® dressing and a muslin bandage covering the Tegaderm® dressing. Alternatively, the stem cell population can be implanted subcutaneously, for example, directly beneath the skin, in body fat, or in the peritoneum.

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 population described herein.

It is also contemplated that a 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, a unit dose comprises about 10 million cells. It is further contemplated that a unit dose comprises about 1000 cells to about 5 million cells. A unit dose of about 100,000 cells, 300,000 cells, or 500,000 cells may be applied. As described herein, a unit dose may be applied topically, particularly if used for wound healing. For example, a unit dose may be applied topically per square centimeter.

If desired, the unit dose can be applied once, twice, three times, or more than once a week. For example, the unit dose can be applied for one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or more. A unit dose containing about 100,000 cells, about 300,000 cells, or about 500,000 cells can be applied twice a week for 8 weeks, preferably on 1 cm ² .

The unit dose may be contained in any suitable container. For example, the unit dose may be contained in a 1 ml vial. In this case, for example, a 0.1 ml vial may be applied to the subject, preferably per square centimeter. The unit dose may additionally be contained in a syringe.

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 ^™ , Hypothermosol ^™ -FRS, or PlasmaLyte. The culture medium of the present invention may 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 may be separated from the carrier prior to administration. For example, the cells may be centrifuged and separated prior to administration to a subject.

The treatment methods and unit doses of the present invention may include the use of live cells. The viability of the mesenchymal stem cell population can be tested by known methods, for example, by staining with Trypan Blue, as described in the experimental section.

The present invention will be further described by the following non-limiting experimental embodiments.

The present invention will be further described by the following non-limiting experimental embodiments.

The sequences used in this article are described in Table 1 below.

Experimental Examples

1. Umbilical cord tissue was cryopreserved before isolation of mesenchymal stem cells

Umbilical cord tissue (the umbilical cord was donated with the mother's informed consent) was processed, and then mesenchymal stem cells were isolated from the umbilical cord amniotic membrane, as follows.

1.1 Cleaning of umbilical cord tissue samples:

a. Remove the scalpel from the protective cover.

b. Hold the umbilical cord firmly with tweezers and cut it into 10cm long pieces with a scalpel. Put the unusable umbilical cord back into the original tissue cup.

c. Move the 10 cm long umbilical cord segment to a new 150 mm culture dish. The 150 mm culture dish can be used instead of the cup.

d. Place the tweezers and scalpel on the lid of a 150mm culture dish.

e. Remove 25 ml of Plasmalyte A (Baxter, Catalog # 2B2543Q) using a 30 ml syringe. Hold the syringe at a 45° angle with one hand and dispense Plasmalyte A directly into the umbilical cord tissue.

f. Hold the culture plate at a slight angle and remove the Plasmalyte A using a 30ml syringe and blunt needle.

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

h. Repeat the wash step, using a new dish each time, as needed. Make sure all blood clots on the surface are removed. If tissue cleaning is required, use more Plasmalyte A.

i. Place the tissue into a newly labeled tissue culture dish to continue cutting the tissue. Place 20 ml of Plasmalyte A into the culture medium so that the tissue does not dry out while cutting.

j. Cut the umbilical cord into equal slices of approximately 1 cm, producing a total of 10 slices.

k. Further cut each 1cm slice into smaller pieces, each slice is about 0.3cm x 0.3cm to 0.5cm x 0.5cm.

l. Remove all Plasmalyte A from the plate.

m. Use the 30ml syringe in the original Plasmalyte A bag to draw 25ml of Plasmalyte A and dispense it directly onto the umbilical cord tissue piece.

n. Hold the culture plate at an angle to collect all the Plasmalyte A used to wash the tissue side and remove it with a syringe and blunt needle.

o. Repeat the rinse cycle once more. No lumps should remain.

Note: If the umbilical cord is not frozen immediately, store the umbilical cord tissue in Plasmalyte A until it can be frozen.

1.2 Cryopreservation of umbilical cord tissue:

a. Prepare freezing storage solution:

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

ii. Label a 150ml transfer bag with "Tissue Freezing Solution" and connect the plasma transfer device to the port using aseptic technique.

iii. Use the 30ml syringe in the original Plasmalyte A bag to remove 30ml of Plasmalyte A and transfer it to the transfer bag labeled "Tissue Freezing Solution", which contains the time and date the solution was prepared.

iv. Remove 15 ml of 5% human serum albumin using a 20 ml syringe and transfer it to the labeled transfer bag.

v. Add 5ml DMSO into the transfer bag.

vi. Mix thoroughly and record the mixing of the frozen solution.

b. Remove Plasmalyte A from tissue before adding freezing solution.

c. Using a 60ml syringe, draw all 50ml of the frozen solution into the syringe and add approximately 30ml of the frozen solution to the 150mm cell culture dish containing the umbilical cord tissue. Place a blunt needle on the syringe to keep it sterile.

d. Rotate the culture plate containing tissue and freezing solution every minute for a total of 10 minutes.

e. Pick out 8 randomly selected sections with tweezers and place them in each of four 4ml cryovials. Pick out 4 randomly selected sections and place them in a 1.8ml cryovial. The sections should be free of blood clots.

f. Fill the remaining cryovials into individual cryovials containing umbilical cord tissue, with the 4 ml tubes filled to the 3.6 ml line and the 1.8 ml Nunc vials filled to the 1.8 ml line.

g. Label one Bactec Lytic/10-Anaerobic/F and one Bactec Plus Aerobic/F bottle with the tissue ID.

h. Use a syringe and blunt needle to remove 20ml of frozen solution from the culture plate, wipe the Bactec vial with an alcohol swab, replace the blunt needle with an 18g needle, and inoculate the aerobic and anaerobic Bactec bottles with 10ml each.

i. Start the speed control refrigerator.

j. After controlled freezing is complete, place the unit in a liquid nitrogen freezer with continuous temperature monitoring until further use.

2. Isolation of interstitial umbilical cord lining stem cells from umbilical cord tissue

2.1. Preparation of culture medium for processing umbilical cord tissue MSCs:

a. To make 500 ml of PTT-6 (medium/growth medium), add the following in the order listed:

i. 250ml of DMEM

ii. 118ml M171

iii. 118ml DMEM F12

iv. 12.5ml FBS (final concentration is 2.5%)

v. 1ml of EGF (final concentration is 10ng/ml)

vi. 0.175ml of insulin (final concentration is 5μg/ml)

The volumes of components i to vi above yield a final volume of 499.675 ml of medium. If no other components are added to the medium, the remaining 0.325 ml (added to a volume of 500 ml) can be, for example, any one of components i to vi, meaning DMEM, M171, DMEM/F12, or FBS. Alternatively, of course, the concentration of the stock solution of EGF or insulin can be adjusted so that the total volume of the medium is 500 ml. Alternatively, an antibiotic stock solution, such as penicillin-streptomycin-amphotericin, can be added to give a final volume of 500 ml. The medium may also be supplemented with 0.325 ml of one or more of the following supplements: adenine, hydrocortisone, 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3) to achieve a total volume of 500 ml of medium.

vii. Label the "PTT-6" culture flask medium preparation date, the initial operator, and the phrase "expired" after the expiration date. The expiration date is the earliest expiration date of any of the components or 1 month from the preparation date, whichever comes first.

b. To prepare wash medium (Ham's buffered saline solution (HBSS) without calcium or magnesium and containing 5% FBS), add 2.5 ml FBS to 47.5 ml HBSS in a 50 ml centrifuge tube. Label the tube "Wash Medium" and include the original operator and the date the medium was prepared.

c. Test the sterility of all media using Bactec Lytic/10-Anaerobic/F (Becton Dickinson & Company) and Bactec Pluc+Aerobic/F (Becton Dickinson & Company). Inject 20 ml of the prepared media into each bottle.

2.2 Thawing the umbilical cord tissue to collect MSCs:

a. Once the operator is ready to process the sample in a clean room, begin thawing. Do not thaw more than 1 vial at a time unless the vials are from the same donor.

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

c. Prepare 10 ml of wash medium consisting of 70% to 90% PlasmaLyte A in a cleanroom biosafety cabinet. Sterilize the solution by filtering it through a 0.2-μm syringe filter attached to a 10 ml syringe and refrigerate the solution until use.

d. Attach the processing label to the 50ml conical tube.

e. Make sure the water bath temperature is 36-38℃.

f. Remove the vial of tissue from the liquid nitrogen storage tank and quickly thaw it in a 37℃ water bath filled with 1L of sterile water. The vial holder of the Mr.Frosty Nalgene Cryo 1℃ Cryo Container floats with the vial and can be used as a floating rack when thawing samples.

g. Remove the vial from the water bath and spray it with 70% isopropyl alcohol solution. A good time to pull the vial out of the water bath is when small ice floes are visible in the vial - it is recommended that the internal temperature of the vial be less than 37°C.

h. Place the vial into the transfer channel and alert the clean room technician.

