WO2019199234A1 - Procédé d'induction ou d'amélioration des propriétés de cicatrisation de cellules souches mésenchymateuses - Google Patents

Procédé d'induction ou d'amélioration des propriétés de cicatrisation de cellules souches mésenchymateuses Download PDF

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WO2019199234A1
WO2019199234A1 PCT/SG2019/050204 SG2019050204W WO2019199234A1 WO 2019199234 A1 WO2019199234 A1 WO 2019199234A1 SG 2019050204 W SG2019050204 W SG 2019050204W WO 2019199234 A1 WO2019199234 A1 WO 2019199234A1
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mesenchymal stem
cell population
stem cell
cells
umbilical cord
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PCT/SG2019/050204
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English (en)
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Toan Thang Phan
Gavin TAN
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Cellresearch Corporation Pte. Ltd.
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Priority to JP2020555404A priority Critical patent/JP2021526125A/ja
Priority to AU2019250653A priority patent/AU2019250653A1/en
Priority to KR1020207030469A priority patent/KR20200143399A/ko
Priority to SG11202009687PA priority patent/SG11202009687PA/en
Priority to BR112020020092-1A priority patent/BR112020020092A2/pt
Priority to EP19784508.4A priority patent/EP3775163A4/fr
Application filed by Cellresearch Corporation Pte. Ltd. filed Critical Cellresearch Corporation Pte. Ltd.
Priority to CN201980039206.6A priority patent/CN112262211A/zh
Priority to CA3095490A priority patent/CA3095490A1/fr
Publication of WO2019199234A1 publication Critical patent/WO2019199234A1/fr
Priority to ZA2020/05967A priority patent/ZA202005967B/en
Priority to PH12020551658A priority patent/PH12020551658A1/en
Priority to JP2024035680A priority patent/JP2024069365A/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/395—Thyroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • the present invention relates to a method of inducing or improving wound healing properties of a mesenchymal stem cell population.
  • the invention is also directed to a cell culture medium suitable for inducing or improving wound healing properties of mesenchymal stem cells and/or suitable for isolating a mesenchymal stem cell population.
  • the invention is also directed to a pharmaceutical composition and uses of the isolated mesenchymal stem cell population.
  • the invention is also directed to methods of treating a disease or disorder comprising administering a mesenchymal stem cell population or a pharmaceutical composition containing such a mesenchymal stem cell population of the invention to a subject in need thereof.
  • the invention is also directed to an extremely homogenous and well-defined mesenchymal stem cell population, for example of the umbilical cord or of the placenta.
  • MSCs human mesenchymal stem cells derived from the amniotic membrane of the umbilical cord (umbilical cord lining (CL-MSCs), umbilical cord blood (CB-MSCs), placenta (P-MSCs), and Wharton’s jelly (WJ-MSCs)
  • CL-MSCs amniotic membrane of the umbilical cord
  • CB-MSCs umbilical cord blood
  • P-MSCs placenta
  • WJ-MSCs Wharton’s jelly
  • mesenchymal stem cells of the amniotic membrane can easily be obtained using the protocol as described in US patent application 2006/0078993 and International patent application W02006/019357, it would be of advantage for clinical trials with these cord lining MSC to have at hand a method that allows to isolate a population of these cord lining MSC’s that is highly homogenous and can thus be used for clinical trials. In addition, it would be an advantage to have at hand a method that induces or improves wound healing properties of a mesenchymal stem cell population in general.
  • the invention provides a method of inducing or improving wound healing properties of a mesenchymal stem cell population, method comprising cultivating the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum).
  • DMEM Denbecco’s modified eagle medium
  • F12 Haam’s F12 Medium
  • M171 Medium 171
  • FBS Fetal Bovine Serum
  • the mesenchymal stem cell population may be a mesenchymal stem cell population of the umbilical cord, a placental mesenchymal stem cell population, a mesenchymal stem cell population of the cord blood, a mesenchymal stem cell population of the bone marrow, or an adipose-tissue derived mesenchymal stem cell population.
