CN112262211A - Method for inducing or improving wound healing property 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 culturing the mesenchymal stem cell population in a medium comprising DMEM (dulbecco modified eagle medium), F12 (ham F12 medium), M171 (medium 171) and FBS (fetal bovine serum). The present invention also relates to a population of mesenchymal stem cells, wherein at least about 90% or more of the cells of the population 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 the mesenchymal stem cell population.

Description

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

Cross reference to related patent applications

This application claims priority to U.S. provisional application No.62/656,531 filed on 12.4.2018, the contents of which are incorporated herein by reference in their entirety for all purposes.

Technical Field

The present invention relates to a method of inducing or improving wound healing properties of a mesenchymal stem cell population. The invention also relates to a cell culture medium suitable for inducing or improving wound healing properties of mesenchymal stem cells and/or for isolating a population of mesenchymal stem cells. The invention also relates to a pharmaceutical composition and application of the separated mesenchymal stem cell population. The present invention also relates to a method of treating a disease or disorder, the method comprising administering to a subject in need thereof a mesenchymal stem cell population of the invention or a pharmaceutical composition comprising such a mesenchymal stem cell population. The invention also relates to a population of highly homogeneous (homogenes) and well-defined mesenchymal stem cells, such as the umbilical cord or placenta.

Background

Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord were first reported in U.S. patent application 2006/0078993 (granted U.S. patents 9,085,755, 9,737,568 and 9,844,571) and in the corresponding international patent application WO 2006/019357. Since then, umbilical cord tissue has received much attention as a source of pluripotent cells; due to the wide availability of umbilical cords, and in particular stem cells isolated from the amniotic membrane of the umbilical cord (also known as "cord lining stem cells"), have been considered as an excellent alternative source of cells for regenerative medicine. See Jeschke et al, U.S. Cord Lining membranes and Wharton's Jelly-Derived Mesenchyl Stem Cells, the Siliaries 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 immunoregulatory capacity of Human Mesenchymal STEM Cells (MSC) Derived from the amniotic membrane of umbilical cord (cord lining (CL-MSC), umbilical cord blood (CB-MSC), placenta (P-MSC), AND Wharton's jelly (WJ-MSC)) (Stubbendaorf et al, Immunological Properties of extracellular Human sensory cell Derived from Tissue, STEM CELLS AND DEVOPMENT Volume 22, Number 19,2013, 2619-. Stubbbendarf et al concluded that the extra-embryonic gestational tissue-derived MSC population showed different potential to evade immune responses and exert immunomodulatory effects. The authors also found that since CL-MSCs show low immunogenicity, they show the most promising potential for cell-based therapies, but they also show enhanced proliferation and migration potential, future studies should focus on the best disease model in which CL-MSCs can be administered.

Although amniotic mesenchymal stem cells may be readily obtained using the protocol as described in us patent application 2006/0078993 and international patent application WO2006/019357, it would be advantageous for clinical trials using these umbilical cord-lined MSCs to have a method that allows isolation of populations of these umbilical cord-lined MSCs that are highly homogeneous and therefore capable of being used in clinical trials. Furthermore, it would be advantageous to have a method of inducing or improving the wound healing properties of a general mesenchymal stem cell population.

Accordingly, it is an object of the present invention to provide a method of inducing or improving wound healing properties of a mesenchymal stem cell population. Another object is to isolate a population of mesenchymal stem cells from the amniotic membrane of umbilical cord that meets this need. It is therefore a further object of the present invention to provide a highly homogeneous population of mesenchymal stem cells.

Disclosure of Invention

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

In a first aspect, the present invention provides a method of inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a medium comprising DMEM (dulbecco modified eagle medium), F12 (hamm F12 medium), M171 (medium 171) and FBS (fetal bovine serum). The mesenchymal stem cell population may be a umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, a cord blood mesenchymal stem cell population, a bone marrow mesenchymal stem cell population or a adipose-tissue derived mesenchymal stem cell population.

In a second aspect, the present invention provides an isolated population of mesenchymal stem cells, wherein at least about 90% or more of the cells of the population express each of the following markers: CD73, CD90, and CD 105. Preferably, the isolated population of mesenchymal stem cells lacks expression of the following markers: CD34, CD45 and HLA-DR. In an embodiment of this second aspect, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated population of mesenchymal stem cells express each of CD73, CD90 and CD 105. Furthermore, in these embodiments of the second aspect, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated population of mesenchymal stem cells lack expression of the markers CD34, CD45 and HLA-DR. The mesenchymal stem cell population may be obtained by the method of inducing or improving wound healing properties of the first aspect. Thus, the method of the first aspect may also be a method of isolating a population of mesenchymal stem cells.

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

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

i.250ml of DMEM

ii.118ml M171

iii.118ml DMEM/F12

iv.12.5ml Fetal Bovine Serum (FBS) to give a final concentration of 2.5% (v/v).

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

In a sixth aspect, the present invention provides a method of isolating a population of mesenchymal stem cells, the method comprising culturing the population of mesenchymal stem cells in a medium prepared by the method of the fourth aspect.

In a seventh aspect, the present invention provides 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).

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

Drawings

The invention will be better understood by reference to the detailed description of the invention when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

FIG. 1 shows a technical information table of Dulbecco's modified eagle medium (Dulbecco's modified eagle medium) of Lonza, which includes the product catalog number of DMEM used for preparing an exemplary example of the medium of the present invention (PTT-6) in the experimental section;

FIG. 2 shows a technical information table of the Ham F12 medium (Ham's F12 medium) from Lonza;

FIG. 3 shows a technical information table of the DMEM: F12(1:1) medium from Lonza, which includes the catalog number of the DMEM: F12(1:1) medium used to prepare an illustrative example of the medium of the present invention (PTT-6) in the experimental section;

FIG. 4 shows a technical information table of M171 medium from Life Technologies Corporation, which includes the product catalog number of M171 medium used to prepare an illustrative example of the inventive medium (PTT-6) in the experimental section;

FIG. 5 shows a list of ingredients, including commercial supplier and catalog numbers for the ingredients used to prepare PTT-6 medium in the experimental section;

fig. 6A-C show the results of flow cytometry experiments in which mesenchymal stem cells isolated from umbilical cord were analyzed for expression of mesenchymal stem cell markers CD73, CD90, and CD 105. For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different media, followed by subculturing of the mesenchymal stem cells in the respective media. In these experiments, the following three media were used: a) 90% (v/v) DMEM supplemented with 10% FBS (v/v), b) PTT-4 medium described in U.S. patent application US 2008/0248005 and corresponding international patent application WO2007/046775, consisting of 90% (v/v) CMRL1066 and 10% (v/v) FBS (see paragraph [0183] of WO2007/046775) and c) PTT-6 medium of the present invention, the composition of which is described herein. In this flow cytometry analysis, two different samples of umbilical cord lining mesenchymal stem cell (CLMC) populations were analyzed in each of the three media employed. The results are shown in fig. 6A to 6C.

In more detail, fig. 6A shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in DMEM/10% FBS, fig. 6B shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in PTT-4, and fig. 6C shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in PTT-6.

Fig. 7A-B show the results of flow cytometry experiments in which mesenchymal stem cells isolated from umbilical cord were analyzed for expression of stem cell markers (CD73, CD90 and CD105, CD34, CD45 and HLA-DR (human leukocyte antigen-antigen D-associated) defining the suitability of pluripotent human mesenchymal stem cells for use in a cytotherapeutic approach) and compared to expression of these markers for bone marrow mesenchymal stem cells. For this experiment, mesenchymal stem cells of the amniotic membrane of umbilical cord were isolated from umbilical cord tissue by culturing the umbilical cord tissue in the PTT-6 medium of the present invention, and the mesenchymal stem cells were isolated from human bone marrow using standard procedures.

Figure 7A shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 and lacking expression of CD34, CD45 and HLA-DR after isolation from umbilical cord tissue and culture in PPT-6 medium, while figure 7B shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90 and CD105 and lacking expression of CD34, CD45 and HLA-DR.

FIG. 8 shows an experimental setup in which the dark grey wells, standards reconstituted with PTT-4 medium and corresponding samples from MSCs cultured in PTT-4; light grey wells, standards reconstituted with PTT-6 medium and corresponding samples from MSCs cultured in PTT-6. The italicized labeled sample was the control supernatant, which was assayed as part of the stored sample replicate assay.

Figure 9 shows a singleplex measurement of TGF β 1. It can be seen that cultures CL-MSC and WJ-MSC produced more TGF β 1 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 cultures produced approximately equal amounts of TGF β 1. All error bars are standard deviations of three measurements.

FIG. 10A shows multiplex measurements of PDGF-AA. It can be seen that cultures CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produced more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of three measurements.

Figure 10B shows multiple measurements of VEGF. It can be seen that culture CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produced more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three measurements.

FIG. 10C shows multiple measurements of Ang-1. It can be seen that culture CL-MSC and WJ-MSC cultures produced much more Ang-1 when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC do not produce substantially any Ang-1. All error bars are standard deviations of three measurements.

Fig. 11 shows multiple measurements of HGF. It can be seen that cultures CL-MSC and WJ-MSC cultures produced much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC produced essentially no HGF. All error bars are standard deviations of three measurements.

FIG. 12 shows multiplex measurements of PDGF-AA. It can be seen that culture CL-MSC and WJ-MSC cultures produced more PDGF-AA when grown in PTT-4 than when grown in PTT-6. Cultures AT-MSC and BM-MSC produced equal amounts of PDGF-AA in both media. All error bars are standard deviations of three measurements.

Figure 13A shows multiple measurements of VEGF. It can be seen that culture CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produced more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three measurements.

FIG. 13B shows multiple measurements of Ang-1. It can be seen that culture CL-MSC and WJ-MSC cultures produced much more Ang-1 when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC do not produce substantially any Ang-1. All error bars are standard deviations of three measurements.

Fig. 13C shows multiple measurements of HGF. It can be seen that cultures CL-MSC and WJ-MSC cultures produced much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC produced essentially no HGF. All error bars are standard deviations of three measurements.

Fig. 14 shows multiplex measurements of bFGF. As can be seen, cultures of CL-MSC and WJ-MSC produced more bFGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC produced equal amounts of bFGF when cultured in PTT-4 and PTT-6. All error bars are standard deviations of three measurements.

Figure 15 summarizes TGF β 1 measurements for 5 different experiments (170328, 170804, 170814, 180105, 180226). The Mean Fluorescence Intensity (MFI) of the TGF β standard curve measured for each experiment is depicted in the lower left panel. The MFI of the TGF-beta standard curves obtained in PTT-4 and PTT-6 medium are shown in the upper panel. The lower right panel depicts that cultures CL-MSC and WJ-MSC produce more TGF β 1 when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produce equal amounts of TGF-1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from different measurements for experiments 170328, 170804, 170814, 180105, 180226.

Fig. 16 summarizes Ang-1 measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The Mean Fluorescence Intensity (MFI) of the Ang-1 standard curve measured for each experiment is depicted in the lower left panel. The MFI of the Ang-1 standard curves obtained in PTT-4 and PTT-6 medium is shown in the upper panel. The lower right panel depicts that cultures CL-MSC and WJ-MSC produced more Ang-1 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 cultures produced substantially equal amounts of Ang-1. All error bars are standard deviations from different measurements for experiments 170602, 170511, 170414, 170224, 180105, 180226.

FIG. 17 summarizes PDGF-BB measurements from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The Mean Fluorescence Intensity (MFI) of the PDGF-BB standard curve measured for each experiment is depicted in the lower left panel. The MFI of the PDGF-BB standard curves obtained in PTT-4 and PTT-6 medium are shown in the upper panel. Notably, PDGF-BB was not detected in all experiments.

FIG. 18 summarizes PDGF-AA measurements from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The Mean Fluorescence Intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the lower left panel. The MFI of the PDGF-AA standard curves obtained in PTT-4 and PTT-6 medium are shown in the upper panel. The lower right panel depicts cultures CL-MSC, AT-MSC and BM-MSC, and WJ-MSC cultures produce slightly more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of the measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

FIG. 19 summarizes IL-10 measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The Mean Fluorescence Intensity (MFI) of the IL-10 standard curve measured for each experiment is depicted in the lower left panel. The MFI of the IL-10 standard curves obtained in PTT-4 and PTT-6 medium is shown in the upper panel. Notably, IL-10 was not detected in all experiments.

Figure 20 summarizes VEGF measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The Mean Fluorescence Intensity (MFI) of the VEGF standard curve measured for each experiment is depicted in the lower left panel. The MFI of the VEGF standard curves obtained in PTT-4 and PTT-6 medium are shown in the upper panel. The lower right panel depicts cultures CL-MSC, AT-MSC and BM-MSC, and WJ-MSC produce more VEGF when grown in PTT-6 than in PTT-4. All error bars are standard deviations of different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

Fig. 21 summarizes HGF measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226). The Mean Fluorescence Intensity (MFI) of the HGF standard curve measured for each experiment is depicted in the lower left panel. MFI of HGF standard curves obtained in PTT-4 and PTT-6 media are shown in the upper panel. The lower right panel depicts cultures CL-MSC and WJ-MSC produce more HGF when grown in PTT-6 than in PTT-4. On the other hand, cultures AT-MSC and BM-MSC did not produce as much HGF as other cultures. All error bars are standard deviations of different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

FIG. 22: singleplex measurement of TGF β 1. The Mean Fluorescence Intensity (MFI) of the TGF β 1 standard curve measured for each experiment is depicted in the left panel. As can be seen from the right panel, all CL-MSCs, WJ-MSCs and placental MSCs produced more TGF β 1 when grown in PTT-6 than when grown in PTT-4 or DMEM/F12 (referred to as DMEM only in FIG. 22).

FIG. 23 summarizes the assayed PDGF-BB measurements in the supernatants of CL-MSCs, WJ-MSCs and placental MSCs cultured in PTT-6, PTT-4 or DMEM/F12. The Mean Fluorescence Intensity (MFI) of the PDGF-BB standard curve measured for each experiment is depicted in the left panel. Notably, PDGF-BB was not detected in all experiments.

FIG. 24 summarizes the measurements of IL-10 in supernatants of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM/F12 that were analyzed. The Mean Fluorescence Intensity (MFI) of the VEGF standard curve measured for each experiment is depicted in the left panel. S6 represents the minimum standard used in the determination. Any sample below this is considered to be below the detection limit. As can be seen from the right panel, all CL-MSCs, WJ-MSCs and placental MSCs produced detectable levels of IL-10 when grown in PTT-6, whereas little or no IL-10 was detected when MSCs were grown in PTT-4 or DMEM/F12.