2.3 Preparation of tissue for processing:

a. Umbilical cord tissue processing should be performed in an environmentally monitored (EM) clean room. The entire room and operating cabinet should be cleaned at the end of each shift.

b. Prepare/clean the biosafety cabinet.

c. Perform viable bacterial particle counts when working in a biosafety cabinet.

d. Assemble all necessary supplies in a biosafety cabinet, inspecting each package for damage and expiration date. When handling syringes, serological pipettes, sterile tweezers, scalpels, tissue trays, and needles, make sure not to touch any surfaces that would come in contact with sterile items. Only syringe barrels, tubing, plunger tips, and/or the outside of needle caps or sheaths may be safely handled. Discard supplies if surfaces have been touched or come in contact with non-sterile surfaces.

e. Record the batch numbers and expiration dates (if necessary) of all reagents and supplies to be used.

f. Clean thawed vials with lint-free wipes soaked in 70% alcohol before transferring to the biosafety cabinet to receive thawed vials.

g. Using the aspiration needle with the syringe, remove as much liquid as possible from the vial. Avoid aspirating tissue.

h. Use sterile tweezers and transfer the tissue to a sterile 100mm culture dish.

i. Add 5 ml of wash medium aliquot to the tissue aliquot.

j. Vortex the contents for 15-30 seconds, then remove the rinse medium using a pipette or syringe with an aspiration needle. Repeat this rinse process twice.

k. Add 2 ml of wash medium to the tissue to prevent the tissue from drying out.

2.4. Initiation of MSC growth from tissues:

a. Mark the MSC lot number or umbilical cord tissue ID and the date the growth started on the bottom of the 6-well culture dish "Growth 1". If using a 60mm tissue culture dish, divide the dish into 4 quadrants by drawing a grid on the bottom of the dish.

b. Using sterile, disposable tweezers, place a 3 x 3mm to 5 x 5mm tissue into each well. If using a 60mm tissue culture dish, place the tissue in the middle of each quadrant, keeping the tissues separated (more than 1cm apart).

c. Fill each well with 3 ml of PTT-6.

d. Use an aspirator needle attached to a 30 ml syringe to remove enough medium to barely cover the tissue. Do not tilt the culture plate. The aspirator needle does not touch the bottom of the culture well.

e. Use an inverted optical microscope to observe cell growth every day (24±6hrs). A real-time cell culture imaging system can be used instead of an optical microscope.

f. Change the culture medium daily. Be sure to equilibrate the medium to room temperature before use.

i. Aspirate the culture medium.

ii. Add 3ml of PTT-6.

iii. Aspirate until the medium barely covers the tissue.

g. When cell growth is observed in the tissue, transfer the tissue to a new 6-well dish using the same procedure as 4.a to 4.e above, except label the dish "Growth 2". Maintain cell growth in the "Growth 1" dish by adding 2 ml of PTT-6 to each well. Observe cell confluency daily. Change the medium every 2-3 days (be sure to equilibrate the medium to room temperature before use).

h. When cell growth is observed in the "Growth 2" plate, repeat steps 4.a to 4.e except label the plate "Growth 3". Maintain cell growth in the "Growth 2" plate by adding 2 ml of PTT-6 to each well. Observe cell confluence daily. Change the medium every 2-3 days (be sure to equilibrate the medium to room temperature before use).

i. When growth is observed in the "Growth 3" plate, discard the tissue. If the tissue is very small and does not appear to interfere with cell growth, discard the tissue at the time of subculture.

j. When the cells reach 40-50% full, observe the cells daily to prevent overexpansion.

k. When the cells reach 70-80% confluence, subculture the cells. Do not allow the cells to expand beyond 80% confluence.

The size of the tissue explants is about 1-3 mm, and the tissue explants/cell culture medium is carried out in a 175 mm square culture dish. The average number of mesenchymal stem cells collected from the explants is usually about 4,000-6,000 cells/explant. Accordingly, when the mesenchymal stem cells grow 48 explants at the same time, about 300,000 cells can be obtained at the time of collection. Those 300,000 mesenchymal stem cells collected from the explants can then be used for subculture, which is to inoculate 300,000 cells in a 175 ^cm2 cell culture bottle as described in Example 2.5 below (this can be called the 1st passage). The mesenchymal stem cells obtained from this passage 1 can then be used again to inoculate a 175 ^cm2 cell culture flask (passage 2) and expand the cells as described in Example 2.5 below. The cells obtained from passages 1 and 2 can be "banked" using cryopreservation, wherein the mesenchymal stem cells obtained after passage 2 can be considered to represent a master cell bank, which will be used for further expansion of mesenchymal stem cells, for example, in a bioreactor as described in Example 2.7 below.

2.5. Subculturing MSCs in cell culture dishes

a. Perform viable counts while working in a biosafety cabinet. Equilibrate all media to room temperature before use.

b. When the cell growth reaches about 70-80% full, subculture the cells.

i. Remove PTT-6 from the culture dish.

ii. Rinse with HBSS without calcium or magnesium.

iii. Add 0.2 ml of 1X TrypLE-EDTA and rotate for 1-2 minutes.

iv. Tilt the dish 30-45° to allow the cells to move downward by gravity flow. Tap the side of the dish to speed up detachment.

v. Add 1 ml of PTT-6. Gently pipette up and down and 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.

vi. Centrifuge at 1200 rpm for 10 minutes.

vii. Remove the supernatant and resuspend the cells with 5ml PTT-6.

c. Subculture of MSCs

i. Aliquot 50 μl of the cell suspension and analyze TNC and viability using the trypan blue exclusion assay.

ii. Count the cells using a hemacytometer. Expect to count 20-100 cells per square unit. If the count is higher than 100, dilute the original sample 1:5 and repeat the trypan blue method using a hemacytometer.

iii. Calculate live cells/ml and total live cells:

1. Live cells/ml = live cell count x dilution rate x 10 ⁴

2. Total viable cells = viable cell count x dilution rate x total volume x 10 ⁴

iv. Calculate % survival rate:

1. % survival rate = live cell count x 100/(live cell count + dead cell count)

v. Dilute the cell suspension to 1.0 x 10 ⁶ cells/ml:

1. "X" volume = total viable cells/10 ⁶ cells/ml

2. For example, if the total viable cell count is 1.0 x 10 ⁷ ;

3. "X" = ^107/106 cells/ml or 10ml, therefore, by adding 5ml ^to the cell suspension (which is 5ml), the total cell volume is brought to 10ml.

vi. If the cell suspension is less than 10 ⁶ cells/ml, determine the volume required to inoculate 2 x 10 ⁶ cells per 150 mm dish or 175 cm ² flask.

1. Volume required for 2 x 10 ⁶ cells = 2 x 10 ⁶ cells ÷ viable cells/ml

2. For example, if the viable cells/ml is 8 x 10 ⁵ cells/ml, then 2 x 10 ⁶ cells ÷ 8 x 10 ⁵ cells/ml or 2.5 ml is required.

vii. Leave 0.5ml for MSC marker analysis.

viii. Inoculate 2 x 10 ⁶ cells per 150 mm dish or 175 cm ² flask containing 30 ml of PTT-6.

ix. Observe cell attachment, colony formation, and confluence every three days. 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 replace the optical microscope.

x. Change the culture medium every 2-3 days.

2.6 Cryopreservation of MSCs

a. Perform viable bacterial particle counts when working in a biosafety cabinet.

b. When cells reach 70-80% confluency, detach cells using 2 ml 1X TrypLE-EDTA per 150 mm dish or 175 ^cm2 flask.

i. Remove PTT-6 from the culture dish.

ii. Wash with 5 ml HBSS or PBS without calcium or magnesium.

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

iv. Tilt the dish 30-45° to allow the cells to move downward by gravity flow. Tap the side of the dish to speed up detachment.

v. Add 10ml PTT-6 to inactivate TrypLE. Mix well to separate cell clumps.

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

vii. Centrifuge at 1200 rpm for 10 minutes.

viii. Aspirate the medium and resuspend with 10ml PTT-6.

ix. Aliquot 50 μl and determine total viable cell count and % viability as above. Cell counts should be performed within 15 minutes as cells may begin to clump.

c. Prepare cells for cryopreservation

i. Prepare cell suspension medium and cryopreservation medium, and freeze cells.