  • the invention provides an isolated mesenchymal stem population, wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD 105.
  • the isolated mesenchymal stem population lack expression of the following markers: CD34, CD45 and HLA-DR.
  • 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 cells of the isolated mesenchymal stem cell population express each of CD73, CD90 and CD 105.
  • 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 cells of the isolated mesenchymal stem cell population preferably lack expression of the markers CD34, CD45 and HLA-DR.
  • the mesenchymal stem cell population may be obtained by a method of inducing or improving wound healing properties of the first aspect.
  • the method of the first aspect can also be a method of isolating a mesenchymal stem cell population.
  • the invention provides a pharmaceutical composition containing a mammalian cell of (the second aspect of) the invention.
  • the invention provides a method of making a culture medium for either inducing or improving wound healing properties of a mesenchymal stem cell population or for isolating a mesenchymal stem cell population, the method comprising mixing to obtain a final volume of 500 ml culture medium:
  • Fetal Bovine Serum iv. 12.5 ml Fetal Bovine Serum (FBS) to obtain a final concentration of 2.5% (v/v).
  • the invention provides a cell culture medium obtainable by the method of the fourth aspect.
  • the invention provides a method of isolating a mesenchymal stem cell population, comprising cultivating the mesenchymal stem cell population in the culture medium prepared by the method of the fourth aspect.
  • the invention provides a cell culture medium comprising:
  • the invention provides the use of a cell culture medium of the seventh aspect for inducing or improving wound healing properties of a mesenchymal stem cell population or for isolating the mesenchymal stem cell population.
  • Fig. 1 shows the technical information sheet of Lonza for Dulbecco’s modified eagle medium, including the catalogue number of the DMEM used for the making of the illustrative example of a medium of the invention (PTT-6) in the Experimental Section;
  • Fig. 2 shows the technical information sheet of Lonza for Ham’s F12 medium
  • FIG. 3 shows the technical information sheet of Lonza for DMEM:Fl2 (1: 1) medium, including the catalogue number of the DMEM:Fl2 (1: 1) medium used for the making of the illustrative example of a medium of the invention (PTT-6) in the Experimental Section;
  • Fig. 4 shows the technical information sheet of Life Technologies Corporation for M 171 medium, including the catalogue number of the M 171 medium used for the making of the illustrative example of a medium of the invention (PTT-6) in the Experimental Section;
  • Fig. 5 shows the list of ingredients, including their commercial supplier and the catalogue number that have been used in the Experimental Section forthe making of the medium PTT-6.
  • Figs. 6A-C show the results of flow cytometry experiments in which mesenchymal stem cells isolated from the umbilical cord have been analysed for the expression of the mesenchymal stem cell markers CD73, CD90 and CD105.
  • mesenchymal stem cells were isolated from umbilical cord tissue by cultivation of the umbilical cord tissue in three different cultivation media, followed by subculturing of the mesenchymal stem cells in the respective medium.
  • the three following culture media were used in these experiments: a) 90% (v/v/ DMEM supplemented with 10 % FBS (v/v), b) the culture medium PTT-4 described in US patent application US 2008/0248005 and the corresponding International patent application W02007/046775 that consist of 90% (v/v) CMRL1066, and 10% (v/v) FBS (see paragraph [0183] of W02007/046775 and c) the culture medium of the present invention PTT-6 the composition of which is described herein.
  • PTT-6 the composition of which is described herein.
  • Fig. 6A shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and cultivation in DMEM/lO% FBS
  • Fig. 6B shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD 105 after isolation from umbilical cord tissue and cultivation in PTT-4
  • Fig.6C shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and cultivation in PTT-6.
  • Figs. 7A-B show the results of flow cytometry experiments in which mesenchymal stem cells isolated from the umbilical cord have been analysed for their expression of stem cells markers (CD73, CD90 and CD105, CD34, CD45 and HLA-DR (Human Leukocyte Antigen - antigen D Related) that are used for defining the suitability of multipotent human mesenchymal stem cells for cellular therapy and compared to the expression of these markers by bone marrow mesenchymal stem cells.