FIG. 25 summarizes the measurements of VEGF in the supernatants of CL-MSCs, WJ-MSCs and placental MSCs cultured in PTT-6, PTT-4 or DMEM/F12 that were analyzed. The Mean Fluorescence Intensity (MFI) of the VEGF standard curve measured for each experiment is depicted in the left panel. S1 represents the highest standard used in the determination. Any sample above this is considered to be speculative (over-concentrated). As can be seen from the right panel, all CL-MSCs, WJ-MSCs and placental MSCs produced much higher levels of VEGF when grown in PTT-6 than when MSCs were grown in PTT-4 or DMEM/F12.

Fig. 26 summarizes the multiplex measurements of bFGF. The Mean Fluorescence Intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the left panel. As can be seen from the right panel, cultured CL-MSCs and WJ-MSCs produce more bFGF when grown in PTT-6 than in PTT-4. It can be seen that all CL-MSCs, WJ-MSCs and placental MSCs produced much lower levels of bFGF when grown in PTT-6 than when MSCs were grown in PTT-4 or DMEM/F12.

FIG. 27 summarizes PDGF-AA measurements. The Mean Fluorescence Intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the left panel. S6 represents the minimum standard used in the determination. Any sample below this is considered to be below the detection limit. It can be seen that all CL-MSCs, WJ-MSCs and placental MSCs produced higher levels of PDGF-AS when grown in PTT-6 than when MSCs were grown in PTT-4 or DMEM/F12.

FIG. 28 summarizes the measurement of Ang-1. The Mean Fluorescence Intensity (MFI) of the Ang-1 standard curve measured for each experiment is depicted in the left panel. S1 represents the highest standard used in the determination. Any sample above this is considered to be speculative (over-concentrated). The right panel depicts that all CL-MSCs, WJ-MSCs and placental MSCs produce much higher levels of Ang-1 when grown in PTT-6 than MSCs when grown in PTT-4 or DMEM/F12.

Fig. 29 summarizes HGF measurements. The Mean Fluorescence Intensity (MFI) of the HGF standard curve measured for each experiment is depicted in the left panel. The right panel depicts that all CL-MSCs, WJ-MSCs and placental MSCs produce much higher levels of Ang-1 when grown in PTT-6 than MSCs when grown in PTT-4 or DMEM/F12.

Detailed Description

As mentioned above, in a first aspect, the present invention relates to a method of inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a medium comprising DMEM (dulbecco modified eagle medium), F12 (hamm F12 medium), M171 (medium 171) and FBS (fetal bovine serum). It has surprisingly been found in the present application that the use of such a medium has the effect of inducing or improving the wound healing properties of a broad population of mesenchymal stem cells, irrespective 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 the mesenchymal stem cell population is caused by the ability of the culture medium of the present invention to increase expression and/or secretion of at least one, two, three or all four of angiopoietin 1(Ang-1), TGF- β 1, VEGF and HGF from the mesenchymal stem cell population. See experimental section which shows that the mesenchymal stem cell population of umbilical cord amniotic membrane cultured in the PTT-6 medium of the present invention expresses and/or secretes angiopoietin 1(Ang-1), TGF- β 1, VEGF and HGF increased relative to the culture of such mesenchymal stem cell population in the medium (PTT-4), which medium (PTT-4) has been used in U.S. patent application US 2008/0248005 and in the corresponding international patent application WO2007/046775 for the isolation of umbilical cord amniotic membrane mesenchymal stem cell population, which was shown to have excellent wound healing properties in U.S. patent application US 2008/0248005 and in international patent application WO2007/046775 (see examples 23-26 of WO2007/046775 which shows that such umbilical cord amniotic mesenchymal stem cell population (UCMC) alleviates full-layer burns (example 23), partial cortical wounds (example 24), non-healing radiation wounds (example 25) and non-healing diabetic wounds and non-healing diabetic foot wounds (example 26)). As shown in the experimental section herein, culturing in media containing DMEM (dulbecco modified eagle medium), F12 (ham F12 medium), M171 (medium 171) and FBS (fetal bovine serum) increased the amount of angiopoietin 1(Ang-1), TGF- β 1, VEGF and HGF in not only the umbilical cord amniotic mesenchymal stem cell population but also mesenchymal stem cell populations of other compartments of the umbilical cord (such as walton's jelly) or (adjacent) compartments (such as placenta). It is therefore believed that the present application provides a universal teaching of inducing or improving wound healing properties of a given mesenchymal stem cell population by culturing a mesenchymal stem cell population in a culture medium of the present invention, such as medium PTT-6.

In this context, the finding of the present invention is that a combined increase in the amount of Ang-1, TGF- β 1, VEGF and/or HGF produced by a population of mesenchymal stem cells will improve or will improve the wound healing properties of such a population of stem cells, and that by a composition/solution containing three or four of Ang-1, TGF- β 1, VEGF or HGF as the sole wound healing proteins, also a simulation of the wound healing properties of the stem cell population is opened.

In this context, it is noted that the involvement of angiopoietin 1(Ang-1), TGF-. beta.1, VEGF and HGF proteins in the wound healing process is known to those skilled in the art. For the involvement of angiogenin 1 in wound healing, see, for example, Li et al, Stem Cell Research & Therapy 2013,4:113, "sensory Stem cells modified with genetic engineering of genetic engineering around and health" or Bitto et al, "angioengineering-1 gene transfer around and health of the genetic diagnostic with creating VEGF expression", Clinical Science May 14,2008,114 (707) 718. In the studies of Li et al, the angiopoietin-1gene was inserted into bone marrow mesenchymal stem cells, and the results indicate "Ang 1-MSC significantly improved wound healing with increased epidermal and dermal regeneration and enhanced angiogenesis compared to MSC, Ad-Ang1, or pseudo-therapy". Notably, Li et al authors mention that Mesenchymal Stem Cells (MSCs) alone do not produce sufficient Ang-1, and for this reason, authors insert the Ang 1-gene into MSCs to produce genetically modified cells. In contrast to Li studies, the present application has surprisingly found that culturing "native" mesenchymal stem cells in a medium such as PTT-6 can provide conditions under which, for example, umbilical cord tissue mesenchymal stem cells (i.e., a population of mesenchymal stem cells cultured in PTT-6) produce increased levels of Ang-1 and thus render the mesenchymal stem cells suitable for use in wound healing or further improve their wound healing properties. This means that the present invention provides the advantage that instead of genetically modifying naturally occurring mesenchymal stem cells to induce wound healing properties in the mesenchymal stem cells (which is not only laborious, but also not a preferred option for therapeutic applications due to the inherent risks of gene therapy), the wound healing properties of naturally occurring mesenchymal stem cells are induced or enhanced by "simply" culturing a population of mesenchymal stem cells in a culture medium of the present invention. The method is simpler, safer and more cost-effective.

For the involvement of other proteins such as Hepatocyte Growth Factor (HGF) in wound Healing, particularly in the Healing of chronic/non-Healing wounds, see, e.g., Yoshida et al, "differentiation of regenerative Growth Factor Leads to regulated wound Healing Associated with secreted wound Healing and Granulation Tissue formulation" J.invest.Dermatol.120: 335-; li, Jin-Feng et al, "HGF additives round Hearing by Promoting the differentiation of epidemic Cells through β 1-Integrain/ILK pathway," BioMed Research International 2013(2013), 470418 or Conway et al, "Hepatocyte growth factor regulation: An integral part of our roads brand cyclic". Wound Reg (2007) 15683-.

For The involvement of Vascular Endothelial Growth Factor (VEGF) in Wound Healing, particularly in The Healing of chronic/non-Healing wounds, see, e.g., 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.2009May 15; 153(2):347-358.

For The involvement of Transforming Growth Factor Beta (including TGF-. beta.1, TGF-. beta.2, and TGF-. beta.3) In Wound Healing, particularly In The Healing of chronic/non-Healing wounds, see, e.g., Ramirez et al, "The Role of TGFb signalling In Wound Healing" Advances In Wound Healing Care, Volume 3, Number 7,2013, 482. 491 or Pakyari et al, Critical Role of transformation Growth Factor Beta In Difference phase of Wound Healing, Advances In Wound Healing 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 culturing in the culture medium of the present invention provides for the isolation of a population of mesenchymal stem cells, such as a population of umbilical cord amniotic mesenchymal stem cells, in which greater than 90% or even 99% or more of the cells are positive for the three mesenchymal stem cell markers CD73, CD90 and at the same time these stem cells lack the expression of CD34, CD45 and HLA-DR (see experimental part), meaning that 99% or even more of the cells of the population express the stem cell markers CD73, CD90 and CD105 and do not express the markers CD34, CD45 and HLA-DR. Such a highly homogeneous and well-defined cell population is an ideal candidate for clinical trials and cell-based therapies, as it for example fully complies with the generally accepted standards for human mesenchymal stem cells for cell therapy, for example defined by: dominici et al, "minor criteria for defining porous structural cells, the International Society for Cellular Therapy status", Cytotherapy (2006) Vol.8, No.4, 315-. In addition, using a bioreactor such as a Quantum Cell Expansion System, a large number of mesenchymal stem cells, such as 3 to 7 billion mesenchymal stem cells per run, can be obtained (see also experimental section). Thus, the present invention provides the further advantage of providing the number of stem cells required for therapeutic applications, such as use in wound healing, in a cost-effective manner. Furthermore, all components used to prepare the media of the invention are commercially available in GMP quality. Therefore, the present invention opens the way for GMP production of a highly homogeneous mesenchymal stem cell population, such as a mesenchymal stem cell population of placenta tissue or umbilical cord tissue, such as a mesenchymal stem cell population of umbilical cord amnion or a mesenchymal stem cell population of Wharton's jelly.

The mesenchymal stem cell population that is made suitable for wound healing (by inducing wound healing properties or by improving wound healing properties in a population that has no wound healing properties prior to being subjected to the culture process of the present invention) may be any suitable mesenchymal stem cell known in the art, for example, an adult stem cell population or a neonatal stem cell. The population of mesenchymal stem cells may be derived from any mammalian tissue or compartment/body part known to contain mesenchymal stem cells. In illustrative examples, the population of mesenchymal stem cells may be a population of umbilical cord mesenchymal stem cells (these are examples of neonatal stem cells), a population of placental mesenchymal stem cells (another example of neonatal stem cells), a population of mesenchymal stem cells at the umbilical cord-placental junction (another example of a population of neonatal stem cells), a population of umbilical cord blood mesenchymal stem cells (yet another example of neonatal stem cells), a population of bone marrow mesenchymal stem cells (which may be a population of adult stem cells), or a population of adipose-derived mesenchymal stem cells (yet another example of a population of adult stem cells).

The umbilical cord mesenchymal stem cell population may be derived from any compartment of 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 Wharton's Jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord Mixed (MC) mesenchymal stem cell population, which means a mesenchymal stem cell population comprising stem cells of two or more of these compartments. Mesenchymal stem cells of these compartments and isolation therefrom are known to the person skilled in the art and are described by way of example by: subramanian et al, "synthetic Characterization of Cells from the values components of the Human inner skin by the wheel's Jelly components of the Best resources of the clinical non-viable therapeutic Stem Cells", PLoS ONE 10(6), e0127992,2015 and references cited therein, Van Pham et al, "Isolation and promotion of a biological code Tissue derived biological Stem Cells for clinical applications", Tissue Bank (2016)17:289 Cells 302, 2016. The umbilical cord mixed mesenchymal stem cell population may be obtained by, for example, removing arteries and veins of umbilical cord tissue, fragmenting the remaining tissue and Wharton's jelly, and culturing the umbilical cord tissue (tissue isolate) in the culture medium of the present invention. Also by the methods described in Schugar et al, "High harvest yield, High expansion, and phenotyp stability of CD146 sensory bacterial from cellular private human biological sample tissue, journal of biomedicine & biological technology, 2009; 2009:789526 "(cultured in serum-supplemented DMEM with 10% fetal bovine serum, 10% horse serum and 1% penicillin/streptomycin) whole umbilical cord tissue with intact umbilical cord blood vessels as tissue-separated pieces was cultured to obtain umbilical cord mixed mesenchymal stem cell populations. In this context, it should be noted that the expression of "J Vis exp.2017" by, for example, Beravoluu et al, "Isolation and Characterization of Mesenchyl structural Cells from Human Umbilical Cord and Fetal plant. (122) 55224 it is described that mesenchymal stem cell populations can be isolated at the umbilical cord-placental junction.

In light of the above, it is noted herein that the mesenchymal stem cell population cultured in the medium comprising DMEM (dulbecco modified eagle medium), F12 (ham F12 medium), M171 (medium 171) and FBS (fetal bovine serum) to induce or improve its wound healing properties in the present invention may be isolated from its natural environment before being cultured in the medium of the present invention. Such methods are particularly useful for mesenchymal stem cell populations that are not readily isolatable by tissue isolates, such as umbilical cord blood mesenchymal stem cell populations or bone marrow mesenchymal stem cell populations. However, this method may also be used for umbilical cord mesenchymal stem cell populations, placental mesenchymal stem cell populations, or adipose-tissue derived mesenchymal stem cell populations. Such stem cell populations, such as the population Of Wharton's Jelly mesenchymal stem Cells, may be first isolated as described by Subramanian et al, 2015, PLoS ONE (supra) or International patent application WO 2004/072273, "Progenitor Cells From Wharton's Jelly Of Human Umbilical Cord" above, and then cultured in the medium Of the present invention comprising DMEM (Dulbecco's modified eagle's medium), F12 (Hamm F12 medium), M171 (medium 171) and FBS (fetal bovine serum). It is also possible, for example, as described in European patent application EP 1288293, Talwadekar et al, "replacement and preservation of Cord Tissue MSCs with Cord Blood AB Plasma" Biomed Res J2014; 126-; (122) 55224 a population of placental mesenchymal stem cells is isolated from a placenta and subsequently cultured in the medium of the present invention as described. Similarly, it can be as described in Schneider et al, "adopsoe-derived sensory stem cells from free sexual activity and recovered fast area reactive sources" Eur J Med Res.2017; 22:17 adipose-tissue derived mesenchymal stem cell populations were isolated as described in the references cited therein and subsequently cultured in the media of the present invention (see also experimental section). As another illustrative example, one may also first look like Beralvolumu et al, "Isolation and Characterization of Mesenchyl structural Cells from Human Umbilical Cord and Fetal plant," J Vis exp.2017; (122) 55224 a population of mesenchymal stem cells at the umbilical cord-placental junction is isolated and subsequently cultured in the medium of the invention.

Alternatively, and particularly for mesenchymal stem cells that can be isolated using tissue isolates, a population of mesenchymal stem cells can be isolated directly from its native tissue environment by culturing native tissue in a cell culture medium of the invention. Such methodologies are particularly useful for culturing a population of mesenchymal stem cells from umbilical cord tissue, placental tissue (which may, for example, comprise or be placental amniotic membrane), or umbilical cord-placental junction.