2.7. Subculture (expansion) of MSCs in a Quantum bioreactor (Terumo BTC, Inc.)

MSCs can also be expanded using the Quantum bioreactor. The starting cell number for expansion in the Quantum bioreactor should range from 20 to 30 million cells per run. At harvest, the typical yield per run is 300 to 700 million MSCs. The bioreactor is operated according to the manufacturer's protocol. The resulting mesenchymal stem cells are typically stored frozen (see below) and used as a working cell bank.

Materials/reagents:

1. Quantum expansion kit

2. Quantum waste bag

3. Quantum culture medium bag

4. Quantum sample bag

5. PTT-6

6. PBS

7. Fibronectin

8. TrypLE

9. 3ml syringe

10. Glucose test strips

11. Lactate test paper

12. 60ml cell culture dish or equivalent

13. Medical grade 5% CO ₂ gas mixture

14. 50ml Combi-tip

equipment:

1. Biosafety Cabinet

2. Glucose meter (Bayer Healthcare/Ascensia Contour Blood Glucose Meter)

3. Lactate Plus (Nova Biomedical)

4. Peristaltic pump with head

5. Centrifuge, Eppendorf 5810

6. Sterile tube connector

7. M4 repeating pipette

8. RF sealing machine

program:

1. Prepare the Quantum Bioreactor

a) Start the Quantum bioreactor

b) Coating bioreactor:

1) Prepare the fibronectin solution in a biosafety cabinet.

15min)。 1) Allow the freeze-dried fibronectin to acclimate to room temperature (at room temperature

15min).

2) Add 5 ml of sterile distilled water; do not swirl or stir.

3) Allow fibronectin to dissolve in the solution for 30 minutes.

4) Use a 10ml syringe connected to an 18g needle to transfer the fibronectin solution into a Ccell injection bag containing 95ml PBS.

2) Connect the bag to the "reagent" line.

3) Check for bubbles (you can use "Remove IC Air" or "Remove EC Air" to remove bubbles and use "Purge" as the injection source).

4) Open or set up the program for coating the bioreactor (Table A. Steps 3-5).

5) Run the program.

6) While the program is running and coating the bioreactor, prepare a media bag with 4L of PTT-6 media.

7) Connect the medium bag to the IC medium line with a sterile tubing connector.

8) When the bioreactor coating step is completed, separate the cell sample bag for the fibronectin solution using an RF sealer.

c) Wash away excess fibronectin.

d) Use culture medium to regulate the bioreactor.

2. Cultivating cells in a Quantum bioreactor

a) Load and attach cells in a uniform suspension.

b) Feeding and culturing cells

1) Select the medium flow rate to feed the cells.

2) Daily sampling of lactate and glucose.

3) Adjust the media flow rate as the lactate level increases. The actual maximum lactate tolerance will be determined by the original culture bottle of cells. Determine if there is enough PTT-6 media in the media bag. Replace the PTT-6 media bag with fresh PTT-6 media bag as needed.

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.

3. Harvesting cells from the Quantum bioreactor

a) When the lactate concentration has not decreased, harvest the cells after the last lactate and glucose sampling.

b) Collect cells:

1) Connect the cell injection bag containing 100ml TrypLE to the "reagent" line using a sterile tubing connector.

2) Make sure there is enough PBS in the PBS bag. If not, connect a new bag with at least 1.7 liters of PBS to the "clean" line using a sterile tubing connector.

3) Run the collection program.

4. Cryopreservation of cells

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

2) Resuspend with 25 ml of cold cell suspension. Count cells using a Sysmex or Biorad Cell Counter. Attach the cell count record to the corresponding Quantum Processing Batch Record.

3) Adjust the cell concentration to 2x10 ⁷ /ml.

4) Add an equal volume of cryopreservation solution and mix thoroughly (do not shake or vortex).

5) Using a repeating pipette, add 1 ml of the frozen cell suspension to each 1.8 ml vial. Freeze using the CRF program, as described in SOP D6.100 CB Freeze, using Controlled Rate Freezers.

6) Store the vials in the designated liquid nitrogen storage space.

7) Attach the CRF operation report to the corresponding MSC P3-Quantum processing batch record sheet.

3. Analysis of stem cell marker expression in mesenchymal umbilical cord lining stem cell populations isolated from umbilical cord tissue using different culture media

To express the mesenchymal stem cell markers CD73, CD90, and CD105, flow cytometry was performed to analyze mesenchymal stem cells isolated from the umbilical cord.

For these experiments, umbilical cord tissue was cultured in three different culture media, and mesenchymal stem cells were isolated from the umbilical cord tissue, and then the mesenchymal stem cells were subcultured in individual culture media, as described in Example 2.

The following three media were used in these experiments: a) 90% v/v/DMEM supplemented with 10% FBS (v/v), b) medium PTT-4 as described in U.S. Patent Application No. 2008/0248005 and corresponding International Patent Application No. WO 2007/046775, which consists of 90% (v/v) CMRL1066 and 10% (v/v) FBS (see paragraph [0183] of WO 2007/046775, and c) medium PTT-6 of the present invention, whose composition is as described herein. In this flow cytometric analysis, two different samples of the umbilical cord lining mesenchymal stem cell (CLMC) population were analyzed for each of the three media used.

The following methods were used for flow cytometry analysis.

Materials and methods

program

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

1. Culture the explant tissue samples in a cell culture plate and immerse them in each culture medium, and then maintain them in a CO ₂ cell culture incubator at 37°C, as described in Example 2.

2. Change the culture medium every 3 days.

3. Monitor cell outgrowth of tissue culture explants under light microscopy.

4. When the cells are approximately 70% confluent, trypsinize the cells (0.0125% trypsin/0.05% EDTA) to separate them from the culture plates and use them for flow cytometry experiments.

b) Trypsinization of cells for experiments

1. Remove the culture medium from the cell culture dish.

2. Gently rinse with sterile 1X PBS to remove trace amounts of FBS, as FBS will interfere with the enzymatic action of trypsin.

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

4. Observe the cells under a microscope to ensure they are detached. Add complete medium containing FBS (DMEM containing 10% FBS) to neutralize trypsin.

5. Use a pipette to transfer the cells in the culture medium to the wall of the culture dish to break up the cell clumps. Collect the cell suspension and transfer it to a 50ml centrifuge tube.

6. Add sterile 1X PBS to the cell culture dish and rinse. Collect the cell suspension into the same centrifuge tube.

7. Centrifuge at 1800rpm for 10mins.

8. Discard the supernatant and resuspend the cell pellet with PBA medium.

c) Count cells

1. Make sure the hemocytometer and its coverslip are clean and dry, preferably by cleaning with 70% ethanol and drying before wiping with Kim wipes (lint-free paper).

2. Aliquot a small amount of the cell suspension into a microcentrifuge tube and remove it from the BSC cabinet.

3. Use an equal volume of trypan blue to stain the cells in the suspension, such as adding 500μl of trypan blue to 500μl of cell suspension (dilution ratio = 2X, which produces a 0.2% trypan blue solution).

4. Avoid exposing cells to trypan blue for more than 30 minutes because trypan blue is toxic and will lead to an increase in non-viable cells, resulting in cell count errors.

5. Add 20μl of the cell suspension mixture into each chamber of the hemocytometer and observe under an optical microscope.

a. Count the number of viable cells (bright cells; non-viable cells take up trypan blue and are therefore dark) in each quadrant of the hemacytometer, a total of 8 quadrants in the upper and lower chambers.

The total cell count is (mean number of cells/quadrant) x 10 ⁴ cells/ml.

d) Stained cells

i. Preparation before cell staining

●The cell suspension was divided into 3 tubes (CD73, CD90, CD105) and 2 tubes (negative control group) in duplicate, each containing 50,000 cells.

ii. Staining with primary antibody (Ab)

●Add 1μl [0.5mg/ml Ab] of primary antibody to 100ul of cell suspension. Incubate at 4℃ for 45min.

●Add PBA to 1ml.

●Centrifuge at 8000rpm for 5mins at 4℃.

●Remove the supernatant.

●Add 1 ml of PBA and resuspend the cell pellet.

●Centrifuge at 8000rpm for 5mins at 4℃.

●Remove the supernatant.

●Resuspend in 100ul PBA.

iii. Stain with secondary antibody in dark place

●Add 1ul [0.5mg/ml ab] of secondary antibody to 100ul of cell suspension. Incubate at 4℃ for 30min.

●Add PBA to 1ml.

●Centrifuge at 8000rpm for 5mins at 4℃.

●Remove the supernatant.

●Add 1 ml of PBA and resuspend the cell pellet.