  • stem cells markers CD73, CD90 and CD105, CD34, CD45 and HLA-DR (Human Leukocyte Antigen - antigen D Related) that are used for defining the suitability of multipotent human mesenchymal stem cells for cellular therapy and compared to the expression of these markers by bone marrow mesenchymal stem cells.
  • the mesenchymal stem cells of the aminotic membrane of the umbilical cord were isolated from umbilical cord tissue by cultivation of the umbilical cord tissue in the culture medium of the present invention PTT-6 while the bone marrow mesenchymal stem cells were isolated from human bone marrow using a standard protocol.
  • Fig. 7A shows the percentage of isolated mesenchymal cord lining stem cells that express the stem cell markers CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR after isolation from umbilical cord tissue and cultivation in PTT-6 medium while Fig. 7B shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA- DR.
  • Fig. 8 shows a set up of the experiments with dark grey wells, standards reconstituted with PTT-4 medium and corresponding samples from MSCs cultured in PTT-4; Light grey wells, standards reconstituted with PTT-6 medium and corresponding samples from MSCs cultured in PTT-6. Samples in italic are control supernatants that are being tested as part of recurrent testing of stored samples.
  • Fig. 9 shows singleplex measurement of TGFpl. As can be seen cultures CL- MSC and WJ- MSC produce more TGFP 1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced more or less equal amounts of TGFpl when grown in PTT-6 or PTT-4. All error bars are standard deviation from triplicate measurements. [0027] Fig.lOA shows multiplex measurement of PDGF-AA. As can be seen cultures CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produce more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviation from triplicate measurements.
  • Fig.lOB shows multiplex measurement of VEGF.
  • cultures CL- MSC, WJ-MSC, AT-MSC and BM-MSC cultures produce more VEGF when grown in PTT- 6 than when grown in PTT-4. All error bars are standard deviation from triplicate measurements.
  • Fig. 10C shows multiplex measurement of Ang- 1. As can be seen cultures CL-
  • Fig. 11 shows multiplex measurement of HGF. As can be seen cultures CL- MSC and WJ-MSC cultures produce much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC essentially did not produce any HGF. All error bars are standard deviation from triplicate measurements.
  • Fig. 12 shows multiplex measurement of PDGF-AA.
  • cultures CL-MSC and WJ-MSC cultures produce 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 culture media. All error bars are standard deviation from triplicate measurements.
  • Fig. 13A shows multiplex measurement of VEGF. As can be seen cultures CL-
  • MSC, WJ-MSC, AT-MSC and BM-MSC cultures produce more VEGF when grown in PTT- 6 than when grown in PTT-4. All error bars are standard deviation from triplicate measurements.
  • Fig. 13B shows multiplex measurement of Ang-l.
  • cultures CL- MSC and WJ-MSC cultures produce much more Ang-l when grown in PTT-6 than when grown in PTT-4.
  • Cultures AT-MSC and BM-MSC essentially did not produce any Ang-l. All error bars are standard deviation from triplicate measurements.
  • Fig. 13C shows multiplex measurement of HGF. As can be seen cultures CL- MSC and WJ-MSC cultures produce much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC essentially did not produce any HGF. All error bars are standard deviation from triplicate measurements.
  • Fig. 14 shows multiplex measurement of bFGF.
  • cultures CL- MSC and WJ-MSC cultures produce 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 deviation from triplicate measurements.
  • Fig. 15 summarizes measurement of TGFp i over 5 different experiments (170328, 170804, 170814, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the TGFB standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the TGFB standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that cultures CL-MSC and WJ- MSC produce more TGFP 1 when grown in PTT-6 than when grown in PTT-4.
  • AT-MSC and BM-MSC cultures produced equal amounts of TGFpl when grown in PTT-6 or PTT-4. All error bars are standard deviation from different measurements for the experiments 170328, 170804, 170814, 180105, 180226.