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

In one embodiment, the culture medium of the invention allows isolation of a mesenchymal stem cell population from an amniotic membrane under conditions that allow for proliferation of mesenchymal stem/progenitor cells without differentiation of said mesenchymal stem/progenitor cells. Thus, following isolation of the mesenchymal stem cells from amniotic membrane as described herein, the isolated mesenchymal stem/progenitor cell population has the ability to differentiate into a variety of cell types, for example as described in us patent application 2006/0078993, us patent 9,085,755, international patent application WO2006/019357, us patent 8,287,854 or WO 2007/046775. For example, as described in U.S. patent application 2006/0078993, umbilical cord amniotic mesenchymal stem cells have a fusiform shape, expressing the following genes: POU5f1, Bmi-1, Leukemia Inhibitory Factor (LIF), and secretes 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, tenocytes, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, adipocytes, mucin-producing cells, cells derived from endocrine glands such as insulin-producing cells (e.g., β -islet cells) or neuroectodermal cells. The isolated stem cells of the present invention may be differentiated in vitro for subsequent use of the differentiated cells for medical purposes. An illustrative example of such a method is the differentiation of mesenchymal stem cells into insulin-producing beta-islet cells, which can then be administered to a patient suffering from an insulin deficiency, such as diabetes, for example by implantation (see also WO2007/046775 in this respect). Alternatively, the mesenchymal stem cells of the invention may be used in their undifferentiated state for cell-based therapy, for example, for wound healing purposes, such as for the treatment of burns or chronic diabetic wounds. In these therapeutic applications, the mesenchymal stem cells of the invention can either promote wound healing by interacting with the surrounding diseased tissue or differentiate into corresponding skin cells (see for example again WO 2007/046775).

In light of the above disclosure, it is noted herein that such mesenchymal stem cell populations described herein may be isolated and cultured from (i.e., derived from) any umbilical cord tissue, so long as the umbilical cord tissue contains an amniotic membrane (also referred to as "cord lining"). Thus, as described in the experimental part of the present application, the mesenchymal stem cell population may be isolated from (a part of) the whole umbilical cord. Thus, in addition to the amniotic membrane, the umbilical cord tissue may comprise any other tissue/component of the umbilical cord. For example, as shown in figure 16 of U.S. patent application 2006/0078993 or international patent application WO2006/019357, the amniotic membrane of the umbilical cord is the outermost portion of the umbilical cord, covering the umbilical cord. In addition, the umbilical cord contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-depleted blood away from the fetus). For protection and mechanical support, these three vessels are embedded in gordonia gel, a gelatinous substance made primarily of mucopolysaccharides. Thus, umbilical cord tissue for use in the present invention may also include the one vein, the two arteries, and Wharton's jelly. The use of such a whole (intact) part of the umbilical cord has the following advantages: the amniotic membrane need not be separated from the other components of the umbilical cord. This reduces the separation steps and thus makes the method of the invention simpler, faster, less prone to error and more economical-all of which are important aspects of GMP production required for mesenchymal stem cell therapeutic applications. Thus, isolation of mesenchymal stem cells may begin by tissue isolation bulk (tissue explantation), and if larger quantities of mesenchymal stem cells are required, e.g. for clinical trials, the isolated mesenchymal stem cells may be subsequently subjected to subsequent subculture (culture). Alternatively, the amniotic membrane may be first isolated from the other components of the umbilical cord, and then the mesenchymal umbilical cord lining stem cells may be isolated from the amniotic membrane by culturing the amniotic membrane in the medium of the present invention. The culturing may also be performed by tissue isolation block method, optionally followed by subculturing of the isolated mesenchymal stem cells.

In this context, the term "tissue isolate" or "tissue isolate method" is used in its conventional meaning in the art and refers to a method in which once a tissue (e.g., placental tissue or umbilical cord tissue) is harvested, the tissue or a portion of the tissue is placed in a cell culture dish containing culture (growth) medium and, over time, stem cells migrate from the tissue to the surface of the dish. These primary stem cells can then be further expanded by micropropagation (subculture) as described herein and transferred to fresh dishes. In this context, it should be noted that in terms of producing cells for therapeutic purposes, in the first step of isolating/obtaining the mesenchymal stem cell population of the present invention (e.g., umbilical cord mesenchymal stem cells such as amniotic membrane or Wharton's jelly mesenchymal stem cells), a master cell bank of the isolated mesenchymal stem cells is obtained, whereas in subsequent subcultures a working cell bank may 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 the expression of each of the markers CD34, CD45 and HLA-DR) is used in clinical trials or as an approved therapeutic agent, the cell population of the working cell bank is typically used for this purpose. The stem cell population of the isolation step (which may constitute a master cell bank) and the stem cell population of the subculture step (which may constitute a working cell bank) may each be stored, for example, in a cryopreserved form.

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

Herein, "inducing or improving the wound healing properties of the mesenchymal stem cell population" means the ability of the culture medium to increase or initiate (induce) the expression and/or secretion of at least one of Ang-1, TGF- β 1, VEGF and HGF proteins by the mesenchymal stem cell population. As mentioned previously, all four of these proteins are known to be involved in wound healing. The "inducing or improving wound healing properties" is evaluated relative to culturing a population of mesenchymal stem cells in a reference (culture) medium such as: the medium PTT-4 (consisting of 90% (v/v) CMRL1066 and 10% (v/v) FBS) which has been used in US patent application US 2008/0248005 and in the corresponding international patent application WO2007/046775 for the isolation and culture of umbilical cord amniotic mesenchymal stem cell populations, which have been shown to have excellent wound healing properties in US patent application US 2008/0248005 and in international patent application WO 2007/046775. When cultured in the medium of the present invention, the population of mesenchymal stem cells will secrete at least one of the four marker proteins Ang-1, TGF- β 1, VEGF and HGF in greater amounts (corresponding to higher secretion levels or higher concentrations) into the supernatant/medium than the population of mesenchymal stem cells cultured in the reference medium, with consequent increase in wound healing properties of the population of mesenchymal stem cells. Wound healing properties of the stem cell population are induced if during the culturing in the reference medium, it is observed that the mesenchymal stem cell population does not (detectably) secrete any of the four marker proteins, whereas during or after the culturing of the mesenchymal stem cell population in the medium of the invention, it is observed that at least one of the four marker proteins is detectably secreted. And, when expression or secretion of at least two or at least three or all four of the four marker proteins Ang-1, TGF- β 1, VEGF and HGF is increased, wound healing properties of the mesenchymal stem cell population are improved, relative to culturing the stem cell population in a reference medium. The secretion of the four marker proteins into the culture medium (and thus the production of these factors by the stem cell population) can be measured/determined in any suitable way, for example, by determining the amount of protein by commercially available antibodies/immunoassays (see experimental section). Such measurements can be made 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 developed in 1969, which is a modified basal Eagle's Medium (BME) (see FIG. 1 showing a data sheet for DMEM available from Lonza). The original DMEM formulation contained 1000mg/L glucose and was first reported for use in culturing embryonic mouse cells. Since then, DMEM has become a standard medium for cell culture, and is commercially available from a variety of sources such as ThermoFisher Scientific (Cat. No. 11965-084), Sigma Aldrich (Cat. No. D5546), or Lonza, to name a few suppliers. Thus, any commercially available DMEM may be used in the present invention. In a preferred embodiment, the DMEM used herein is DMEM medium available from Lonza under catalog number 12-604F. This medium was DMEM supplemented with 4.5g/L glucose and L-glutamine. In another preferred embodiment, DMEM used herein is DMEM medium from Sigma Aldrich, catalog number D5546, which contains 1000mg/L glucose and sodium bicarbonate, but no L-glutamine.

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

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

By "M171" is meant culture medium 171 that has been developed as a basal medium for culturing the growth of normal human mammary epithelial cells (see fig. 4, which shows a data sheet for M171 medium from Life Technologies Corporation). This basic medium is also widely used and is commercially available from, for example, ThermoFisher Scientific or, for example, Life Technologies Corporation (Cat. No. M171500). Any commercially available M171 medium can be used in the present invention. In a preferred embodiment, the M171 medium used herein is M171 medium available from Life Technologies Corporation under catalog number M171500.

"FBS" means fetal bovine serum (which is also known as "fetal bovine (calf) serum"), the portion of blood that remains after the blood has naturally coagulated and then centrifuged to remove any remaining red blood cells. Fetal bovine serum is the most widely used serum supplement in eukaryotic cell culture in vitro because it has very low levels of antibodies and contains more growth factors, making it versatile in many different cell culture applications. Preferably, FBS is obtained from a member of the International Serum Industry Association (ISIA) which is primarily concerned with ensuring the safety and safe use of serum and animal derived products by proper traceability of origin, authenticity of the label and proper standardization and supervision. Suppliers of FBS as members of ISIA include Abattoid bases Company, Animal Technologies Inc., Biomin Biotechnology LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific, and Life Science Production, to name a few. In a presently preferred embodiment, FBS is available from GE Healthcare under product catalog number A15-151.

Referring now to the culture medium of the present invention, the culture medium may comprise DMEM at a final concentration of about 55 to 65% (v/v), F12 at a final concentration of about 5 to 15% (v/v), M171 at a final concentration of about 15 to 30% (v/v) and PBS at a final concentration of about 1 to 8% (v/v) for induction or improvement of wound healing properties of mesenchymal stem cells, or for isolation or culture of mesenchymal stem cells. As used herein, the numerical value of "% (v/v)" refers to the volume of a single component relative to the final volume of the medium. This means, for example, that 1 liter of medium contains about 550 to 650ml of DMEM if DMEM is present in the medium at a final concentration of about 55 to 65% (v/v).

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

In addition to the above components, the culture medium may comprise supplements that are advantageous for culturing the mesenchymal umbilical cord lining stem cells. For example, the culture medium of the present invention may comprise Epidermal Growth Factor (EGF). If present, EGF may be present in the medium at a final concentration of about 1ng/ml to about 20 ng/ml. In some such embodiments, the medium may comprise EGF at a final concentration of about 10 ng/ml.

The culture medium of the invention may also comprise insulin. Insulin, if present, may be present at a final concentration of about 1 to 10 μ g/ml. In some such embodiments, the medium may comprise insulin at a final concentration of about 5 μ g/ml.

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

In one embodiment of the method of the invention, a tissue, such as an umbilical cord tissue or a placenta, may be cultured until a suitable number of (primary) mesenchymal stem cells, such as umbilical cord lining stem cells, Wharton's jelly or placental stem cells, have grown from the tissue. In typical embodiments, umbilical cord tissue is cultured until the cell outgrowth of mesenchymal stem cells of the respective tissue reaches about 70% to about 80% confluence. It is to be noted here that the term "fusion" is used in its conventional meaning in the field of cell culture and is intended as an estimate/indicator of the number of adherent cells in a culture dish or flask, referring to the proportion of the surface covered by the cells. For example, 50% confluency means that about half of the surface is covered and there is still room for cell growth. 100% confluency means that the surface is completely covered by cells and no more room remains for growth of a monolayer of cells.

Once a suitable number of primary cells (mesenchymal stem cells) have been obtained from the corresponding tissue by the tissue isolation block method, the mesenchymal stem cells are removed from the culture vessel used for culture. By doing so, a master cell bank containing (primary) isolated mesenchymal stem cells, e.g. umbilical cord or placenta, may be obtained. Typically, since such mesenchymal stem cells are adherent cells, the cells are harvested using standard enzymatic treatments. For example, the enzymatic treatment may comprise a tryptic digestion as described in international U.S. patent application 2006/0078993, international patent application WO2006/019357 or international patent application WO2007/046775, meaning that the outgrown cells may be harvested by tryptic digestion (0.125% trypsin/0.05% EDTA) for further amplification. If the harvested mesenchymal stem cells are used, for example, to generate a master cell bank, the cells may also be cryopreserved and stored for further use, as described below.

Once harvested, the mesenchymal stem cells may be transferred to a culture vessel for subculture. Subculture or culture is also carried out if a population of mesenchymal stem cells has been previously isolated from its natural environment (as described above, such isolated stem cells for use in the methods of the invention may be from umbilical cord blood, bone marrow or adipose tissue, but also from umbilical cord tissue or placental tissue) (these two terms are used interchangeably hereinafter). The subculture can also be started from frozen primary cells, i.e. from the master cell bank. For subculture, any suitable number of cells may be seeded in a culture vessel such as a cell culture plate. For this purpose, mesenchymal cells may be cultured, for example, at about 0.5 x10⁶Individual cell/ml to about 5.0 x10⁶The individual cells/ml are suspended in a medium suitable for subculture (most conveniently, the medium of the present invention). In one embodiment, the cells are cultured at about 1.0x10⁶Individual cells/ml were suspended for subculture. The subculture can be carried out both in simple culture flasks and in multi-layer systems which can be stacked in an incubator, for example, such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA). Alternatively, the subculture can also be carried out in a closed self-contained system, such as a bioreactor. Bioreactors of different designs are known to those skilled in the art, for example parallel plate, hollow fiber or microfluidic bioreactors. See, e.g., Sensebe et al, "Production of sensory cells recording to good manufacturing practices: a review". An illustrative example of a commercially available hollow fiber bioreactor is

Cell Expansion System (Terumo BCT, Inc), which has been exemplifiedSuch as for expanding Mesenchymal stem Cells for clinical trials (see Hanley et al, Efficient Manufacturing of Therapeutic Mesenchymal Cells Using the Quantum Cell Expansion System, cytology.2014august; 16(8): 1048-. Another example of a commercially available bioreactor that can be used to subculture the mesenchymal stem Cell population of the present invention is the Xuri Cell Expansion System available from GE healthcare. If a working cell bank for therapeutic applications is to be produced under GMP conditions and a large number of cells are required, for example, in

The culture of mesenchymal stem Cell populations in the automated System of the Cell Expansion System is particularly beneficial.

Subculturing of the mesenchymal stem cells of the invention takes place in the medium of the invention. Thus, the culture medium of the invention may be used for the isolation of a population of mesenchymal stem cells from, for example, placental amniotic membrane or from Wharton's jelly of umbilical cord and for the subsequent culture of isolated primary cells by subculture. Also for subculture, mesenchymal stem cells may be cultured until a suitable amount of cells are grown. In exemplary embodiments, the mesenchymal stem cells are subcultured until the mesenchymal stem cells reach about 70% to about 80% confluence.

The isolation/culture of the mesenchymal stem cell population may be performed under standard conditions for culturing mammalian cells. Generally, the method of isolating a mesenchymal stem cell population of the present invention is generally performed under the following conditions (temperature, atmosphere): the conditions are typically used to culture cells of the species from which the cells are derived. For example, human umbilical cord tissue and mesenchymal umbilical cord lining stem cells are typically 5% CO at 37 ℃ respectively₂Culturing in a normal atmosphere. In this context, it is noted that in the present invention, the mesenchymal cell population may be derived from any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, wherein in one embodiment mesenchymal stem cells of human origin are preferred.