●Centrifuge at 8000rpm for 5mins at 4℃.

●Remove the supernatant.

●Resuspend with 200-300ul PBA and perform flow cytometry analysis.

● Transfer cells to FACS tubes and perform BD FACS CANDO flow cytometry reading.

Figures 1a to 1c show the results of flow cytometry experiments. Figure 1a shows that the umbilical cord tissue was isolated and cultured in DMEM/10% After FBS, the percentage of isolated interstitial umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105, FIG. 1b shows the percentage of isolated interstitial umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after being isolated from umbilical cord tissue and cultured in PTT-4, and FIG. 1c shows the percentage of isolated interstitial umbilical cord lining stem cells expressing stem cell markers CD73, CD90, and CD105 after being isolated from umbilical cord tissue and cultured in PTT-6. As can be seen in Figure a , the population isolated using DMEM/10% FBS as the culture medium has approximately 75% CD73+ cells, 78% 90+ cells, and 80% CD105+ cells (average of two experiments), whereas after the umbilical cord tissue was isolated/cultured in PTT-4 medium (see Figure b ), the numbers of CD73-positive, CD90-positive, and CD105-positive mesenchymal stem cells were approximately 87% (CD73+ cells), 93% (CD90+ cells), and 86% (CD105+ cells) (average of two experiments). The purity of all three markers (CD73, CD90, CD105) of the mesenchymal stem cell population obtained by culturing in the PTT-6 medium of the present invention is at least 99.0%, which means that the purity of this cell population is significantly higher than that of the cells cultured in the PTT-4 medium or DMEM/10% FBS. In addition, and even more importantly, the mesenchymal stem cell population obtained by culturing in PTT-6 is essentially 100% pure and is a defined stem cell population. This makes the stem cell population of the present invention an ideal candidate for stem cell therapy. Therefore, this mesenchymal umbilical cord lining stem cell population can become the gold standard for this stem cell therapy method.

The results shown in Figure 6 were further confirmed by the flow cytometry analysis results shown in Figures 2a and 2b . Figure 2a shows the percentage of isolated stromal cord lining stem cells (cord amniotic stromal stem cells) expressing stem cell markers CD73, CD90, and CD105 and lacking CD34, CD45, and HLA-DR after isolation from umbilical cord tissue and culture in PTT-6 medium. As shown in FIG. 2a , the mesenchymal stem cell population contained 97.5% viable cells, 100% of which expressed each of CD73, CD90, and CD105 (see the “CD73+CD90+” and “CD73+CD105+” columns), while 99.2% of the stem cell population did not express CD45 and 100% of the stem cell population did not express CD34 and HLA-DR (see the “CD34-CD45-” and “CD34-HLA-DR-” columns). Therefore, the mesenchymal stem cell population obtained by culturing in PTT-6 is essentially 100% pure and is a defined stem cell population that meets the criteria that mesenchymal stem cells must meet for use in cell therapy (95% or more of the stem cell population express CD73, CD90, and CD105, and 98% or more of the stem cell population lacks expression of CD34, CD45, and HLA-DR, see Sensebe et al., “Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra). It should be noted that the amniotic mesenchymal stem cells 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 standards for mesenchymal stem cells in cell therapy.

Figure 2b shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90, and CD105 and lacking CD34, CD45, and HLA-DR. As shown in Figure 2b , the bone marrow mesenchymal stem cell population contained 94.3% live cells, of which 100% expressed each of CD73, CD90, and CD105 (see "CD73+CD90+" and "CD73+CD105+" columns), while only 62.8% of the bone marrow stem cell population lacked CD45 and 99.9% of the stem cell population lacked CD34 and HLA-DR (see "CD34-CD45-" and "CD34-HLA-DR-" columns). Therefore, the bone marrow mesenchymal stem cells, which are regarded as the gold standard of mesenchymal stem cells, are far inferior to the homogeneous/pure (umbilical cord amniotic membrane) mesenchymal stem cell population of this application in terms of stem cell markers. This finding also shows that the stem cell population of the present invention can be an ideal candidate for stem cell therapy and can become the gold standard for this stem cell therapy method.

4. Analysis of wound healing marker protein secretion from mesenchymal stem cell populations isolated from cultured in the medium of the present invention

Based on the highly significant results (essentially 100% pure and defined MSC populations were obtained by culturing in PTT-6), each isolated MSC population was cultured in PTT-6 and analyzed for secretion of wound healing marker proteins and compared with the results obtained by culturing in PTT-4 medium (used as a reference medium).

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

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

- WJ-MSC. This WJ-MSC population was isolated from human umbilical cord WJ explants (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.

- Adipose tissue-derived mesenchymal stem cells (AT-MSC). This AT-MSC population was isolated from donor skin tissue after abdominoplasty as a tissue explant as described by 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 (cultured in DMEM supplemented with 5% penicillin/streptomycin and 10% FBS).

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

- Placental mesenchymal stem cells (PT-MSC). This PT-MSC population was isolated from the placenta using the method 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.

Method for culturing isolated MSCs

●5 million MSCs from each source were plated in 100mm tissue culture dishes containing DMEM/F12/10% FCS for 24 hours.

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

●Discard the culture medium and wash the cells with PBS.

●Add 10ml DMEM to the culture medium for 24 hours.

●Discard the culture medium and add 5ml DMEM to the culture medium

●After 24 hours of culture, the conditioned medium was collected, the cell debris was removed by centrifugation, and the supernatant was aliquoted into tubes and stored at -80°C. The secretion of marker proteins was then analyzed using a cytokine assay.

Cytokine test of PTT-6 and PTT-4 culture medium supernatants of MSCs derived from CL-MSC, WJ-MSC, bone marrow MSC, and adipose MSC

Cytokine detection was performed using MSC supernatant. Luminex 200 and Xponent software were used for measurement and analysis.

The purpose of this experiment is to determine the relative levels of multiple (PDGF-AA, PDGF-BB, IL-10, VEGF, Ang-1, and HGF), TGFβ1 single, and bFGF2 single cytokines in the cell culture supernatant. The supernatants are (MSC, mesenchymal stem cells; CL, umbilical cord lining; WJ, Wattenberg jelly; AT, adipose tissue; BM, bone marrow):

˙CL-MSCs were cultured in PTT-4.

˙WJ-MSCs were cultured in PTT-4.

˙AT-MSCs were cultured in PTT-4.

˙BM-MSCs were cultured in PTT-4.

˙CL-MSCs were cultured in PTT-6.

˙WJ-MSCs were cultured in PTT-6.

˙AT-MSCs were cultured in PTT-6.

˙BM-MSCs were cultured in PTT-6.

Each sample was tested in triplicate (3 wells), except for the sample supplied to 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 assay (cells were not cultured in PTT-6 or PTT-4 to prepare the conditioned medium for CR001A, CR001C, CR001D, and CR001G).

The purpose of this experiment is to generate cytokine profiles of MSCs cultured in PTT-4 or PTT-6 and compare the profiles of MSCs from different tissue sources (umbilical cord lining, Valdenza jelly, adipose tissue, and bone marrow). This profile will reveal which stem cell populations grow in which culture medium and secrete more cytokines of interest to promote wound healing.

All culture dishes were set up as described in Figure 5. The abbreviations used are as follows: MSC, mesenchymal stem cell; CL, umbilical cord lining; WJ, Walton's jelly; AT, adipose tissue; BM, bone marrow.

Multiple analysis

Multiple information:

R&D Systems/Bio-techne cat.# LXSAHM. This kit is lot # L123680, which expires on 08/28/18, and has the following analytes:

˙Ang-1, angiopoietin.

˙VEGF, vascular endothelial growth factor.

˙PDGF-AA, platelet-derived growth factor (PDGF-AA means disulfide-bonded homodimers composed of the A chain, while PDGF-BB is composed of the B homodimer. R&D indicates that PDGF-BB antibodies can also detect PDGF-AB heterodimers).

˙PDGF-BB.

˙HGF, hepatocyte growth factor.

˙IL-10, interleukin-10.

TGFβ1 single information: R&D Systems/Bio-techne:

˙Basic kit, cat.# LTGM00, lot # P156217, delivered 02/27/18, expired 08/30/18.

˙TGFβ1 component, cat.# LTGM100, lot # P161760, delivered on 02/27/18, expired on 11/27/19.

bFGF2 single information (used on March 19th, 2018): eBioscience/Thermo:

˙Base Kit, cat.# EPX010-10420-901, lot # 172174000, expires 01/31/20.