  • Fig. 16 summarizes measurement of Ang-l over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the Ang-l standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the Ang-l standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that cultures CL- MSC and WJ- MSC produce more Ang- 1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced essentially equal amounts of Ang-l when grown in PTT-6 or PTT-4. All error bars are standard deviation from different measurements for the experiments 170602, 170511, 170414, 170224, 180105, 180226.
  • Fig. 17 summarizes measurement of PDGF-BB over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the PDGF-BB standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the PDGF-BB standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs. Notably, in none of the experiments PDGF-BB has been detected.
  • Fig. 18 summarizes measurement of PDGF-AA over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the PDGF-AA standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the PDGF-AA standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that 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.
  • Fig. 19 summarizes measurement of IL-10 over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the IL-10 standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the IL-10 standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs. Notably, in none of the experiments IL-10 has been detected.
  • Fig. 20 summarizes measurement of VEGF over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the VEGF standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the VEGF standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that cultures CL- MSC, AT-MSC and BM-MSC and WJ-MSC produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviation from different measurements for the experiments 170602, 170511, 170414, 170224, 180105, 180226.
  • Fig. 21 summarizes measurement of HGF over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the HGF standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the HGF standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that cultures CL-MSC, and WJ- MSC produce more HGF when grown in PTT-6 than when grown in PTT-4.
  • cultures AT-MSC and BM-MSC did not produce as much HGF as the other cultures. All error bars are standard deviation from different measurements for the experiments 170602, 170511, 170414, 170224, 180105, 180226.
  • FIG. 22 Singleplex measurement of TGFP 1 .
  • Mean fluorescent intensity (MFI) measured for the standard TGFP 1 curves across experiments is depicted in the graph on the left-hand side As can be seen the graph on the right-hand sidall of CL-MSC, WJ- MSC and placental MSC produce more TGFP 1 when grown in PTT-6 than when grown in PTT-4 or DMEM/F12 (referred to only as DMEM in Fig. 22).
  • MFI Mean fluorescent intensity
  • Figure 23 Summarizes measurement of PDGF-BB in the analysed supernatants of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM/F12. Mean fluorescent intensity (MFI) measured for the PDGF-BB standard curves across experiments is depicted in the graph on the left-hand side. Notably, in none of the experiments PDGF-BB has been detected.
  • MFI Mean fluorescent intensity
  • FIG. 24 Summarizes measurement of IL-10 in the analysed supernatants of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM/F12.
  • Mean fluorescent intensity (MFI) measured for the VEGF standard curves across experiments is depicted in the graph on the left-hand side.
  • S6 denotes the lowest standard used in the assay. Any samples that fall below are considered below detection.
  • all of CL-MSC, WJ- MSC and placental MSC produce detectable levels of IL- 10 when grown in PTT-6 while little or no IL- 10 were detected when the MSC’ s were grown in PTT-4 or DMEM/F12
  • Figure 25 Summarizes measurement of VEGF in the analysed supernatants of
  • FIG. 26 Summarizes multiplex measurement of bFGF.
  • Mean fluorescent intensity (MFI) measured for the PDGF-AA standard curves across experiments is depicted in the graph on the left-hand side.
  • MFI Mean fluorescent intensity
  • CL-MSC and WJ-MSC produce more bFGF when grown in PTT-6 than when grown in PTT-4.
  • all of CL-MSC, WJ- MSC and placental MSC produce much lower levels of bFGF when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • Figure 27 Summarizes measurement of PDGF-AA. Mean fluorescent intensity
  • MFI measured for the PDGF-AA standard curves across experiments is depicted in the graph on the left-hand side.
  • S6 denotes the lowest standard used in the assay. Any samples that fall below are considered below detection.
  • all of CL-MSC, WJ- MSC and placental MSC produce higher levels of PDGF-AS when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • FIG. 28 Summarizes measurement of Ang-l.