Once the desired/appropriate number of mesenchymal stem cells are obtained from culture or subculture, the mesenchymal stem cells are harvested by removing them from the culture vessel used for subculture. Harvesting of the mesenchymal stem cells is typically performed again by enzymatic treatment, including trypsinization of the cells. The isolated mesenchymal stem cells are then collected and used directly or stored for further use. Preservation is usually performed by cryopreservation. The term "cryopreservation" is used herein in its conventional sense to describe the process of preserving mesenchymal stem cells by cooling to a low sub-zero temperature, such as (typically) -80 ℃ or-196 ℃ (boiling point of liquid nitrogen). Cryopreservation may be performed as known to those skilled in the art, and may include the use of cryoprotectants such as dimethyl sulfoxide (DMSO) or glycerol, which slow the formation of ice crystals in the umbilical cord cells.

The isolated mesenchymal stem cell population obtained by the culturing and/or isolating method of the present invention is highly defined and homogeneous. In typical embodiments of the method, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD 105. Further, in these embodiments, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45 and HLA-DR. In particular embodiments, about 97% or more, about 98% or more, or about 99% or more of the isolated population of mesenchymal stem cells express CD73, CD90, and CD105, while lacking in the expression of CD34, CD45, and HLA-DR.

Thus, in light of the above disclosure, the present invention also relates to a population of mesenchymal stem cells (e.g., isolated from the amniotic membrane of Wharton's jelly or umbilical cord) such as a population of placental mesenchymal stem cells or a population of umbilical cord mesenchymal stem cells, wherein at least about 90% or more of the cells of the population of stem cells express each of the following markers: CD73, CD90, and CD 105. In preferred embodiments, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the isolated population of mesenchymal stem cells are CD73+, CD90+, and CD105+, with the meaning that this percentage of the isolated population of cells expresses each of CD73, CD90, and CD105 (see experimental section of this application). Furthermore, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45 and HLA-DR. In particular embodiments, about 97% or more, about 98% or more, or about 99% or more of the isolated population of mesenchymal stem cells express CD73, CD90, and CD105, while lacking in the expression of CD34, CD45, and HLA-DR. Such highly homogeneous umbilical cord amniotic membrane-derived mesenchymal stem cell populations are reported herein for the first time and meet the criteria for mesenchymal stem cells for cell therapy (see also experimental section and e.g., Sensebe et al, "Production of therapeutic structural/stem cells recording to good manufacturing practices: a review", supra). It is noted in this context that the mesenchymal stem cell population may be obtained by the isolation method of the present invention, but may also be obtained by different methods such as cell sorting, if desired. In one embodiment of such a population of umbilical cord mesenchymal stem cells of the invention, wherein at least about 91% or more of the cells of the stem cell population express CD73, CD90, and CD105 and lack the expression of CD34, CD45, and HLA-DR, excluding a population of mesenchymal stem cells isolated from the amniotic membrane of umbilical cord.

As noted above, the present invention also relates to a pharmaceutical composition comprising a population of mesenchymal stem cells described herein, wherein at least about 90% or more of the cells of the population of stem cells express each of the following markers: CD73, CD90, and CD105, and optionally lack expression of CD34, CD45, and HLA-DR. The pharmaceutical composition may comprise any pharmaceutically acceptable excipient and may be formulated for any desired pharmaceutical route of administration. The pharmaceutical composition may, for example, be suitable for systemic or topical application. In a related aspect, the invention also provides a pharmaceutical composition containing three or four of Ang-1, TGF- β 1, VEGF or HGF as the only wound healing proteins. Such pharmaceutical compositions may be formulated, for example, as liquids or as lyophilisates/freeze-dried formulations by using pharmaceutically suitable buffers, such as 0.9% saline, ringer's solution or Phosphate Buffered Saline (PBS).

In another aspect, the present invention relates to a method for preparing a medium for inducing or improving wound healing properties and/or for isolating a population of mesenchymal stem cells, wherein the method comprises mixing to obtain a medium with a final volume of 500 ml:

i.250ml of DMEM

ii.118ml M171

iii.118ml DMEM/F12

iv.12.5ml Fetal Bovine Serum (FBS) to give a final concentration of 2.5% (v/v).

As described above, DMEM/F12 medium was a 1:1 mixture of DMEM and ham F12 medium. Thus 118ml DMEM/F12 medium contained 59ml DMEM and 59ml F12. Therefore, when this method for preparing a medium is employed, the final concentration (v/v) in the total volume of 500ml is as follows:

DMEM: 250ml +59ml 309ml, corresponding to 309/500 ═ 61.8% (v/v)

M171: 118ml, corresponding to 118/500 ═ 23.6% (v/v)

F12: 59ml, corresponding to 59/500 ═ 11.8% (v/v).

Embodiments of the method of preparing a culture medium further comprise adding:

v.1ml EGF stock (5. mu.g/ml) to give a final EGF concentration of 10ng/ml, and

insulin 0.175ml stock solution (14.28mg/ml) to give a final insulin concentration of 5 μ g/ml.

It is to be noted here that in these embodiments, the volumes of these components i to vi described above are mixed to give a final volume of 499.675ml of medium. If no further components are added to the medium, the remaining 0.325ml (added to a volume of 500 ml) may be, for example, any of components i to iv, which means may be DMEM, M171, DMEM/F12 or FBS. Alternatively, the concentration of the EGF or insulin stock solution may of course be adjusted so that the total volume of the medium is 500 ml. Furthermore, it should also be noted that the components i to iv do not have to be added in the order in which they are listed, but of course any order of mixing the components may be used to obtain the medium of the invention. This means, for example, that M171 and DMEM/F12 can be mixed together and then combined with DMEM and FBS to achieve a final concentration as 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 embodiments, the method further comprises adding to DMEM a volume of 0.325ml of one or more of the following supplements: adenine, hydrocortisone, 3', 5-triiodo-L-thyronine sodium salt (T3), thereby obtaining a total volume of 500ml of medium. In this embodiment, the final concentration of these supplements in DMEM may be as follows:

about 0.05 to 0.1. mu.g/ml adenine, for example about 0.025. mu.g/ml adenine,

about 1 to 10. mu.g/ml hydrocortisone,

about 0.5 to 5ng/ml 3,3 ', 5-triiodo-L-thyronine sodium salt (T3), for example 1.36ng/ml3, 3', 5-triiodo-L-thyronine sodium salt (T3).

In accordance with the above disclosure, the present invention also relates to a cell culture medium obtainable or obtained by the method of preparing a culture medium described herein.

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

Accordingly, the present invention also relates to 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).

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

In addition, the medium may further comprise Epidermal Growth Factor (EGF) at a final concentration of about 1ng/ml to about 20 ng/ml. In some embodiments, the medium comprises EGF at a final concentration of about 10 ng/ml. The media described herein can further comprise insulin at a final concentration of about 1 μ g/ml to about 10 μ g/ml. In such embodiments, the medium may comprise insulin at a final concentration of about 5 μ g/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 some embodiments, the medium comprises all three of adenine, hydrocortisone, and 3, 3', 5-triiodo-L-thyronine sodium salt (T3). If present, the medium may comprise adenine at a final concentration of about 0.01 to about 0.1. mu.g/ml adenine or about 0.05 to about 0.1. mu.g/ml adenine, hydrocortisone at a final concentration of about 0.1 to about 10. mu.g/ml hydrocortisone or about 1 to about 10. mu.g/ml hydrocortisone, and/or 3, 3', 5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 to about 5 ng/ml.

In an embodiment of the cell culture medium, 500ml of the cell culture medium of the invention comprises:

i.250ml of DMEM

ii.118ml M171

iii.118ml DMEM/F12

iv.12.5ml Fetal Bovine Serum (FBS) (final concentration 2.5%).

In further embodiments, the cell culture medium may further comprise:

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

insulin at a final concentration of 5. mu.g/ml.

Insulin and EGF may be added to the medium using a stock solution selected so that the total volume of the medium does not exceed 500 ml.

In a particular example, components i to vi of the medium of the invention are the components shown in fig. 5, meaning that these components are obtained from the respective manufacturers under the catalog numbers shown in fig. 5. The medium obtained by mixing the components i to vi shown in FIG. 5 is also referred to herein as "PTT-6". It should also be noted in this context that the components i to vi as well as any other components (such as any other commercial supplier's antibiotics) can be used for preparing the medium of the invention.

Furthermore, the cell culture medium of the invention may comprise adenine at a final concentration of about 0.01 to about 0.1 μ g/ml adenine or about 0.05 to about 0.1 μ g/ml adenine, hydrocortisone at a final concentration of about 0.1 to about 10 μ g/ml, about 0.5 to about 10 μ g/ml or about 1 to about 10 μ g/ml hydrocortisone and/or 3, 3', 5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.1 to about 5ng/ml or about 0.5 to about 5 ng/ml.

Finally, the present invention also provides a method of treating a non-human mammal (such as a cat, a dog, a horse, to name a few) or a human patient suffering from a disease or a condition comprising administering to the non-human mammal or human patient a population of mesenchymal stem cells disclosed herein or a pharmaceutical composition comprising a population of stem cells disclosed herein. The disease may be any disease or condition, in particular any disease or condition in which wound healing is desired/required. The subject (patient or non-human mammal) has a wound caused by a burn, a bite, a trauma, surgery or a disease such as a skin disorder or a metabolic disorder. As an example of such a metabolic disorder, the patient may for example suffer from type I or type II diabetes, as well as from chronic foot ulcers.For treating a subject, the mesenchymal stem cell population of the invention may be administered in any suitable manner, including for example, but not limited to, topical administration, by implantation or by injection. In principle, any local mode of administration is intended herein. Administration of the mesenchymal stem cell population may be carried out by means of a syringe. However, the mesenchymal stem cells may also be contacted in a cream, ointment, gel, suspension, or any other suitable substance prior to application of the mesenchymal stem cells to the subject. The stem cell population may then be placed directly on a wound such as a burn or diabetic wound, for example (see international patent application WO 2007/046775). After its application to a subject, a dressing (e.g., using a dressing) is utilized

Dressing) and covering the same

An elastic bandage (crepe bandage) of the dressing may hold the mesenchymal stem cell population in place. Alternatively, the population of stem cells may also be implanted subcutaneously, for example directly under the skin, in body fat or in the peritoneum.

The 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 a population of mesenchymal stem cells described herein.

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

If desired, the unit dose may be administered once, twice, three times or more weekly. For example, the unit dose may be administered for one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more weeks. A unit dose containing about 100,000 cells, about 300,000 cells or about 500,000 cells may be applied twice a week for 8 weeks, preferably at 1cm²The application is as follows.

The unit dose may be contained in any suitable container. For example, the unit dose may be contained in a 1ml vial. In such cases, for example, 0.1ml of the vial may be applied to the subject, preferably at each square centimeter. The unit dose may optionally be contained in a syringe.

In a unit dose of the invention, the cells can be contacted with a pharmaceutically acceptable carrier (e.g., a liquid carrier). The carrier may be any known carrier, such as HypoThermosol^TM、Hypothermosol^TM-FRS or PlasmaLyte. The culture medium of the present invention 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, cells can be centrifuged and separated prior to administration to a subject.

The therapeutic methods and unit doses of the invention may comprise the use of living cells. Viability of the mesenchymal stem cell population is determined using known methods, for example trypan Blue staining as described in the experimental section.

The invention is further illustrated by the following non-limiting experimental examples.

The invention is further illustrated by the following non-limiting experimental examples.

The sequences used herein are depicted in table 1 below.

TABLE 1 sequences of proteins used herein

Experimental examples

1. Cryopreservation of umbilical cord tissue prior to isolation of mesenchymal stem cells

Umbilical cord tissue (umbilical cord donated with the mother's informed consent) is processed for subsequent isolation of mesenchymal stem cells from the amniotic membrane of the umbilical cord, as described below.

1.1 washing of umbilical cord tissue samples:

a. and taking down the scalpel from the protective cover.

b. The umbilical cord was clamped with forceps and cut into 10 cm long sections with a scalpel. The unusable umbilical cord was replaced in the original tissue cup.

c. The 10 cm long cord section was transferred to a new 150mm petri dish. A 150mm petri dish may be used instead of the cup.

d. The lid of the 150mm dish was used as a place to place forceps and scalpel.

e. 25ml of plasmyte A (Baxter, cat #2B2543Q) was removed with a 30ml syringe. The Plasmalyte a was dispensed directly onto the umbilical cord tissue with one hand holding the syringe at a 45 ° angle.

f. The dish was tilted slightly and the Plasmalyte A was removed using a 30ml syringe and a blunt needle.

g. The used Plasmalyte a was collected in a 300ml transfer bag which was used as a trash receptacle and discarded in a biohazard bin.

h. The washing procedure was repeated, using a new petri dish for each wash, if necessary. Ensuring that all blood clots on the surface have been removed. If tissue washing is required, more Plasmalyte A can be used.

i. The tissue is placed in a new labeled tissue culture dish to continue cutting the tissue. To this dish 20ml of Plasmalyte A was added so that the tissue did not dry out when cut.

j. The cord was cut into equal length cuts of about 1cm for a total of 10 cuts.

k. Each 1cm cut was then cut into smaller pieces, each cut being about 0.3cm by 0.3cm to 0.5cm by 0.5 cm.

Remove any Plasmalyte a in the petri dish.

m. take 25ml of Plasmalyte a from the original Plasmalyte a bag using a 30ml syringe and dispense directly onto the umbilical cord tissue block.

n. the dish was held at an angle to concentrate all Plasmalyte a used for washing the tissue to one side and then removed with a syringe and a blunt needle.

Repeat the wash again. No clots should be left behind.

Note that: if the cord is not frozen immediately, the cord tissue is stored in Plasmalyte A until ready to be frozen.

1.2 cryopreservation of umbilical cord tissue:

a. preparing a cryopreservation solution:

i. 50ml of a refrigerating fluid consisting of 60% Plasmalyte A, 30% human serum albumin (5%) and 10% dimethyl sulfoxide (DMSO) was prepared.

One 150ml transfer bag was labeled "tissue cryo-fluid" and the plasma transfer device was connected to the port using sterile technique.

Take 30ml of Plasmalyte a from the original Plasmalyte a bag with a 30ml syringe and transfer it into a "tissue freezing fluid" labeled transfer bag with the date and time of solution preparation.

15ml of 5% human serum albumin was taken with a 20ml syringe and transferred to the labelled transfer bag.

v. add 5ml DMSO to the transfer bag.