˙bFGF2 component, cat.# EPX01A-12074-901, lot # 169751102, expires on 12/31/19.

bFGF2 single information (used on March 22nd, 2018): eBioscience/Thermo:

˙Base Kit, cat.# EPX010-10420-901, lot # 172174000, expires 01/31/20.

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

Multitasking information:

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

˙Ang-1, angiopoietin.

˙VEGF, vascular endothelial growth factor.

˙PDGF-AA, platelet-derived growth factor 2.

˙PDGF-BB.

˙HGF, hepatocyte growth factor.

˙IL-10, interleukin-10.

˙bFGF, basic fibroblast growth factor.

Data input

The raw data is exported in PDF and Excel formats. Data in Excel format is used for data processing.

program

Cytokine assays of MSC supernatants were performed according to the detailed method information. As part of this experiment, one modification to the method was made: Standard 8 from the multiplex set was no longer used. The reason for discontinuing the use of Standard 8 is that the R&D Systems method itself uses only Standards 1 to 6. In addition, Standard 8 was validated in ClinImmune, where only two of the six analytes included in 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 media, standard curves were constructed with PTT-6 and PTT-4. Test samples grown in PTT-6 or PTT-4 were extrapolated from the relevant standard curves.

The results were extrapolated from analyte-specific standard curves from Luminex software, generated by the same software: the analysis algorithm was set in Logistic 5P Weighted analysis with weighting of 1/y2.

Samples

1. PTT-4 and PTT-6 culture medium (not exposed to MSCs).

2. To test the supernatant of MSCs.

3. Any: supernatant of CL-MSCs from different donors CR001A, C, D, and G.

Experimental results excerpt

TGFβ1 single test

˙Use 1 of 3 equal parts of the sample - as shown in Figure 4. All error bars are standard deviations of three replicate measurements.

Figure 4 : Single assays of TGFβ1. It can be seen that when CL-MSCs and WJ-MSCs were cultured, more TGFβ1 was produced when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC culture media produced more or less equal amounts of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations of three assays.

First multiplex test

˙Use 1 of 3 aliquots.

˙PDGF-BB and IL-10 were not detected in any samples.

The data are shown in Figures 5 and 11.

FIG5 : FIG5A is a multiplex assay of PDGF-AA. It can be seen that when CL-MSC, WJ-MSC, AT-MSC, and BM-MSC medium are cultured, more PDGF-AA is produced when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of three determinations. FIG5B is a multiplex assay of VEGF. It can be seen that when CL-MSC, WJ-MSC, AT-MSC, and BM-MSC medium are cultured, more VEGF is produced when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three determinations. FIG5C is a multiplex assay of Ang-1. It can be seen that when CL-MSC and WJ-MSC medium are cultured, more Ang-1 is produced when grown in PTT-6 than when grown in PTT-4. Cultured AT-MSC and BM-MSC produced virtually no Ang-1. All error bars are standard deviations of three determinations.

Figure 6 : Multiplex assays of HGF. It can be seen that when CL-MSCs and WJ-MSCs were cultured in PTT-6, more HGF was produced than when they were grown in PTT-4. AT-MSCs and BM-MSCs did not produce virtually any HGF. All error bars are standard deviations of three determinations.

Multiple tests (including bFGF)

˙Use 3 of 3 equal parts. The data are shown in Figures 7-9.

Figure 7 : Multiplex determination of PDGF-AA. It can be seen that when CL-MSCs and WJ-MSCs were cultured in PTT-4, they produced more PDGF-AA than those grown in PTT-6. AT-MSCs and BM-MSCs cultured in both media produced the same amount of PDGF-AA. All error bars are standard deviations of three determinations.

FIG8 : FIG8A is a multiplex assay for VEGF. It can be seen that when CL-MSC, WJ-MSC, AT-MSC, and BM-MSC medium are cultured, more VEGF is produced when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three determinations. FIG8B is a multiplex assay for Ang-1 multiplex assay. It can be seen that when CL-MSC and WJ-MSC medium are cultured, more Ang-1 is produced when grown in PTT-6 than when grown in PTT-4. Culturing AT-MSC and BM-MSC does not produce essentially any Ang-1. All error bars are standard deviations of three determinations. FIG8C is a multiplex assay for HGF. It can be seen that when CL-MSC and WJ-MSC medium are cultured, more HGF is produced when grown in PTT-6 than when grown in PTT-4. Cultured AT-MSC and BM-MSC produced virtually no HGF. All error bars are standard deviations of three determinations.

Figure 9 : Multiplex determination of bFGF. It can be seen that when CL-MSC and WJ-MSC were cultured in PTT-6, they produced more bFGF than when they were grown in PTT-4. When AT-MSC and BM-MSC were cultured in PTT-6 and PTT-4, they produced the same amount of bFGF. All error bars are standard deviations of three determinations.

˙It should be noted that the abundance of bFGF samples was very low, at or near the detection limit.

Figures 10 to 21 illustrate excerpts of data obtained from different experiments.

Figure 10: Excerpt of TGFβ1 assay results from 5 different experiments (170328, 170804, 170814, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the TGFβ standard curve in the experiment. The upper graph shows the MFI of the TGFβ standard curve taken from PTT-4 and PTT-6 medium. The lower right graph shows that when CL-MSC and WJ-MSC are cultured, more TGFβ1 is produced when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC culture media produce equivalent amounts of TGFβ1 when grown in PTT-6 or PTT-4. All error bars are standard deviations of different measurements from experiments 170328, 170804, 170814, 180105, and 180226.

Figure 11: Extract of Ang-1 assay results from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the Ang-1 standard curve in the experiment. The upper graph shows the MFI of the Ang-1 standard curve taken from PTT-4 and PTT-6 culture media. The lower right graph shows that when CL-MSC and WJ-MSC are cultured, more Ang-1 is produced when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC culture media produce essentially the same amount of Ang-1 when grown in PTT-6 or PTT-4. All error bars are standard deviations of different measured values from experiments 170602, 170511, 170414, 170224, 180105, and 180226.

Figure 12: Extract of PDGF-BB assay results from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the PDGF-BB standard curve in the experiment. The upper graph shows the MFI of the PDGF-BB standard curve taken from the PTT-4 and PTT-6 culture media. It is worth noting that PDGF-BB was not detected in any experiment.

Figure 13: Extract of PDGF-AA assay results from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the PDGF-AA standard curve in the experiment. The upper graph shows the MFI of the PDGF-AA standard curve taken from PTT-4 and PTT-6 medium. The lower right graph shows that when CL-MSC, AT-MSC, BM-MSC, and WJ-MSC were cultured in medium, slightly more PDGF-AA was produced when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of the measurements from experiments 170602, 170511, 170414, 170224, 180105, and 180226.

Figure 14: Extract of IL-10 assay results from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the IL-10 standard curve in the experiment. The upper graph shows the MFI of the IL-10 standard curve taken from PTT-4 and PTT-6 culture media. It is worth noting that IL-10 was not detected in any experiment.

Figure 15: Extract of VEGF measurement results from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left side of the figure depicts the mean fluorescence intensity (MFI) measured by the VEGF standard curve in the experiment. The upper figure shows the MFI of the VEGF standard curve taken from PTT-4 and PTT-6 medium. The lower right side of the figure shows that when CL-MSC, AT-MSC, BM-MSC, and WJ-MSC were cultured in PTT-6 medium, more VEGF was produced than when grown in PTT-4. All error bars are standard deviations of different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

Figure 16: Excerpt of HGF measurement results from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The lower left graph depicts the mean fluorescence intensity (MFI) measured by the HGF standard curve in the experiment. The upper graph shows the MFI of the HGF standard curve taken from PTT-4 and PTT-6 medium. The lower right graph shows that when CL-MSC and WJ-MSC are cultured, they produce more HGF when grown in PTT-6 than when grown in PTT-4. On the other hand, AT-MSC and BM-MSC cultured do not produce as much HGF as in the other media. All error bars are standard deviations of different measured values from experiments 170602, 170511, 170414, 170224, 180105, and 180226.

Cytokine assay of supernatants of MSCs cultured in PTT-6 and PTT-4 medium or DMEM/F12-CL-MSC, WJ-MSC, and placenta MSC

Cytokine detection was performed using MSC supernatant. The assay and analysis were performed as described above.

The purpose of this experiment is to determine 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. Supernatants were obtained from mesenchymal stem cells from the umbilical cord lining (CL), Walton's jelly (WJ), and placenta. Mesenchymal stem cells were cultured in PTT-6, PPT-4, or DMEM/F12 medium.

˙CL-MSCs were cultured in PTT-4.

˙WJ-MSCs were cultured in PTT-4.

˙Placental MSCs cultured in PTT-4.