  • Mean fluorescent intensity (MFI) measured for the Ang- 1 standard curves across experiments is depicted in the graph on the left-hand side.
  • S 1 denotes the highest standard used in the assay. Any samples that fall above are considered extrapolated (too concentrated).
  • the graph on the right-hand side depicts that all of CL-MSC, WJ- MSC and placental MSC produce much higher levels of Ang-l when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • FIG. 29 Summarizes measurement of HGF.
  • Mean fluorescent intensity (MFI) measured for the HGF standard curves across experiments is depicted in the graph on the left-hand side.
  • the graph on the right-hand side depicts that all of CL-MSC, WJ- MSC and placental MSC produce much higher levels of Ang-l when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • the invention is directed to a method of inducing or improving wound healing properties of a mesenchymal stem cell population, the method comprising cultivating the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum).
  • DMEM Denbecco’s modified eagle medium
  • F12 Haam’s F12 Medium
  • M171 Medium 171
  • FBS Fetal Bovine Serum
  • the induction or improvement of the wound healing properties of the mesenchymal stem cell population is caused by the ability of the medium of the present invention to increase the expression and/or secretion of at least one, two, three or all four of Angiopoietin 1 (Ang-l), TGF-pl, VEGF, and HGF by the mesenchymal stem cell population.
  • Ang-l Angiopoietin 1
  • TGF-pl TGF-pl
  • VEGF vascular endothelial growth factor 1
  • Examples 23-26 of WO 2007/046775 showing that such a mesenchymal stem cell population of the amniotic membrane of the umbilical cord (UCMC) alleviate full thickness bums (Example 23), partial thickness wounds (Example 24), non-healing radiation wound (Example 25) as well as non healing diabetic wound and non-healing diabetic foot wounds (Example 26)).
  • UCMC amniotic membrane of the umbilical cord
  • the finding of the present invention that a combined increase in the amount of Ang- 1 , TGF-b 1 , VEGF, and/or HGF that a mesenchymal stem cell population produces is to improve or improve the wound healing properties of this stem cell population also open up to mimicking the wound healing properties of the stem cell population by an composition/solution that contains three or four of Ang-l, TGF-b 1 , VEGF, or HGF as the only wound healing proteins.
  • angiopoietin 1 (Ang- 1), TGF-b!, VEGF, and HGF in the wound healing process is known to the person skilled in the art.
  • Angiopoietin 1 for the involvement of Angiopoietin 1 in wound healing, see, for example, Li et al. Stem Cell Research & Therapy 2013, 4: 113 “Mesenchymal stem cells modified with angiopoietin- 1 gene promote wound healing” or Bitto et al,“Angiopoietin- 1 gene transfer improves the impaired wound healing of the genetically diabetic mice without increasing VEGF expression”, Clinical Science May 14, 2008, 114 (12) 707-718.
  • Li et al the angiopoietin- 1 gene was inserted into bone marrow mesenchymal stem cells and the results showed that that“Angl-MSCs significantly promoted wound healing with increased epidermal and dermal regeneration, and enhanced angiogenesis compared with MSCs, Ad- Angl or sham treatment.”
  • Angl-MSCs significantly promoted wound healing with increased epidermal and dermal regeneration, and enhanced angiogenesis compared with MSCs, Ad- Angl or sham treatment.
  • MSCs mesenchymal stem cells
  • cultivation of“natural” mesemchymal stem cells in a culture medium such as PTT-6 provide conditions under which for example, cord tissue mesenchymal stem cells (i.e. a mesenchymal stem cell population that is cultivated in PTT-6) produce increased level of Ang-l and thus render the mesenchymal stem cells suitable for wound healing or further improve their wound healing properties.
  • cord tissue mesenchymal stem cells i.e. a mesenchymal stem cell population that is cultivated in PTT-6
  • the present invention provides the advantage that instead of genetically modifying naturally occurring mesenchymal stems to induce wound healing properties in mesenchymal stem cells (which is not only laboursome but also not a preferred option for therapeutic applications because of the inherent risks of gene therapy) the wound healing properties of naturally occurring mesenchymal stem cells are induced or enhanced by “simple” cultivation of a mesenchymal stem cell population in the culture medium of the invention. This approach is easier, safer and also more cost efficient.