Mixing uniformly and recording the mixing condition of the refrigerating fluid.

b. Plasmalyte a was removed from the tissue before addition of the freezing fluid.

c. Using a 60ml syringe, a total of 50ml of the freezing fluid was drawn into the syringe, and about 30ml of the freezing fluid was added to a 150mm cell culture dish containing umbilical cord tissue. A blunt needle was placed over the syringe to maintain sterility.

d. The petri dish containing the tissue and the freezing fluid was rotated for 10 minutes per minute.

e. 8 randomly selected sections were selected with forceps and placed into each of 4ml frozen vials. 4 randomly selected sections were selected and placed into a 1.8ml frozen vial. These sections should be free of blood clots.

f. The remaining freezing fluid was added to each frozen vial containing umbilical cord tissue, with 4ml tubes added to a 3.6ml filling line and 1.8ml Nunc vials added to a 1.8ml filling line.

g. One Bactec Lytic/10-Anaerobic/F vial and one Bactec Plus Aerobic/F vial were labeled with tissue IDs.

h. After removing 20ml of the cryo-fluid from the petri dish with a syringe and blunt needle, wiping the Bactec vial with an alcohol cotton swab, the blunt needle was converted to an 18g needle and inoculated with aerobic and anaerobic Bactec vials, 10ml per vial.

i. The control rate freezer is started.

j. After the controlled rate freezing was completed, the device was placed in a liquid nitrogen freezer for continuous temperature monitoring until further use.

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

2.1 preparation of media for processing MSCs from umbilical cord tissue:

a. the following were mixed in the order listed to prepare 500ml of PTT-6 (culture/growth medium):

i.DMEM，250ml

ii.M171 118ml

iii.DMEM F12 118ml

12.5ml of FBS (final concentration: 2.5%)

EGF 1ml (final concentration 10ng/ml)

Insulin 0.175ml (final concentration 5. mu.g/ml)

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

The bottle is marked with "PTT-6", the date of preparation of the medium, the initials of the operator, and the expiry date after the phrase "due". By expiration date is meant the earliest expiration date of any component, or 1 month from the preparation date, whichever comes first.

b. In a 50ml centrifuge tube, 2.5ml of FBS was added to 47.5ml of HBSS to prepare a rinsing medium (Hank's buffered saline solution (HBSS) containing no calcium or magnesium with 5% FBS). The tubes are marked with "rinsing medium", the operator's initials and the date of preparation of the medium.

c. All media were aseptically tested using Bactec Lytic/10-Anaerobic/F (Becton Dickinson & Company) and Bactec Pluc + Aerobic/F (Becton Dickinson & Company). 20ml of the prepared medium was injected into each flask.

2.2 thawing umbilical cord tissue for MSC harvest:

a. thawing is initiated when the operator is ready to process the sample in the clean room. Unless these vials are from the same donor, the number of vials per thaw must not exceed 1.

b. The water bath was wiped with disinfectant, then 70% isopropyl alcohol, and 1L of sterile water was injected into the water bath. The water bath was heated to 36-38 ℃.

c. 10mL of a rinse medium consisting of 70% to 90% PlasmaLyte A was prepared under a biosafety cabinet in a clean room. The solution was sterile filtered using a 0.2- μm syringe filter attached to a 10ml syringe and kept cold until use.

d. The handling label was attached to a 50ml conical tube.

e. The water bath temperature was confirmed to be 36-38 ℃.

f. The tissue vials were removed from the liquid nitrogen reservoir and quickly thawed in a 37 ℃ water bath containing 1L of sterile water. The vial holder for the mr. frosty NalgeneCryo 1 ℃ frozen container floats with the vial and can be used as a floating holder when thawing the sample.

g. The vial was removed from the water bath and sprayed with 70% isopropanol solution. A good time to remove the vial from the water bath was when small ice could be seen floating in the vial-suggesting that the internal temperature of the vial was below 37 ℃.

h. The vial is placed in the transfer lane and the clean room process technician is alerted.

2.3 preparation of tissue treatment:

a. umbilical cord tissue processing should be performed in a clean room for Environmental Monitoring (EM). At the end of each shift, the entire room and security hood (hood) is cleaned.

b. The biosafety cabinet is prepared/cleaned.

c. The presence (viable) particle count is performed while working in a biosafety cabinet.

d. All necessary supplies are assembled in a biosafety cabinet, and the packaging of each supply is checked for breakage and expiration date. When taking syringes, seropipettes, sterile forceps, scalpels, tissue plates and needles, it is ensured that no surface that will come into contact with the sterile product is touched. Only the syringe barrel, tubing, plunger head and/or the exterior of the needle cap or sheath are safely accessible. The supply is discarded if the surface has been touched or contacted with a non-sterile surface.

e. The lot number and expiration date (if applicable) of all reagents and supplies to be used are recorded.

f. The thawed vials were received by cleaning the vials with lint free cloth soaked with 70% alcohol and then transferred to a biosafety cabinet.

g. As much liquid as possible is taken out of the vial using a pipette needle with a syringe. Avoiding aspiration of tissue.

h. Using sterile forceps, the tissue was transferred to a sterile 100mm petri dish.

i. To the tissue fragments, 5ml of rinse medium was added in equal portions.

j. The contents are spun for 15-30 seconds and the rinse medium is then removed with a pipette or syringe with a pipette needle. This rinsing process was repeated twice.

k. 2mL of rinse medium was added to the tissue to avoid drying of the tissue.

2.4 initiation of the self-organized outgrowth of MSCs:

a. the 6-well plate bottom is labeled "outgrowth 1", MSC lot number or umbilical cord tissue ID and date to start outgrowth. If a 60mm tissue culture dish is used, the plate is divided into 4 quadrants by drawing a grid at the bottom of the dish.

b. One 3x 3mm to 5x 5mm tissue was placed into each well using sterile disposable forceps. If a 60mm tissue culture dish is used, tissue is placed in the middle of each quadrant to keep the tissues separated (by more than 1cm from each other).

c. 3ml of PTT-6 was added to each well.

d. Using a pipette needle attached to a 30ml syringe, enough media was aspirated to cover the tissue barely. The plate is not tilted. Without the need for a pipette needle to contact the bottom of the well.

e. Halo of cell growth was observed daily (24 ± 6 hours) using an inverted light microscope. A real-time cell culture imaging system may be used instead of an optical microscope.

f. The medium was changed daily. The medium was allowed to equilibrate to room temperature before use.

i. The medium was aspirated off.

Add 3ml of PTT-6.

Aspirate until the tissue is nearly submerged in culture medium.

g. When outgrowth of cells from the tissue was observed, the tissue was transplanted into a new 6-well plate using the same procedure as described above under 4.a to 4.e, except that "outgrowth 2" was marked on the plate. The halo of cell growth in the "outgrowth 1" plate was maintained by adding 2ml of PTT6 to each well. Fusion was observed daily. The medium was changed every 2-3 days (ensuring that the medium was equilibrated to room temperature before use).

h. When a halo of cell growth is observed in the "outgrowth 2" plate, steps 4.a to 4.e are repeated, except that "outgrowth 3" is marked on the plate. The halo of cell growth in the "outgrowth 2" plate was maintained by adding 2ml of PTT-6 to each well. Fusion was observed daily. The medium was changed every 2-3 days (ensuring that the medium was equilibrated to room temperature before use).

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

j. When the cells reached 40-50% confluence, the cells were observed daily to prevent over-expansion.

k. When the cells reached 70-80% confluence, the cells were subcultured. Cells were not allowed to expand to more than 80% confluence.

In the case where the size of the tissue isolation block is about 1-3mm, and the tissue isolation block method/cell culture is performed in a culture dish of 175 mm square, the average number of mesenchymal stem cells harvested from the isolation block is generally about 4,000 and 6,000 cells/isolation block. Thus, when mesenchymal stem cells were simultaneously grown from 48 isolated pieces, about 300,000 cells could be obtained at the time of harvest. The 300,000 cells were then seeded at 175cm by plating them as described in example 2.5 below²In the cell culture flask (this may be referred to as passage 1), the 300,000 mesenchymal stem cells collected from the isolated block were used for subculture. The mesenchymal stem cells obtained from this passage 1 can then be reseeded at 175cm²Cells were grown in cell culture flasks (passage 2) and as described in example 2.5 below. The cells obtained from passage 1 and passage 2 may be preserved by freezing into a "bank" in which the mesenchymal stem cells obtained after passage 2 are considered to represent the master cell bank which will be used for further expansion of the mesenchymal stem cells, for example in a bioreactor as described in example 2.7 below.

2.5. Subculturing MSCs in cell culture dishes

a. The counting of the existing particles is carried out while working in the biosafety cabinet. All media were equilibrated to room temperature before use.

b. When the halo of cell growth reached about 70-80% confluence, the cells were subcultured.

i. PTT-6 was removed from the culture dish.

Rinsing with HBSS without calcium or magnesium.

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

Tilt the dish 30-45 ° and move the cells downward by gravity flow. Separation was accelerated by tapping gently on the sides of the plate.

v.1ml of PTT-6 was added. Pipet gently up and down and then transfer the cells into a15 ml centrifuge tube. A clean pipette tip was used for each well. Cells from all 6 wells can be pooled into a single 15ml tube.

Centrifuge at 1200rpm for 10 minutes.

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

c. Subculturing the MSC.

i. Cell suspensions were aliquoted in 50 μ l and analyzed for TNC and viability by Trypan Blue Exclusion Assay (Trypan Blue Exclusion Assay).

Count cells using a hemocytometer. It is expected to count 20-100 cells/square. If the count is above 100, the original sample is diluted 1:5 and trypan blue is repeated using a hemocytometer.

Calculate surviving cells/ml and total surviving cells:

1. viable cell/ml ═ viable cell count × dilution factor × 10⁴

2. Total viable cells count × dilution factor × total volume × 10⁴

Calculating% survival:

1. survival rate ═ viable cell count × 100/(viable cell count + dead cell count)

v. dilution of the cell suspension to 1.0X10⁶Individual cell/ml

1. "X" volume-Total viable cells/10⁶Individual cell/ml

2. For example, if the total viable cell count is 1.0X10⁷；

3、“X”＝10⁷/10⁶Individual cells/ml or 10ml, therefore, by thinning5ml of the cell suspension (i.e., 5ml) was added to make the total cell volume 10 ml.

If the cell suspension is less than 10⁶Per ml, 150mm dish or 175cm each is determined²Inoculation in bottles 2X 10⁶The volume required for each cell.

1、2×10⁶The volume required for each cell was 2X 10⁶Individual cell ÷ viable cell/ml

2. For example, if the viable cells/ml is 8X 10⁵One cell/ml, 2X 10 is required⁶Individual cells/8X 10⁵Individual cells/ml or 2.5 ml.

Leave 0.5ml for MSC marker analysis.

In each 150mm dish or 175cm of 30ml PTT-6²Inoculation in bottles 2X 10⁶And (4) cells.

Every three days cells were observed for adherence, colony formation and fusion. When the cells reached 40-50% confluence, the cells were observed every 1-2 days to prevent over-expansion. Cells were not allowed to expand to more than 80% confluence. A real-time cell culture monitoring system may be used instead of an optical microscope.

x. medium was changed every 2-3 days.

2.6 cryopreservation of MSC cells

a. The counting of the existing particles is carried out while working in the biosafety cabinet.

b. When the cells reached 70-80% confluence, 150mm dishes or 175cm per cell²The flask used 2ml of 1X TrypLE-EDTA to isolate the cells.

i. PTT-6 was removed from the culture dish.

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

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

Tilt the dish 30-45 ° and move the cells downward by gravity flow. A gentle tap on the side of the dish helps to speed up the separation.

v. addition of 10ml of PTT-6 to inactivate TrypLE. Mix well to separate the cell mass.

Cells were transferred to 15ml centrifuge tubes using a Pasteur pipette.

Centrifuge at 1200rpm for 10 minutes.

Medium was aspirated and resuspended with 10ml of PTT-6.

Aliquots were 50 μ l and total viable cell number and% survival determined as described above. Since cells may begin to form clumps, cell counting needs to be completed within 15 minutes.

c. Cells were prepared for cryopreservation.

i. Preparing cell suspension medium and cryopreservation medium, and freezing cells

2.7. Subculturing (expanding) MSCs in Quantum bioreactors (Terumo BTC, Inc.)

Quantum bioreactors, which can be used to amplify MSCs, can also be employed. The starting number of cells for expansion in the Quantum bioreactor should be 2 to 3 million cells per run. The yield per run at harvest is typically 3 to 7 billion MSCs. The bioreactor was operated according to the manufacturer's instructions. The mesenchymal stem cells thus obtained are usually cryopreserved (see below) and used as a working cell bank.

Materials/reagents:

1. quantum amplification device

2. Quantum waste bag

3. Quantum culture medium bag

4. Quantum entering bag

5、PTT-6

6、PBS

7. Fibronectin

8、TrypLE

9. 3ml syringe

10. Glucose test paper

11. Lactic acid test paper

12. 60ml cell culture plate or equivalent

13. Medical grade 5% CO₂Gas mixture

14、50ml Combi-tip

Equipment:

1. biological safety cabinet

2. Blood glucose meter (Bayer Healthcare/Ascensia Contour glucose meter)

3、Lactate Plus(Nova Biomedical)

4. Peristaltic pump with head (Peristtical pump)

5. Centrifuge, Eppendorf 5810

6. Aseptic tube connector

7. M4 repeat pipettor

8. RF sealer

The process is as follows:

1. preparation of Quantum bioreactor

a) Starting Quantum bioreactor

b) Coating the bioreactor:

1) fibronectin solutions were prepared in biosafety cabinets.

1) Acclimatization of lyophilized fibronectin to room temperature (. gtoreq.15 min at room temperature)

2) 5ml of sterile distilled water was added; not rotating or agitating

3) Fibronectin was allowed to form a solution for 30 minutes.

4) The fibronectin solution was transferred to cells containing 95ml PBS into bags using a 10ml syringe with an 18g needle attached.

2) Connecting bags to 'reagent' lines

3) The bubbles were examined (they could be removed by using "remove IC air" or "remove EC air" and using "scrubbing" as the entry source.

4) The procedure for coating the bioreactor was opened or set up (FIG. 1, steps 3-5).

5) Running program

6) When the program was run to coat the bioreactor, a media bag with 4L of PTT-6 media was prepared.

7) The media bag is connected to the IC media line using a sterile tubing connector.

8) When the bioreactor coating step was completed, the cells for the fibronectin solution were isolated into the bag using an RF sealer.

c) Washing away excess fibronectin

d) The bioreactor is conditioned with a culture medium.

2. Culturing cells in Quantum bioreactors

a) Cells were loaded and attached with a homogeneous Suspension (uniformity Suspension):

b) feeding and culturing cells

1) The flow rate of the medium was selected to feed the cells.

2) Samples of lactic acid and glucose were taken daily.

3) The flow rate of the medium was adjusted as the lactic acid level increased. The actual maximum tolerated lactate concentration will be determined from the cell-derived culture flask culture. It was determined whether there was sufficient PTT-6 medium in the media bag. If necessary, the PTT-6 medium bag is replaced with a new PTT-6 medium bag.

4) When the flow rate reached the desired value, the lactic acid level was measured every 8-12 hours. If lactate levels do not decrease or if lactate levels continue to increase, cells are harvested.