˙CL-MSCs were cultured in PTT-6.

˙WJ-MSCs were cultured in PTT-6.

˙Placental MSCs cultured in PTT-6.

˙CL-MSCs were cultured in DMEM/F12.

˙WJ-MSCs were cultured in DMEM/F12.

Each sample was tested in triplicate, except for the placental supernatant sample. The purpose of this experiment was to generate cytokine profiles from MSCs cultured in PTT-4 or PTT-6 and to compare the profiles of MSCs from different tissue sources (endothelium, Valdenza jelly, and placental MSCs). Cytokine assays were performed as described above. This profile will reveal which stem cell populations grown in which culture medium will secrete more of the cytokine of interest to promote wound healing.

Figure 17 : Single measurement of TGFβ1. The left side of the figure depicts the mean fluorescence intensity (MFI) measured by the TGFβ1 standard curve in the experiment. It can be seen in the right figure that CL-MSC, WJ-MSC, and placental MSC grown in PTT-6 produce more TGFβ1 than those grown in PTT-4 or DMEM/F12 (only DMEM in Figure 17).

Figure 18 : Extracts of PDGF-BB assays of supernatants from CL-MSC, WJ-MSC, and placental MSC cultured in PTT-6, PTT-4, or DMEM/F12. The left side of the figure depicts the mean fluorescence intensity (MFI) measured by the PDGF-BB standard curve in the experiments. Of note, PDGF-BB was not detected in any of the experiments.

Figure 19 : IL-10 assay of supernatants extracted from CL-MSC, WJ-MSC, and placental MSC cultured in PTT-6, PTT-4, or DMEM/F12. The left panel depicts the mean fluorescence intensity (MFI) measured by the VEGF standard curve in the experiment. S6 represents the lowest standard used in the experiment. Any sample falling below is considered below the detection range. It can be seen in the right panel that all CL-MSC, WJ-MSC, and placental MSC produced detectable levels of IL-10 when grown in PTT-6, while little or no IL-10 was detected when MSCs were grown in PTT-4 or DMEM/F12.

Figure 20 : VEGF assay of supernatants extracted from CL-MSC, WJ-MSC, and placental MSC cultured in PTT-6, PTT-4, or DMEM/F12. The left panel depicts the mean fluorescence intensity (MFI) measured by the VEGF standard curve in the experiment. S1 represents the highest standard used in the experiment. Any samples falling above it are considered extrapolated (too concentrated). It can be seen in the right panel that all CL-MSC, WJ-MSC, and placental MSC produce higher levels of VEGF when grown in PTT-6 compared to when the MSC are grown in PTT-4 or DMEM/F12.

Figure 21 : Multiplexed measurement of bFGF. The left panel depicts the mean fluorescence intensity (MFI) measured by the PDGF-AA standard curve in the experiment. It can be seen in the right panel that CL-MSC and WJ-MSC cultured when grown in PTT-6 produced more bFGF than when grown in PTT-4. It can be seen that all CL-MSC, WJ-MSC, and placental MSC produced lower levels of bFGF when grown in PTT-6 compared to when MSC were grown in PTT-4 or DMEM/F12.

Figure 22 : Excerpt of PDGF-AA assay. The left panel depicts the mean fluorescence intensity (MFI) measured by the PDGF-AA standard curve in the experiment. S6 represents the lowest standard used in the experiment. Any sample falling below is considered below the detection range. 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.

Figure 23 : Excerpt of Ang-1 measurement. The left panel depicts the mean fluorescence intensity (MFI) measured by the Ang-1 standard curve in the experiment. S1 represents the highest standard used in the experiment. Any sample falling above it is considered extrapolated (too concentrated). In the right panel, all CL-MSCs, WJ-MSCs, and placental MSCs produced higher levels of Ang-1 when grown in PTT-6 compared to MSCs grown in PTT-4 or DMEM/F12.

Figure 24 : Extracted HGF measurements. The left panel depicts the mean fluorescence intensity (MFI) measured by the HGF standard curve in the experiment. In the right panel, all CL-MSCs, WJ-MSCs, and placental MSCs produced higher levels of Ang-1 when grown in PTT-6 compared to MSCs grown in PTT-4 or DMEM/F12.

The following conclusions can be drawn from the above experiments. When mesenchymal stem cells, especially those isolated from the umbilical cord compartment or from the placenta, are cultured in PTT-6 medium, the secretion of factors such as angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF by the mesenchymal stem cell population is significantly increased compared to their production in PTT-4 medium or commercially available medium (such as DMEM/F12). It is worth noting that PTT-6 medium can increase the production/secretion of these factors regardless of the natural environment/mesenchymal stem cell population compartment.

Since PTT-6 medium results in the secretion of Ang-1, TGF-β1, VEGF, and HGF (whose involvement in wound healing is known as discussed herein) by all MSC populations, it is clear that PTT-6 medium has the effect of inducing or improving wound healing properties of a broad range of MSC populations, regardless of whether the natural environment/MSC population compartment was originally derived from MSCs. Again, note that Experiment 4 was conducted with cell populations that were isolated from their natural environment prior to culture in PTT-6.

In addition, culturing mesenchymal stem cells using tissue explants in PTT-6 provided a highly homogeneous mesenchymal stem cell population (containing 97.5% viable cells, 100% of which expressed each of CD73, CD90, and CD105 of the umbilical cord amnion, 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-" columns)). Since the culture of PTT-6 in the Valdennes stromal stem cell population has the same positive effect on the production of cytokines Ang-1, TGF-β1, VEGF, and HGF as the production of these cytokines in the umbilical cord lining stem cells, it can be expected that the culture of Valdennes stromal stem cells in PTT-6 will also produce this highly homogeneous population of stromal Valdennes stromal stem cells. It can also be expected that tissue explants of other umbilical cord compartments (such as the culture of the umbilical cord vessels) will produce a similarly homogeneous population of perivascular (PV) stromal stem cells. Similarly, tissue explants (including placental amniotic membrane) of placental tissue cultured in PTT-6 are expected to produce a similarly homogeneous population of placental mesenchymal stem cells. Therefore, 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 cells of the isolated mesenchymal stem cell population express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

The features of the present invention are also listed in the following items.

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

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

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

4. A method as in 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).

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

6. The method of item 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).

7. A method as in 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.

8. A method as in item 7, wherein the culture medium contains EGF at a final concentration of about 10 ng/ml.

9. A method as in any one of items 1 to 8, wherein the culture medium contains insulin at a final concentration of about 1 μg/ml to 10 μg/ml.

10. A method as in item 9, wherein the culture medium contains insulin at a final concentration of about 5 μg/ml.

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

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

13. A method as described in claim 12 or 13, wherein the culture medium comprises adenine at a final concentration of about 0.01 to about 0.1 μg/ml, hydrogenated corticosterone at a final concentration of about 0.1 to about 10 μg/ml, and/or 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng/ml.

14. A method as described in any one of items 1 to 13, wherein the mesenchymal stem cell population is cultured in a culture medium as defined in any one of items 1 to 13, relative to a reference medium that does not contain all of DMEM (Double's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (medium 171), and FBS (fetal bovine serum), so that the expression and/or secretion of at least one of angiopoietin 1 (Ang-1), TGF-β (especially TGF-β1), VEGF, and HGF of the mesenchymal stem cell population is increased.

15. The method of item 14, wherein the reference medium consists of 90% (v/v) CMRL1066 and 10% (v/v) FBS.

16. A method as in any of the preceding items, wherein the mesenchymal stem cell population is isolated from its natural environment before being cultured in the culture medium defined in any of the preceding items 1 to 13.

17. A method as in any one of items 1 to 15, comprising isolating a population of mesenchymal stem cells from a natural tissue environment by culturing the natural tissue in a cell culture medium as defined in any one of items 1 to 13 above.

18. The method of item 17, wherein the tissue is umbilical cord tissue.

19. The method of item 18, wherein the umbilical cord tissue is selected from the group consisting of whole umbilical cord tissue, tissue containing umbilical cord amnion, tissue containing Valtón's jelly, tissue containing amnion, amnion and Valtón's jelly, separated umbilical cord blood vessels, Valtón's jelly separated from other components of umbilical cord tissue, and separated umbilical cord amnion.

20. The method of item 17, wherein the tissue comprises or is placental amniotic tissue.

21. The method of any one of items 17 to 20 above, wherein the umbilical cord tissue is a piece of intact umbilical cord, a piece of umbilical cord amniotic membrane, or a piece of placental amniotic membrane.

22. A method as in any one of items 19 to 21, comprising culturing umbilical cord tissue or placental amniotic tissue until the cell growth of the amniotic mesenchymal stem cell population reaches about 70-80% confluence.