3. Harvesting cells from Quantum bioreactors

a) When lactate concentration was not reduced, cells were harvested after the last sampling of lactate and glucose.

b) Harvesting cells:

1) the cell entry bag containing 100ml TrypLE was connected to the "reagent" line using a sterile tubing connector.

2) Sufficient PBS was confirmed in the PBS bag. If not, a new bag with at least 1.7 liters of PBS was connected to the "wash" line using a sterile tube connector.

3) Running the Harvest program

4. Cryopreservation of cells

1) Once the cells were harvested, the cells were transferred to a 50ml centrifuge tube to pellet the cells.

2) Resuspend using 25ml of cold cell suspension. Cells were counted using a Sysmex or Biorad cytometer. Cell count reports were appended to the corresponding Quantum treatment batch records.

3) Adjusting the cell concentration to 2X 10⁷/ml

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

5) Using a repeat pipettor, 1ml of the cell suspension in cryopreservative solution was added to each 1.8ml vial. Cryopreservation was performed Using the CRF program as described in SOP D6.100 CB cryoprecipitation Using Controlled Rate Freezers.

6) Vials were stored in designated liquid nitrogen storage spaces.

7) The CRF run report is appended to the table of the corresponding MSC P3-Quantum process batch records.

3. Analyzing stem cells in a mesenchymal umbilical cord lining stem cell population isolated from umbilical cord tissue using different media Expression of the marker

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

For these experiments, mesenchymal stem cells were isolated from umbilical cord tissue by culturing the umbilical cord tissue in three different media, and then subcultured in the corresponding media as shown in example 2.

In these experiments, the following three media were used: a) 90% (v/v) DMEM supplemented with 10% FBS (v/v), b) PTT-4 medium as described in U.S. patent application 2008/0248005 and the corresponding international patent application WO2007/046775, consisting of 90% (v/v) CMRL1066 and 10% (v/v) FBS (see paragraph [0183] of WO2007/046775) and c) PPT-6 medium of the invention, the composition of which is described herein. In this flow cytometry analysis, two different samples of umbilical cord lining mesenchymal stem cell (CLMC) populations were analyzed in each of the three media employed.

The flow cytometry analysis employed the following protocol.

Materials and methods

Name of instrument	Company name	Name of series
			BD FACS CANDO	BD	V07300367
Inverted microscope, CKX41SF	Olympus	4K40846
			Centrifuge, micro-rotating table surface type	Biosan	010213-1201-0003

List of reagents	Company name	Catalog number of products
			10X Trypsin	Biowest	X0930-100
10X PBS	Lonza	17-517Q
			DMEM	Lonza	12-604F
Fetal bovine serum	GE healthcare	A11-151

The process is as follows:

a) isolation and culture of cells from umbilical cord lining membranes

1. Separate tissue samples were incubated in cell culture plates and submerged in the respective media, and then maintained at 37 ℃ CO as described in example 2₂An incubator.

2. The medium was changed every 3 days.

3. The outgrowth of cells from tissue culture isolated pieces was monitored under a light microscope.

4. At about 70% confluence, cells were detached from the plates by trypsinization (0.0125% trypsin/0.05% EDTA) and used for flow cytometry experiments.

b) Trypsin digestion of cells for experiments

1. The medium was removed from the cell culture plate.

2. A gentle rinse with sterile 1X PBS was used to remove traces of FBS, as FBS interferes with the enzymatic action of trypsin.

3.1 Xtrypsin was added to the cell culture plates and incubated at 37 ℃ for 3-5 min.

4. Cells were observed under a microscope and cell shedding was ensured. Trypsin was neutralized by addition of FBS-containing complete medium (DMEM with 10% FBS).

5. The cell pellet is broken by pipetting the cells in the medium against the wall of the culture plate. The cell suspension was collected and transferred to a 50ml centrifuge tube

6. Sterile 1X PBS was added to the plate and rinsed, and the cell suspension was collected into the same centrifuge tube.

7. Centrifuge at 1800rpm for 10 minutes.

8. The supernatant was discarded and the cell pellet resuspended in PBA medium.

c) Counting cells

1. The cytometer and its cover slip are ensured to be clean and dry, preferably washed with 70% ethanol and dried, then wiped with a Kim paper towel (dust free paper).

2. A small amount of suspended cells were aliquoted into a microcentrifuge tube and removed from the BSC hood.

3. The suspension cells are stained with an equal volume of trypan blue, for example 500 μ l of trypan blue (dilution factor 2X, resulting in a 0.2% trypan blue solution) is added to 500 μ l of the suspension.

4. Exposure of cells to trypan blue for more than 30 minutes is avoided since trypan blue is toxic and can lead to an increase in non-viable cells, resulting in erroneous cell counts.

5. Mu.l of the cell suspension mixture was added to each well of the hemocytometer and observed under an optical microscope.

a. The number of viable cells (bright cells; non-viable cells readily absorb trypan blue and are therefore dark) was counted in each quadrant of the cytometer, for a total of 8 quadrants in the upper and lower wells.

Total cell count was expressed as (mean cell number/quadrant). times.10⁴Individual cells/ml are given.

d) Staining cells

i.Preparation before staining cells

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

ii.Staining with Primary anti- (Ab)

Mu.l [0.5mg/ml Ab ] primary antibody was added to 100. mu.l of the cell suspension. Incubate at 4 ℃ for 45 min.

Make up to 1ml with PBA.

Centrifuge at 8000rpm for 5min at 4 ℃.

Remove the supernatant.

Add 1ml PBA and resuspend the pellet.

Centrifuge at 8000rpm for 5min at 4 ℃.

Remove the supernatant.

Resuspend in 100. mu.l PBA.

iii.Staining with a Secondary antibody AbIn the dark

Mu.l [0.5mg/ml ab ] secondary antibody was added to 100. mu.l of the cell suspension. Incubate at 4 ℃ for 30 min.

Make up to 1ml with PBA.

Centrifuge at 8000rpm for 5min at 4 ℃.

Remove the supernatant.

Add 1ml PBA and resuspend the pellet.

Centrifuge at 8000rpm for 5min at 4 ℃.

Remove the supernatant.

Resuspended in 200-300. mu.l PBA for flow cytometry analysis.

Transfer cells to FACS tubes for reading in a BD FACS CANDO flow cytometer.

The results of the flow cytometry analysis are shown in fig. 6a to 6 c. Figure 6a shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in DMEM/10% FBS, figure 6b shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in PTT-4, and figure 6c shows the percentage of isolated mesenchymal umbilical cord lining stem cells expressing the stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and culture in PTT-6. As can be seen from fig. 6a, the isolated cell population cultured with DMEM/10% FBS as the medium had about 75% CD73+ cells, 78% CD90+ cells, and 80% CD105+ cells (average of two experiments), whereas the number of CD 73-positive, CD 90-positive, and CD 105-positive mesenchymal stem cells was about 87% (CD73+ cells), 93% (CD90+ cells), and 86% (CD105+ cells) after the isolation/culture of umbilical cord tissue with PPT-4 medium (see fig. 6b), which are the average of two experiments. With respect to all three markers (CD73, CD90, CD105), the purity of the mesenchymal stem cell population obtained by culture in the PTT-6 medium of the present invention was at least 99.0%, meaning that the purity of the cell population was significantly higher than that of the culture with PPT-4 medium or DMEM/10% FBS. Furthermore and even more importantly, the mesenchymal stem cell population obtained by culture in PTT-6 is essentially a 100% pure and defined stem cell population. This makes the stem cell populations of the present invention ideal candidates for stem cell-based therapies. Thus, this mesenchymal umbilical cord lining stem cell population may become the gold standard for therapeutic approaches based on such stem cells.

The findings shown in fig. 6 are further confirmed by the results of flow cytometry analysis shown in fig. 7a and 7 b. Figure 7a shows the percentage of isolated mesenchymal umbilical cord lining stem cells (mesenchymal stem cells of the umbilical cord amniotic membrane) that express stem cell markers CD73, CD90 and CD105 and lack the expression of CD34, CD45 and HLA-DR after isolation from umbilical cord tissue and culture in PPT-6 medium. As shown in fig. 7a, the mesenchymal stem cell population contained 97.5% of the viable cells, of which 100% expressed each of CD73, CD90 and CD105 (see "CD 73+ CD90 +" and "CD 73+ CD105 +" rows), whereas 99.2% of the stem cell population did not express CD45, and 100% of the stem cell population did not express CD34 and HLA-DR (see "CD 34-CD 45-" and "CD 34-HLA-DR-" rows). Thus, the mesenchymal stem cell population obtained by culturing in PTT-6 medium is essentially a 100% pure and defined population of stem cells that meets the criteria that mesenchymal stem cells to be used for cell therapy should meet (95% or more of the stem cell population express CD73, CD90 and CD105, while 98% or more of the stem cell population lack the expression of CD34, CD45 and HLA-DR, see Senseee et al, "Production of sensory structural/stem cells accessing good manufacturing practices: a review", supra). It is noted here that the amniotic membrane of mesenchymal stem cells of the present invention adheres to plastic under standard culture conditions and differentiates into osteoblasts, adipocytes and chondroblasts in vitro, see us patent 9,085,755, us patent 8,287,854 or WO2007/046775, thus complying with the generally accepted standards for mesenchymal stem cells for cell therapy.

Figure 7b shows the percentage of isolated bone marrow mesenchymal stem cells expressing CD73, CD90, and CD105 and lacking expression of CD34, CD45, and HLA-DR. As shown in fig. 7b, the mesenchymal stem cell population contained 94.3% of the viable cells, of which 100% expressed each of CD73, CD90 and CD105 (see "CD 73+ CD90 +" and "CD 73+ CD105 +" rows), whereas only 62.8% of the mesenchymal stem cell population lacked expression of CD45, and 99.9% of the stem cell population lacked expression of CD34 and HLA-DR (see "CD 34-CD 45-" and "CD 34-HLA-DR-" rows). Thus, the gold-standard bone marrow mesenchymal stem cells considered to be mesenchymal stem cells have much lower homogeneity/purity in terms of stem cell markers than the mesenchymal stem cell population (of the amniotic membrane of umbilical cord) of the present application. This finding also suggests that the stem cell populations of the present invention may be ideal candidates for stem cell-based therapies, and that the stem cell populations may become the gold standard for stem cell-based therapies.

4. Wound healing marker protein secretion fraction for culturing mesenchymal stem cell population isolated in the culture medium of the present invention Analysis of

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

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

Umbilical cord amniotic mesenchymal stem cells (cord lining MSC/CL-MSC). This CL-MSC population was isolated by tissue isolation blocks of human umbilical cord lining membrane (cultured in DMEM supplemented with 10% fetal bovine serum (DMEM/10% FBS)) as described in example 2 of WO 2007/046775.

-mesenchymal stem cells of Wharton's jelly (WJ-MSC). Such as Beravolu et al, "Isolation and Characterization of Mesenchyl structural Cells from Human Umbilical corp and Fetal planta." J Vis exp.2017; (122) 55224, this WJ-MSC population was isolated by tissue isolation of Wharton's jelly from human umbilical cord (cultured in DMEM with 4,500mg/mL glucose and 2mM L-glutamine supplemented with 10% human serum/FBS and antibiotic solution).

-adipose-tissue derived mesenchymal stem cells (AT-MSC). Such as Schneider et al, "advanced-sensitive cell from reactive and reactive fast area sources for regenerating media" Eur J Med Res.2017; 22:17, this AT-MSC population was isolated by tissue isolation block method from adipose tissue donated to skin tissue after abdominoplasty (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). Such as Beravolu et al, "Isolation and Characterization of Mesenchyl structural Cells from Human Umbilical corp and Fetal planta." J Vis exp.2017; (122) 55224, the PT-MSC population is isolated from placenta.

Culture protocol 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 for 24 hours.

Discard the medium and wash the cells with PBS.

10ml of DMEM was added to the culture for 24 hours.

Discard the medium and add 5ml of DMEM to the culture.

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

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

Cytokine assays were performed in MSC supernatants. Measurements and analyses were performed using Luminex 200 and Xponent software.

The objective of this experiment was to measure the relative levels of multiple (PDGF-AA, PDGF-BB, IL-10, VEGF, Ang-1 and HGF), TGF β 1 single and bFGF2 single cytokines in the cell culture supernatants. The supernatant is (MSC, mesenchymal stem cells; CL, umbilical cord lining; WJ, Wharton's jelly; AT, adipose tissue; BM, bone marrow):

CL-MSC cultured in PTT-4

WJ-MSC cultured in PTT-4

AT-MSC cultured in PTT-4

BM-MSC cultured in PTT-4

CL-MSC cultured in PTT-6

WJ-MSC cultured in PTT-6

AT-MSC cultured in PTT-6

BM-MSC cultured in PTT-6

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

The aim of this experiment was to generate cytokine profiles (profiles) of MSCs cultured in PTT-4 or PTT-6 and to compare profiles of MSCs of different tissue origin (umbilical cord lining, gutta percha, adipose tissue and bone marrow). This profile will elucidate which stem cell populations grow in which media will secrete more cytokines of interest in order to promote wound healing.

The plate set-up for all plates is depicted in FIG. 8. Abbreviations used are as follows: MSC, mesenchymal stem cells; CL, umbilical cord lining; WJ, Walton gel; AT, adipose tissue; BM, bone marrow.

Multiplex assays

Multiple information:

r & D System/Bio-technique cat. # LXSAHM. The kit was expired with lot # L123680, 08/28/18, with the following analytes:

ang-1, angiogenin

VEGF, a vascular endothelial growth factor

PDGF-AA, platelet-derived growth factor (PDGF-AA means a disulfide-linked homodimer consisting of the A chain, and PDGF-BB consists of the B homodimer R & D show that PDGF-BB antibodies also detect PDGF-AB heterodimers)

·PDGF-BB

HGF, hepatocyte growth factor

IL-10, interleukin-10

TGF β 1 singleplex information: r & D Systems/Bio-technique):

basal kit, cat, # LTGM00, lot # P156217, 02/27/18 received, 08/30/18 expired.

TGF β 1 component, # LTGM100, lot # P161760, 02/27/18 received, 11/27/19 expired.

bFGF2 singleplex message (used 3, 19, 2018): eBioscience/Thermo:

basic kit, cat. # EPX 010-10420-.

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

bFGF2 singleplex message (used 3 month 22 2018): eBioscience/Thermo:

basic kit, cat. # EPX 010-10420-.

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

Multiple information:

r & D System/Bio-technique cat. # LXSAHM. The kit was expired with lot # L123999, 09/25/18, with the following analytes:

ang-1, angiogenin

VEGF, a 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 entry

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

Procedure

Cytokine assays were performed in MSC supernatants according to detailed protocol information. As part of this experiment, the protocol has a modification: std.8 in the multiplex kit was no longer used. The reason for stopping std.8 is that the R & D system protocol itself uses only standards 1 to 6. In addition, only 2 of the 6 analytes that make up the multiplex were validated at ClinImmune for std.8: 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 falls in the middle region of the standard curve. Since the standards were reconstituted using growth medium, standard curves were constructed with both PTT-6 and PTT-4. Test samples grown in PTT-6 or PTT-4 were extrapolated from the corresponding standard curves.

Results were extrapolated by Luminex software based on an analyte-specific standard curve generated by the same software: the analysis algorithm is set to Logistic 5P Weighted with Weighted analysis, where the weighting is 1/y 2.

Sample (I)

1. PTT-4 and PTT-6 media (not exposed to MSC)

2. Supernatant of MSC to be tested

3. Optionally: supernatants of CL-MSCs from different donors; CR001A, C, D, and G.

Summary of the Experimental results

TGF-beta 1 singleplex assay

Use 1 of aliquot 3-as shown in figure 9. All error bars are standard deviations of three measurements.

FIG. 9: singleplex measurement of TGF β 1. It can be seen that cultures CL-MSC and WJ-MSC produced more TGF β 1 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 cultures produced approximately equal amounts of TGF β 1. All error bars are standard deviations of three measurements.

First multiplex analysis

1 using aliquot 3.

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

The data are depicted in fig. 10 and 11.

FIG. 10: FIG. 10A is a multiplex measurement of PDGF-AA. It can be seen that cultures CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produced more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of three measurements. FIG. 10B is a multiplex measurement of VEGF. It can be seen that culture CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produced more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three measurements. FIG. 10C is a multiplex measurement of Ang-1. It can be seen that culture CL-MSC and WJ-MSC cultures produced more Ang-1 when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC do not produce substantially any Ang-1. All error bars are standard deviations of three measurements.

FIG. 11: multiple measurements of HGF. It can be seen that cultures CL-MSC and WJ-MSC cultures produced much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC produced essentially no HGF. All error bars are standard deviations of three measurements.

Multiplex assay (including bFGF)

3 using aliquot 3. The data are shown in FIGS. 12-14.

FIG. 12: multiplex measurement of PDGF-AA. It can be seen that culture CL-MSC and WJ-MSC cultures produced more PDGF-AA when grown in PTT-4 than when grown in PTT-6. Cultures AT-MSC and BM-MSC produced equal amounts of PDGF-AA in both media. All error bars are standard deviations of three measurements.

FIG. 13: FIG. 13A is a multiplex measurement of VEGF. It can be seen that culture CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produced more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviations of three measurements. FIG. 13B is a multiplex measurement of Ang-1 multiplex assay. It can be seen that culture CL-MSC and WJ-MSC cultures produced much more Ang-1 when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC do not produce substantially any Ang-1. All error bars are standard deviations of three measurements. Fig. 13C is a multiplex measurement of HGF. It can be seen that cultures CL-MSC and WJ-MSC cultures produced much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC produced essentially no HGF. All error bars are standard deviations of three measurements.

FIG. 14: multiplex measurement of bFGF. As can be seen, cultures of CL-MSC and WJ-MSC produced more bFGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC produced equal amounts of bFGF when cultured in PTT-4 and PTT-6. All error bars are standard deviations of three measurements.

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

Fig. 15 to 21 depict an overview of the data obtained in the different experiments.

FIG. 15: TGF β 1 measurements for 5 different experiments (170328, 170804, 170814, 180105, 180226) were summarized. The Mean Fluorescence Intensity (MFI) of the TGF β standard curve measured for each experiment is depicted in the lower left panel. The MFI of the TGF-beta standard curves obtained in PTT-4 and PTT-6 medium are shown in the upper panel. The lower right panel depicts that cultures CL-MSC and WJ-MSC produce more TGF β 1 when grown in PTT-6 than when grown in PTT-4. AT-MSC and BM-MSC cultures produce equal amounts of TGF-1 when grown in PTT-6 or PTT-4. All error bars are standard deviations from different measurements for experiments 170328, 170804, 170814, 180105, 180226.

FIG. 16: ang-1 measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) were summarized. The Mean Fluorescence Intensity (MFI) of the Ang-1 standard curve measured for each experiment is depicted in the lower left panel. The MFI of the Ang-1 standard curves obtained in PTT-4 and PTT-6 medium is shown in the upper panel. The lower right panel depicts that cultures CL-MSC and WJ-MSC produced more Ang-1 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 cultures produced substantially equal amounts of Ang-1. All error bars are standard deviations from different measurements for experiments 170602, 170511, 170414, 170224, 180105, 180226.

FIG. 17: PDGF-BB measurements from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) were summarized. The Mean Fluorescence Intensity (MFI) of the PDGF-BB standard curve measured for each experiment is depicted in the lower left panel. The MFI of the PDGF-BB standard curves obtained in PTT-4 and PTT-6 medium are shown in the upper panel. Notably, PDGF-BB was not detected in all experiments.

FIG. 18: PDGF-AA measurements from 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) were summarized. The Mean Fluorescence Intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the lower left panel. The MFI of the PDGF-AA standard curves obtained in PTT-4 and PTT-6 medium are shown in the upper panel. The lower right panel depicts cultures CL-MSC, AT-MSC and BM-MSC, and WJ-MSC cultures produce slightly more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviations of the measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

FIG. 19: IL-10 measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) were summarized. The Mean Fluorescence Intensity (MFI) of the IL-10 standard curve measured for each experiment is depicted in the lower left panel. The MFI of the IL-10 standard curves obtained in PTT-4 and PTT-6 medium is shown in the upper panel. Notably, IL-10 was not detected in all experiments.

FIG. 20: VEGF measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) were summarized. The Mean Fluorescence Intensity (MFI) of the VEGF standard curve measured for each experiment is depicted in the lower left panel. The MFI of the VEGF standard curves obtained in PTT-4 and PTT-6 medium are shown in the upper panel. The lower right panel depicts cultures CL-MSC, AT-MSC and BM-MSC, and WJ-MSC produce more VEGF when grown in PTT-6 than in PTT-4. All error bars are standard deviations of different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

FIG. 21: HGF measurements for 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226) were summarized. The Mean Fluorescence Intensity (MFI) of the HGF standard curve measured for each experiment is depicted in the lower left panel. MFI of HGF standard curves obtained in PTT-4 and PTT-6 media are shown in the upper panel. The lower right panel depicts cultures CL-MSC and WJ-MSC produce more HGF when grown in PTT-6 than in PTT-4. On the other hand, cultures AT-MSC and BM-MSC did not produce as much HGF as other cultures. All error bars are standard deviations of different measurements from experiments 170602, 170511, 170414, 170224, 180105, 180226.

Cytokine analysis of PTT-6 and PTT-4 media or DMEM/F12 supernatant of CL-MSC, WJ-MSC and placental MSC-derived MSCs

Cytokine assays were performed in MSC supernatants. Measurements and analyses were performed as described above.

The objective of this experiment was to measure the relative levels of multiple (PDGF-AA, PDGF-BB, IL-10, VEGF, Ang-1 and HGF), TGF β 1 single and bFGF2 single cytokines in the cell culture supernatants. The supernatant was obtained from mesenchymal stem cells from umbilical Cord Lining (CL), from Wharton's Jelly (WJ) and from placenta. The mesenchymal stem cells are cultured in PTT-6, PPT-4 or DMEM/F12 medium.

CL-MSC cultured in PTT-4

WJ-MSC cultured in PTT-4

Placental MSCs cultured in PTT-4

CL-MSC cultured in PTT-6

WJ-MSC cultured in PTT-6

Placental MSCs cultured in PTT-6

CL-MSC cultured in DMEM/F12

WJ-MSC cultured in DMEM/F12

All samples, except the supernatant sample of the placenta, were tested in triplicate. The aim of this experiment was to generate cytokine profiles (profiles) of MSCs cultured in PTT-4 or PTT-6 and to compare the profiles of MSCs of different tissue origin (umbilical lining vs. gutta-percha vs. placental MSCs). Cytokine measurements were performed as described above. This profile will elucidate which stem cell populations grow in which media will secrete more cytokines of interest in order to promote wound healing.

FIG. 22: singleplex measurement of TGF β 1. The Mean Fluorescence Intensity (MFI) of the TGF β 1 standard curve measured for each experiment is depicted in the left panel. As can be seen from the right panel, all CL-MSCs, WJ-MSCs and placental MSCs produced more TGF β 1 when grown in PTT-6 than when grown in PTT-4 or DMEM/F12 (referred to as DMEM only in FIG. 22).

FIG. 23: the assayed measurements of PDGF-BB in the supernatants of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM/F12 are summarized. The Mean Fluorescence Intensity (MFI) of the PDGF-BB standard curve measured for each experiment is depicted in the left panel. Notably, PDGF-BB was not detected in all experiments.

FIG. 24: the analyzed measurements of IL-10 in the supernatants of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM/F12 are summarized. The Mean Fluorescence Intensity (MFI) of the VEGF standard curve measured for each experiment is depicted in the left panel. S6 represents the minimum standard used in the determination. Any sample below this is considered to be below the detection limit. As can be seen from the right panel, all CL-MSCs, WJ-MSCs and placental MSCs produced detectable levels of IL-10 when grown in PTT-6, whereas little or no IL-10 was detected when MSCs were grown in PTT-4 or DMEM/F12.

FIG. 25: the analyzed measurements of VEGF in the supernatants of CL-MSCs, WJ-MSCs and placental MSCs cultured in PTT-6, PTT-4 or DMEM/F12 were summarized. The Mean Fluorescence Intensity (MFI) of the VEGF standard curve measured for each experiment is depicted in the left panel. S1 represents the highest standard used in the determination. Any sample above this is considered to be speculative (over-concentrated). As can be seen from the right panel, all CL-MSCs, WJ-MSCs and placental MSCs produced much higher levels of VEGF when grown in PTT-6 than when MSCs were grown in PTT-4 or DMEM/F12.

FIG. 26: multiple measurements of bFGF were summarized. The Mean Fluorescence Intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the left panel. As can be seen from the right panel, cultured CL-MSCs and WJ-MSCs produce more bFGF when grown in PTT-6 than in PTT-4. It can be seen that all CL-MSCs, WJ-MSCs and placental MSCs produced much lower levels of bFGF when grown in PTT-6 than when MSCs were grown in PTT-4 or DMEM/F12.

FIG. 27 is a schematic view showing: the PDGF-AA measurements are summarized. The Mean Fluorescence Intensity (MFI) of the PDGF-AA standard curve measured for each experiment is depicted in the left panel. S6 represents the minimum standard used in the determination. Any sample below this is considered to be below the detection limit. It can be seen that all CL-MSCs, WJ-MSCs and placental MSCs produced higher levels of PDGF-AS when grown in PTT-6 than when MSCs were grown in PTT-4 or DMEM/F12.

FIG. 28: the measurement of Ang-1 is summarized. The Mean Fluorescence Intensity (MFI) of the Ang-1 standard curve measured for each experiment is depicted in the left panel. S1 represents the highest standard used in the determination. Any sample above this is considered to be speculative (over-concentrated). The right panel depicts that all CL-MSCs, WJ-MSCs and placental MSCs produce much higher levels of Ang-1 when grown in PTT-6 than MSCs when grown in PTT-4 or DMEM/F12.

FIG. 29: HGF measurements are summarized. The Mean Fluorescence Intensity (MFI) of the HGF standard curve measured for each experiment is depicted in the left panel. The right panel depicts that all CL-MSCs, WJ-MSCs and placental MSCs produce much higher levels of Ang-1 when grown in PTT-6 than MSCs when grown in PTT-4 or DMEM/F12.

The following conclusions can be drawn from the above experiments. When mesenchymal stem cells, in particular mesenchymal stem cells isolated from the umbilical cord compartment or isolated from the placenta, are cultured in PTT-6 medium, the population of mesenchymal stem cells has a significant increase in secreted factors angiopoietin (Ang-1), TGF- β 1, VEGF and HGF, compared to their production levels in PTT-4 medium or commercially available media such as DMEM/F12. Notably, PTT-6 medium is able to increase the production/secretion of these factors regardless of the natural environment/compartment of the mesenchymal stem cell population.

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

In addition, the culture of the mesenchymal stem cells in PTT-6 by the tissue isolation block method provides a highly homogeneous mesenchymal stem cell population of umbilical cord amniotic membrane (which contains 97.5% of live cells 100% of which express each of CD73, CD90 and CD105, while 99.2% of the stem cell population does not express CD45, and 100% of the stem cell population does not express CD34 and HLA-DR (see "CD 34-CD 45" and "CD 34-HLA-DR" -). Because the culture of the mesenchymal stem cell population of PTT-6 Chinese Tong glue has positive effect on the yield of the cytokines Ang-1, TGF-beta 1, VEGF and HGF, and the positive effect is the same as the positive effect on the cytokines in the umbilical cord lining stem cells, the highly homogeneous mesenchymal Chinese Tong glue stem cell population can be expected to be obtained by culturing the Chinese Tong glue in the PTT-6. Thus, it is also contemplated that tissue isolation blocks of other compartments of the umbilical cord, such as culture of umbilical cord blood vessels, will result in a similarly homogeneous Perivascular (PV) mesenchymal stem cell population. Similarly, it is expected that a population of similarly homogeneous placental mesenchymal stem cells may be produced by tissue isolates of placental tissue cultured in PTT-6, including placental amniotic membrane. Accordingly, the present invention provides a generally applicable method to obtain a population of mesenchymal stem cells, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the isolated population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

The present invention is also characterized by the following items.

1. A method of inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a medium comprising DMEM (dulbecco modified eagle medium), F12 (hamm F12 medium), M171 (medium 171) and FBS (fetal bovine serum).

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

3. The method of clause 2, wherein the population of umbilical cord mesenchymal stem cells is selected from the group consisting of a population of Amniotic Membrane (AM) mesenchymal stem cells, a population of Perivascular (PV) mesenchymal stem cells, a population of gum Wharton's Jelly (WJ) mesenchymal stem cells, a population of umbilical cord amniotic membrane mesenchymal stem cells, and a population of umbilical cord Mixed (MC) mesenchymal stem cells.

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

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

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

7. The method of any one of clauses 1 to 6, wherein the culture medium further comprises Epidermal Growth Factor (EGF) at a final concentration of about 1ng/ml to about 20 ng/ml.

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

9. The method of any one of clauses 1-8, wherein the culture medium comprises insulin at a final concentration of about 1 to 10 μ g/ml.

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

11. The method of any of clauses 1-10, 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 clauses 1 to 11, wherein the culture medium comprises all three of adenine, hydrocortisone, and 3, 3', 5-triiodo-L-thyronine sodium salt (T3).