23. A method as in item 22, comprising removing the mesenchymal stem cell population from a culture container used for culture.

24. A method as in item 23, wherein the removal of the mesenchymal stem cell population from the culture container is performed by enzyme treatment.

25. The method of item 24, wherein the enzyme treatment comprises the action of trypsin.

26. A method as described in any one of items 23 to 25, wherein the mesenchymal stem cell population is transferred to a culture container for subculture for subculture.

27. A method as described in any one of items 1 to 16, wherein the mesenchymal stem cell population is transferred to a culture container for subculture for subculture.

28. The method of item 26 or 27, wherein the mesenchymal cell population is cultured in suspension or subculture at a concentration of 1.0 x 10 ⁶ cells/ml.

29. A method according to item 28, wherein the mesenchymal stem cell population is cultured in a culture medium as defined in any one of items 1 to 13.

30. A method as in item 29, wherein the mesenchymal stem cell population is subcultured until the mesenchymal stem cells reach about 70-80% confluence.

31. A method as claimed in any one of items 26 to 30, wherein the cultivation or subculture is carried out in a self-contained bioreactor.

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

33. The method of any of the preceding items, wherein the culture is carried out in a CO ₂ cell culture incubator at a temperature of 37°C.

34. A method according to claim 33, comprising removing the mesenchymal stem cell population from a culture container used for (sub-)culture.

35. A method as in item 34, wherein the removal of the mesenchymal stem cell population from the culture container is performed by enzyme treatment.

36. The method of item 35, wherein the enzyme treatment comprises the action of trypsin.

37. The method of item 36, further comprising collecting the isolated mesenchymal stem cell population.

38. A method as in 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.

39. A method as in 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).

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.

41. A method as in any of the preceding items, further comprising preserving the isolated stem/progenitor cell population for further use.

42. The method of item 41, wherein the storage is carried out by freezing.

43. An isolated mesenchymal stem cell population, wherein at least about 90% or more of the cells in the stem cell population express each of the following markers: CD73, CD90, and CD105.

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

45. A mesenchymal stem cell population as described in 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.

46. The mesenchymal stem cell population of any one of items 43 to 45, 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.

47. The mesenchymal stem cell population of any one of items 43 to 46, wherein the mesenchymal stem cell population of the umbilical cord is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Waldenjelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and a mixed umbilical cord (MC) mesenchymal stem cell population.

48. A mesenchymal stem cell population as defined in any one of items 43 to 47, wherein the population is obtained using a method defined in any one of items 1 to 42.

49. A mesenchymal stem cell population as defined in any one of items 43 to 48, wherein the population is obtained using a method defined in any one of items 1 to 42.

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

51. A pharmaceutical composition as in item 50, wherein the pharmaceutical composition is suitable for systemic or local application.

52. The pharmaceutical composition of item 50 or 51 further comprises a pharmaceutically acceptable excipient.

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

i. 250ml of DMEM

ii. 118ml M171

iii. 118ml of DMEM/F12

iv. 12.5 ml of fetal bovine serum (FBS) (final concentration is 2.5%).

54. The method of item 53, further comprising adding:

v. 1ml of EGF stock solution (5μg/ml) to achieve a final concentration of 10ng/ml

vi. 0.175ml of insulin stock solution (14.28mg/ml) to achieve a final concentration of 5μg/ml.

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

56. A method as described in claim 55, wherein the final concentration of the supplements in DMEM is as follows: about 0.05 to 0.1 μg/ml of adenine, such as about 0.025 μg/ml of adenine, about 1 to 10 μg/ml of hydrogenated corticosterone, about 0.5 to 5 ng/ml of 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3), such as 1.36 ng/ml of 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3).

57. A cell culture medium obtained by the method of any one of items 53 to 56.

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

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

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

61. A cell culture medium comprising: - 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).

62. A cell culture medium as described in item 61, wherein the culture medium comprises DMEM with a final concentration of about 57.5 to 62.5% (v/v), F12 with a final concentration of about 7.5 to 12.5% (v/v), M171 with a final concentration of about 17.5 to 25.0% (v/v), and FBS with a final concentration of about 1.75 to 3.5% (v/v).

63. A cell culture medium as described in item 62, 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).

64. A cell culture medium as in 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.

65. A cell culture medium as described in any one of items 61 to 65, wherein the culture medium contains EGF at a final concentration of about 10 ng/ml.

66. A cell culture medium as described in any one of items 61 to 65, wherein the culture medium contains insulin at a final concentration of about 1 μg/ml to 10 μg/ml.

67. A cell culture medium as in item 66, wherein the culture medium contains insulin at a final concentration of about 5 μg/ml.

68. The cell culture medium of 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).

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

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, hydrocortisone at a final concentration of about 1 to about 10 μg/ml, and/or 3,3 ' ,5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng/ml.

71. A cell culture medium as in any one of items 61 to 70, wherein 500 ml of the cell culture medium comprises:

i. 250ml of DMEM

ii. 118ml M171

iii. 118ml of DMEM/F12

iv. 12.5 ml of fetal bovine serum (FBS) (final concentration is 2.5%).

72. The cell culture medium as in item 71, further comprising:

v. Final concentration of EGF 10ng/ml

vi. Final concentration of insulin 5μg/ml

vi. 0.175ml of insulin (final concentration is 5μg/ml).

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

74. Use of a cell culture medium as defined in any one of items 61 to 73 for inducing or improving the wound healing properties of a mesenchymal stem cell population.

75. Use of a cell culture medium as defined in any one of items 61 to 73 for isolating a mesenchymal stem cell population.

76. The use of item 74 or 75, wherein the mesenchymal stem cell population is selected from the group consisting of umbilical cord mesenchymal stem cell population, placental mesenchymal stem cell population, umbilical cord blood mesenchymal stem cell population, bone marrow mesenchymal stem cell population, and adipose tissue-derived mesenchymal stem cell population.

77. The use as in 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 Walton's jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and a mixed umbilical cord (MC) mesenchymal stem cell population.

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

79. The use as in item 78, wherein at least about 90% or more of the mesenchymal stem cell population lacks expression of the following markers: CD34, CD45, and HLA-DR.

80. The use of item 79, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, 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.

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

82. A pharmaceutical composition as described in item 81, which is formulated into a liquid or a lyophilized/freeze-dried preparation.

It is obvious to those skilled in the art that various substitutions and improvements can be made to the contents disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in this specification represent the level of ordinary skill in the field to which the invention belongs. All patents and publications are hereby incorporated by reference into this case to the same extent as if each individual publication was specifically and individually incorporated by reference into this case.

The present invention is described herein in an illustrative manner and may be properly implemented in the absence of any element or component, limitation or restriction not specifically disclosed herein. Thus, for example, the terms "comprise", "include", "contain", etc. should be interpreted in a broad and non-restrictive manner. In addition, the terms and expressions used herein are used for illustrative purposes rather than limiting, and are not intended to exclude the use of any equivalents or portions of the features shown and described by those terms and expressions, but it is understood that various modifications may be made within the scope of the present invention. Therefore, it should be understood that although the present invention is specifically disclosed by preferred specific embodiments and any features, those skilled in the art may adopt improvements and variations of the present invention disclosed herein, and those improvements and variations all fall within the scope of the present invention. The present invention is described here in a broad and general manner. Each narrower species and subgeneric grouping falling within the general disclosure also constitutes a part of the present invention. This includes a general description of the invention with a proviso or negative limitation to exclude any subject matter from the genus, regardless of whether the excluded material is specifically described herein. In addition, although features or aspects of the invention are described in terms of Markush groups, it should be understood by those skilled in the art that the invention may also be described in terms of any individual member or subgroup of members of the Markush group. Further specific embodiments of the invention will become apparent from the following claims.