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

14. The method of any one of items 1 to 13, wherein culturing the population of mesenchymal stem cells in the medium of any one of the preceding items 1 to 13 results in an increase in at least one of angiopoietin 1(Ang-1), TGF- β (particularly TGF- β 1), VEGF, and HGF expressed and/or secreted by the population of mesenchymal stem cells relative to a reference medium that does not comprise all DMEM (dulbecco modified eagle medium), F12 (ham F12 medium), M171 (medium 171), and FBS (fetal bovine serum).

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

16. The method of any one of the preceding items, wherein the population of mesenchymal stem cells has been isolated from its natural environment prior to culturing in the medium of any one of the preceding items 1 to 13.

17. The method of any one of items 1 to 15, comprising isolating the population of mesenchymal stem cells from a native tissue environment by culturing the native tissue in the cell culture medium of any one of the preceding items 1 to 13.

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

19. The method of clause 18, wherein the umbilical cord tissue is selected from the group consisting of whole umbilical cord tissue, tissue comprising umbilical cord amniotic membrane, tissue comprising Wharton's jelly, tissue comprising amniotic membrane, amniotic membrane and Wharton's jelly, isolated umbilical cord blood vessels, Wharton's jelly isolated from other components of umbilical cord tissue, and isolated umbilical cord amniotic membrane.

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

21. The method of any one of the preceding items 17-20, wherein the umbilical cord tissue is a portion of the entire umbilical cord, a portion of the amniotic membrane of the umbilical cord, or a portion of the amniotic membrane of the placenta.

22. The method of any of clauses 19-22, comprising culturing the umbilical cord tissue or the placental amniotic membrane tissue until the halo of cell growth of the population of amniotic mesenchymal stem cells reaches about 70-80% confluence.

23. The method of clause 22, which comprises removing the population of mesenchymal stem cells from the culture vessel used for the culturing.

24. The method of clause 23, wherein removing the population of mesenchymal stem cells from the culture container is performed by enzymatic treatment.

25. The method of clause 24, wherein the enzymatic treatment comprises a trypsin digestion.

26. The method of any of clauses 23 to 25, wherein the population of mesenchymal stem cells is transferred to a culture vessel for subculture.

27. The method of any of clauses 1-16, wherein the population of mesenchymal stem cells is transferred to a culture vessel for subculture for culture.

28. The method of clauses 26 or 27, wherein the mesenchymal cell population is 1.0x10⁶Individual cells/ml are suspended for culture or subculture.

29. The method of clause 28, wherein the population of mesenchymal stem cells is subcultured in the medium of any one of clauses 1-13.

30. The method of clause 29, wherein the population of mesenchymal stem cells is subcultured until the mesenchymal stem cells reach about 70-80% confluence.

31. The method of any of clauses 26 to 30, wherein the culturing or subculturing is performed in a self-contained bioreactor.

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

33. The method of any one of the preceding items, wherein the CO is at a temperature of 37 ℃₂Culturing in a cell culture box.

34. The method of clause 33, which comprises removing the population of mesenchymal stem cells from the culture vessel used for the (subculture).

35. The method of clause 34, wherein removing the population of mesenchymal stem cells from the culture container is performed by enzymatic treatment.

36. The method of clause 35, wherein the enzymatic treatment comprises a trypsin digestion.

37. The method of clause 36, further comprising collecting the isolated population of mesenchymal stem cells.

38. The method of any one of the preceding items, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD 105.

39. The method of any one of the preceding items, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells lack expression of: CD34, CD45 and HLA-DR (human leukocyte antigen-antigen D associated).

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

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

42. The method of clause 41, wherein the preservation is by cryopreservation.

43. An isolated population of mesenchymal stem cells, wherein at least about 90% or more of the cells of the population of stem cells express each of the following markers: CD73, CD90, and CD 105.

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

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

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

47. The mesenchymal stem cell population of any of items 43 to 46, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an Amniotic Membrane (AM) mesenchymal stem cell population, a Perivascular (PV) mesenchymal stem cell population, a Wharton's Jelly (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population and an umbilical cord Mixed (MC) mesenchymal stem cell population.

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

49. The mesenchymal stem cell population of any of items 43 to 48, wherein the population was obtained by the method of any one of items 1 to 42.

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

51. The pharmaceutical composition of clause 50, wherein the pharmaceutical composition is suitable for systemic or topical application.

52. The pharmaceutical composition of clauses 50 or 51, further comprising a pharmaceutically acceptable excipient.

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

i.250ml of DMEM

ii.118ml M171

iii.118ml DMEM/F12

iv.12.5ml Fetal Bovine Serum (FBS) (final concentration 2.5%).

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

v.1ml EGF stock (5. mu.g/ml) to give a final concentration of 10ng/ml,

insulin 0.175ml stock solution (14.28mg/ml) to give a final concentration of 5 μ g/ml.

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

56. The method of clause 55, wherein the final concentrations of the supplement in DMEM are as follows:

about 0.05 to 0.1. mu.g/ml adenine, for example about 0.025. mu.g/ml adenine,

about 1 to 10. mu.g/ml hydrocortisone,

about 0.5 to 5ng/ml 3,3 ', 5-triiodo-L-thyronine sodium salt (T3), for example 1.36ng/ml3, 3', 5-triiodo-L-thyronine sodium salt (T3).

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

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

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

60. The method of clause 59, wherein the population of umbilical cord mesenchymal stem cells is selected from the group consisting of a population of Amniotic Membrane (AM) mesenchymal stem cells, a population of Perivascular (PV) mesenchymal stem cells, a population of Wharton's Jelly (WJ) mesenchymal stem cells, a population of umbilical cord amniotic membrane mesenchymal stem cells, and a population of umbilical cord Mixed (MC) mesenchymal stem cells.

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

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

64. The cell culture medium of any one of items 61-62, wherein the medium further comprises Epidermal Growth Factor (EGF) at a final concentration of about 1ng/ml to about 20 ng/ml.

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

66. The cell culture medium of any one of clauses 61-65, wherein the medium comprises insulin at a final concentration of about 1 to 10 μ g/ml.

67. The cell culture medium of clause 66, wherein the medium comprises insulin at a final concentration of about 5 μ g/ml.

68. The cell culture medium of any one of clauses 61 to 67, wherein the 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 clause 68, wherein the medium comprises all three of adenine, hydrocortisone, and 3, 3', 5-triiodo-L-thyronine sodium salt (T3).

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

71. The cell culture medium of any one of items 61-70, wherein 500ml of the cell culture medium comprises:

i.250ml of DMEM

ii.118ml M171

iii.118ml DMEM/F12

iv.12.5ml Fetal Bovine Serum (FBS) (final concentration 2.5%).

72. The cell culture medium of clause 71, further comprising:

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

Insulin at a final concentration of 5. mu.g/ml

Insulin 0.175ml (final concentration 5. mu.g/ml).

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

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

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

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

77. The use of item 76, wherein the population of umbilical cord mesenchymal stem cells is selected from the group consisting of a population of Amniotic Membrane (AM) mesenchymal stem cells, a population of Perivascular (PV) mesenchymal stem cells, a population of Wharton's Jelly (WJ) mesenchymal stem cells, a population of umbilical cord amniotic membrane mesenchymal stem cells, and a population of umbilical cord Mixed (MC) mesenchymal stem cells.

78. The use of any one of clauses 74-77, wherein at least about 90% or more of the cells of the population of mesenchymal stem cells express each of the following markers: CD73, CD90, and CD 105.

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

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

81. A pharmaceutical composition containing three or four of Ang-1, TGF- β 1, VEGF or HGF as the only wound healing proteins.

82. The pharmaceutical composition of item 81, formulated as a liquid or lyophilized/freeze-dried formulation.

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

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

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," and "containing" are to be construed broadly and not restrictively. Furthermore, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Further, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Other embodiments of the invention will be apparent from the following claims.

Claims (58)

1. A method of inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising culturing the mesenchymal stem cell population in a medium comprising DMEM (dulbecco modified eagle medium), F12 (hamm F12 medium), M171 (medium 171) and FBS (fetal bovine serum).

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

3. The method of claim 2, wherein the population of umbilical cord mesenchymal stem cells is selected from the group consisting of a population of Amniotic Membrane (AM) mesenchymal stem cells, a population of Perivascular (PV) mesenchymal stem cells, a population of Wharton's Jelly (WJ) mesenchymal stem cells, a population of umbilical cord amniotic membrane mesenchymal stem cells, and a population of umbilical cord Mixed (MC) mesenchymal stem cells.

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

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

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

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

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

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

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

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

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

14. The method of any one of claims 1 to 13, wherein culturing the population of mesenchymal stem cells in the medium of any one of the preceding claims 1 to 13 results in an increase in at least one of angiopoietin 1(Ang-1), TGF- β (particularly TGF- β 1), VEGF, and HGF expressed and/or secreted by the population of mesenchymal stem cells relative to a reference medium that does not comprise all DMEM (dulbecco modified eagle medium), F12 (hahm F12 medium), M171 (medium 171), and FBS (fetal bovine serum).

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

16. The method of any one of the preceding claims, wherein the population of mesenchymal stem cells has been isolated from its natural environment prior to culturing in the medium of any one of the preceding claims 1-13.

17. The method of any one of claims 1 to 15, comprising isolating the population of mesenchymal stem cells from a native tissue environment by culturing a native tissue in the cell culture medium of any one of the preceding claims 1 to 13.

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

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

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

21. The method of any one of the preceding claims 17 to 20, wherein the umbilical cord tissue is a portion of the entire umbilical cord, a portion of the amniotic membrane of the umbilical cord, or a portion of the placental amniotic membrane.

22. The method of any one of claims 19 to 21, comprising culturing the umbilical cord tissue or the placental amniotic membrane tissue until the halo of cell growth of the population of amniotic mesenchymal stem cells reaches about 70-80% confluence.

23. The method of claim 22, comprising removing the population of mesenchymal stem cells from the culture vessel used for the culturing.

24. The method of claim 23, wherein removing the population of mesenchymal stem cells from the culture vessel is performed by enzymatic treatment.

25. The method of claim 24, wherein the enzymatic treatment comprises a trypsin digestion.

26. The method of any one of claims 23 to 25, wherein the population of mesenchymal stem cells is transferred to a culture vessel for subculture.

27. The method of any one of claims 1 to 16, wherein the population of mesenchymal stem cells is transferred to a culture vessel for subculture for culture.

28. The method of claim 26 or 27, wherein the mesenchymal cell population is at 1.0x10⁶Individual cells/ml are suspended for culture or subculture.

29. The method of claim 28, wherein the population of mesenchymal stem cells is subcultured in the medium of any one of claims 1-13.

30. The method of claim 29, wherein the population of mesenchymal stem cells is subcultured until the mesenchymal stem cells reach about 70-80% confluence.

31. The method of any one of claims 26 to 30, wherein the culturing or subculturing is performed in a self-contained bioreactor.

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

33. The method of any one of the preceding claims, wherein at a temperature of 37 ℃ at CO₂Culturing in a cell culture box.

34. The method of claim 33, comprising removing the population of mesenchymal stem cells from the culture vessel used for the (subculture).

35. The method of claim 34, wherein removing the population of mesenchymal stem cells from the culture vessel is performed by enzymatic treatment.

36. The method of claim 35, wherein the enzymatic treatment comprises a trypsin digestion.

37. The method of claim 36, further comprising collecting the isolated population of mesenchymal stem cells.

38. The method of any one of the preceding claims, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90, and CD 105.

39. The method of any one of the preceding claims, wherein at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the isolated mesenchymal stem cells lack expression of: CD34, CD45 and HLA-DR (human leukocyte antigen-antigen D associated).

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

41. The method of any one of the preceding claims, further comprising preserving the isolated stem/progenitor cell population for further use.

42. The method of claim 41, wherein preservation is by cryopreservation.

43. An isolated population of mesenchymal stem cells, wherein at least about 90% or more of the cells of the population of stem cells express each of the following markers: CD73, CD90, and CD 105.

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

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

46. The population of mesenchymal stem cells of any of claims 43 to 45, wherein the population of mesenchymal stem cells is selected from the group consisting of a population of umbilical cord mesenchymal stem cells, a population of placental mesenchymal stem cells, a population of umbilical cord blood mesenchymal stem cells, a population of bone marrow mesenchymal stem cells and a population of adipose-tissue derived mesenchymal stem cells.

47. The population of mesenchymal stem cells of any one of claims 43 to 46, wherein the population of umbilical cord mesenchymal stem cells is selected from the group consisting of a population of Amniotic Membrane (AM) mesenchymal stem cells, a population of Perivascular (PV) mesenchymal stem cells, a population of Wharton's Jelly (WJ) mesenchymal stem cells, a population of umbilical cord amniotic membrane mesenchymal stem cells and a population of umbilical cord Mixed (MC) mesenchymal stem cells.

48. The mesenchymal stem cell population of any one of claims 43 to 47, wherein the population is obtainable by the method of any one of claims 1 to 42.

49. The mesenchymal stem cell population of any one of claims 43 to 48, wherein the population is obtained by the method of any one of claims 1 to 42.

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

51. The pharmaceutical composition of claim 50, wherein the pharmaceutical composition is suitable for systemic or topical application.

52. The pharmaceutical composition of claim 50 or 51, further comprising a pharmaceutically acceptable excipient.

53. Use of a cell culture medium for inducing or improving wound healing properties of a mesenchymal stem cell population, the medium comprising:

DMEM at a concentration of about 55 to 65% (v/v),

f12 at a concentration of about 5 to 15% (v/v),

m171 at a concentration of about 15 to 30% (v/v) and

FBS at a concentration of about 1 to 8% (v/v).

54. The use of claim 53, wherein the medium comprises DMEM at a concentration of about 57.5 to 62.5% (v/v), F12 at a concentration of about 7.5 to 12.5% (v/v), M171 at a concentration of about 17.5 to 25.0% (v/v), and FBS at a concentration of about 1.75 to 3.5% (v/v).

55. The use of claim 54, wherein said medium comprises DMEM at a concentration of about 61.8% (v/v), F12 at a concentration of about 11.8% (v/v), M171 at a concentration of about 23.6% (v/v), and FBS at a concentration of about 2.5% (v/v).

56. Use of the cell culture medium of any one of claims 53-55 for isolating a population of mesenchymal stem cells.

57. The use of any one of claims 53 to 56, wherein said population of mesenchymal stem cells is selected from the group consisting of a population of umbilical cord mesenchymal stem cells, a population of placental mesenchymal stem cells, a population of umbilical cord blood mesenchymal stem cells, a population of bone marrow mesenchymal stem cells, and a population of adipose-tissue derived mesenchymal stem cells.

58. The use of claim 57, wherein the population of umbilical cord mesenchymal stem cells is selected from the group consisting of a population of Amniotic Membrane (AM) mesenchymal stem cells, a population of Perivascular (PV) mesenchymal stem cells, a population of Wharton's Jelly (WJ) mesenchymal stem cells, a population of umbilical cord amniotic membrane mesenchymal stem cells, and a population of umbilical cord Mixed (MC) mesenchymal stem cells.

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