Claims (48)

A method for improving wound healing properties of mesenchymal stem cell populations, wherein the mesenchymal stem cell population is selected from a group consisting of a mesenchymal stem cell population of the umbilical cord, a mesenchymal stem cell population of the placenta, and a mesenchymal stem cell population of the umbilical cord-placental junction, and the method comprises culturing the mesenchymal stem cell population in a medium containing DMEM (Dulcerative Colitis Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum), wherein the mesenchymal stem cell population is separated from its natural environment before being cultured in the culture medium, wherein the culture medium comprises DMEM at a final concentration of 55 to 65% (v/v), F12 at a final concentration of 5 to 15% (v/v), M171 at a final concentration of 15 to 30% (v/v), and FBS at a final concentration of 1 to 8% (v/v), and wherein the mesenchymal stem cell population is cultured in the culture medium, relative to a medium that does not contain all of DMEM (Dulcerative Colitis Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum) reference medium, so that the expression and/or secretion of at least three of angiopoietin 1 (Ang-1), TGF-β, VEGF, and HGF of the mesenchymal stem cell population is increased. The method of claim 1, 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 Walton's jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and a mixed umbilical cord (MC) mesenchymal stem cell population. The method of claim 1 or 2, wherein the culture medium comprises DMEM at a final concentration of 57.5 to 62.5% (v/v), F12 at a final concentration of 7.5 to 12.5% (v/v), M171 at a final concentration of 17.5 to 25.0% (v/v), and FBS at a final concentration of 1.75 to 3.5% (v/v). The method of claim 3, wherein the culture medium comprises DMEM at a final concentration of 61.8% (v/v), F12 at a final concentration of 11.8% (v/v), M171 at a final concentration of 23.6% (v/v), and FBS at a final concentration of 2.5% (v/v). The method of claim 1 or 2, wherein the culture medium further comprises epidermal growth factor (EGF) at a final concentration of 1 ng/ml to 20 ng/ml. The method of claim 5, wherein the culture medium contains EGF at a final concentration of 10 ng/ml. The method of claim 1 or 2, wherein the culture medium contains insulin at a final concentration of 1 µg/ml to 10 µg/ml. The method of claim 7, wherein the culture medium contains insulin at a final concentration of 5 µg/ml. The method of claim 1 or 2, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3′,5-triiodo-L-thyronine sodium salt (T3). The method of claim 9, wherein the culture medium comprises all three of adenine, hydrocortisone, and 3,3′,5-triiodo-L-thyronine sodium salt (T3). The method of claim 10, wherein the culture medium comprises adenine at a final concentration of 0.01 to 0.1 µg/ml, hydrocortisone at a final concentration of 0.1 to 10 µg/ml, and/or 3, 3′, 5-triiodo-L-thyronine sodium salt (T3) at a final concentration of 0.5 to 5 ng/ml. The method of claim 1 or 2, wherein the TGF-β is TGF-β1. The method of claim 1, wherein the reference medium consists of 90% (v/v) CMRL1066 and 10% (v/v) FBS. The method of claim 1 or 2, wherein the mesenchymal stem cell population has been isolated from a natural tissue environment before being cultured in the culture medium of claim 1, which is achieved by culturing the natural tissue in a cell culture medium as defined in any one of claims 1 to 3 above. The method of claim 14, wherein the tissue is umbilical cord tissue. The method of claim 15, wherein the umbilical cord tissue is selected from the group consisting of whole umbilical cord tissue, tissue containing umbilical cord amnion, tissue containing Valtón jelly, tissue containing amnion, amnion and Valtón jelly, separated umbilical cord blood vessels, Valtón jelly separated from other components of umbilical cord tissue, and separated umbilical cord amnion. The method of claim 14, wherein the tissue comprises or is placental amniotic tissue. The method of claim 14, wherein the umbilical cord tissue is a piece of intact umbilical cord, a piece of umbilical cord amnion, or a piece of placental amnion. The method of claim 16, comprising culturing the umbilical cord tissue or the placental amniotic tissue until the cell growth of the amniotic mesenchymal stem cell population reaches 70-80% confluence. The method of claim 19, comprising removing the mesenchymal stem cell population from a culture container used for culture. The method of claim 20, wherein removing the mesenchymal stem cell population from the culture container is performed by enzyme treatment. The method of claim 21, wherein the enzyme treatment comprises the action of trypsin. The method of claim 20, wherein the mesenchymal stem cell population is transferred to a culture container for subculture. The method of claim 1 or 2, wherein the mesenchymal stem cell population is transferred to a culture container for subculture. The method of claim 23, wherein the mesenchymal cell population is cultured in suspension or subculture at a concentration of 1.0 x 10 ⁶ cells/ml. The method of claim 25, wherein the mesenchymal stem cell population is cultured in the culture medium as defined in claim 1. The method of claim 26, wherein the mesenchymal stem cell population is subcultured until the mesenchymal stem cells reach 70-80% confluence. The method of claim 23, wherein the culturing or subculturing is carried out in a self-contained bioreactor. The method of claim 28, wherein the bioreactor is selected from the group consisting of a parallel plate bioreactor, a hollow fiber bioreactor, and a microfluidic bioreactor. The method of claim 1 or 2, wherein the culturing is carried out in a CO ₂ cell culture incubator at a temperature of 37°C. The method of claim 30, comprising removing the mesenchymal stem cell population from a culture container for (subculture). The method of claim 31, wherein removing the mesenchymal stem cell population from the culture container is performed by enzyme treatment. The method of claim 32, wherein the enzyme treatment comprises the action of trypsin. The method of claim 33, further comprising collecting the isolated mesenchymal stem cell population. The method of claim 1 or 2, wherein at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD105. The method of claim 1 or 2, wherein at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 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). The method of claim 34, wherein 97% or more, 98% or more, or 99% or more of the isolated mesenchymal stem cells express CD73, CD90, and CD105 and lack expression of CD34, CD45, and HLA-DR. The method of claim 1 or 2, further comprising preserving the isolated mesenchymal stem cell population for further use. The method of claim 38, wherein the storage is carried out by freezing. An isolated mesenchymal stem cell population, wherein at least 97% 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 CD34, CD45, and HLA-DR, wherein the mesenchymal stem cell population is selected from a group consisting of an umbilical cord mesenchymal stem cell population and a placental mesenchymal stem cell population, and wherein the umbilical cord mesenchymal stem cell population is selected from a group consisting of a perivascular (PV) mesenchymal stem cell population, a Walton's jelly (WJ) mesenchymal stem cell population, and a mixed umbilical cord (MC) mesenchymal stem cell population. The mesenchymal stem cell population of claim 40, wherein the population is obtained using the method defined in claim 1. A pharmaceutical composition comprising an isolated mesenchymal stem cell population as defined in claim 40. A pharmaceutical composition as claimed in claim 42, wherein the pharmaceutical composition is suitable for systemic or local application. The pharmaceutical composition of claim 42 or 43, further comprising a pharmaceutically acceptable excipient. A use of a cell culture medium, wherein the culture medium comprises: - DMEM with a concentration of 55 to 65% (v/v), - F12 with a concentration of 5 to 15% (v/v), - M171 with a concentration of 15 to 30% (v/v), and - FBS with a concentration of 1 to 8% (v/v), It is used to improve the wound healing properties of a mesenchymal stem cell population, wherein the mesenchymal stem cell population is separated from its natural environment before being cultured in the culture medium, wherein the mesenchymal stem cell population is selected from a group consisting of a mesenchymal stem cell population of the umbilical cord and a mesenchymal stem cell population of the placenta, and wherein the mesenchymal stem cell population of the umbilical cord is selected from a group consisting of a perivascular (PV) mesenchymal stem cell population, a WJ mesenchymal stem cell population, and a mixed umbilical cord (MC) mesenchymal stem cell population, wherein the mesenchymal stem cell population is cultured in the culture medium, relative to a medium without all DMEM (Dulbecco's modified Eagle's medium), F12 (Ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum) reference medium, so that the expression and/or secretion of at least three of angiopoietin 1 (Ang-1), TGF-β, VEGF, and HGF of the mesenchymal stem cell population is increased. The use of claim 45, wherein the culture medium comprises DMEM at a concentration of 57.5 to 62.5% (v/v), F12 at a concentration of 7.5 to 12.5% (v/v), M171 at a concentration of 17.5 to 25.0% (v/v), and FBS at a concentration of 1.75 to 3.5% (v/v). The use of claim 46, wherein the culture medium comprises DMEM at a concentration of 61.8% (v/v), F12 at a concentration of 11.8% (v/v), M171 at a concentration of 23.6% (v/v), and FBS at a concentration of 2.5% (v/v). A use of a cell culture medium, wherein the cell culture medium comprises: - DMEM with a concentration of 55 to 65% (v/v), - F12 with a concentration of 5 to 15% (v/v), - M171 with a concentration of 15 to 30% (v/v), and - FBS with a concentration of 1 to 8% (v/v), for isolating a mesenchymal stem cell population, wherein the mesenchymal stem cell population is selected from a group consisting of a mesenchymal stem cell population of the umbilical cord and a mesenchymal stem cell population of the placenta, The umbilical cord mesenchymal stem cell population is selected from a group consisting of a perivascular (PV) mesenchymal stem cell population, a Walton's jelly (WJ) mesenchymal stem cell population, and a mixed umbilical cord (MC) mesenchymal stem cell population.