  • HGF Hepatocyte Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • TGF-P2, and TGF-P3 in wound healing, in particular healing of chronic/non-healing wounds see for example, Ramirez et al.“The Role of TGFb Signaling in Wound Epithelialization” Advances In Wound Care, Volume 3, Number 7, 2013, 482-491 or Pakyari et al., Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing, Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.
  • the present invention has the further surprising advantage that cultivation in the culture medium of the present invention provides for the isolation of a mesenchymal stem cell population such as an mesenchymal stem cell population of the amniotic membrane of umbilical cord of which more than 90 %, or even 99 % or more of the cells are positive for the three mesenchymal stem cell markers CD73, CD90 and while at the same these stem cells lack expression of CD34, CD45 and HLA-DR (see the Experimental Section), meaning 99 % or even more cells of this population express the stem cell markers CD73, CD90 and CD105 while not expressing the markers CD34, CD45 and HLA-DR.
  • a mesenchymal stem cell population such as an mesenchymal stem cell population of the amniotic membrane of umbilical cord of which more than 90 %, or even 99 % or more of the cells are positive for the three mesenchymal stem cell markers CD73, CD90 and while at the same these stem cells lack expression of CD34, CD45 and HLA-DR (see
  • Such an extremely homogenous and well-defined cell population is the ideal candidate for clinical trials and cell-based therapies since, they for example, fully meet the criteria generally accepted for human mesenchymal stem cells to be used for cellular therapy as defined, for example, by Dominici et al,“Minimal criteria for defining multipotent mesenchymal stromal cells.
  • the present invention provides the further advantage to provide the amounts of stem cells that are needed for therapeutic applications such as their use in wound healing in a cost efficient manner.
  • all components used for making the culture medium of the present invention are commercially available in GMP quality. Accordingly, the present invention opens the route to the GMP production of a highly homogenous mesenchymal stem cell population, for example of placental tissue or umbilical cord tissue, for example, a mesenchymal stem cell population of the amniotic membrane of the umbilical cord or a mesenchymal stem cell population of Wharton’s jelly.
  • the mesenchymal stem cell population that is being rendered suitable for wound healing may be any suitable mesenchymal stem cell known in the art, for example, an adult stem cell population or a neonatal stem cell.
  • the mesenchymal stem cell population may be derived from any mammalian tissue or compartment/body part known to contain mesenchymal stem cells.
  • the mesenchymal stem cell population may be a mesenchymal stem cell population of the umbilical cord (these are examples of neonatal stem cells), a placental mesenchymal stem cell population (also a further example of neonatal stem cells), a mesenchymal stem cell population of the cord- placenta junction (a further example of a neonatal stem cell population), a mesenchymal stem cell population of the cord blood (yet a further example of neonatal stem cells), a mesenchymal stem cell population of the bone marrow (which may be an adult stem cell population), or an adipose-tissue derived mesenchymal stem cell population (yet an another example of an adult stem cell population).
  • the mesenchymal stem cell population of the umbilical cord may be (derived) from any compartment of umbilical cord tissue that contains mesenchymal stem cells.
  • the mesenchymal stem cell population may be a mesenchymal stem cell population of the amnion (AM), a perivascular (PV) mesenchymal stem cell population, a mesenchymal stem cell population of Wharton’s jelly (WJ), a mesenchymal stem cell population of the amniotic membrane of umbilical cord but also a mixed mesenchymal stem cell population of the umbilical cord (MC), meaning a population of mesenchymal stem cells that includes stem cells of two or more of these compartments.
  • AM amnion
  • PV perivascular
  • WJ mesenchymal stem cell population of Wharton’s jelly
  • MC mixed mesenchymal stem cell population of the umbilical cord
  • a mixed mesenchymal stem cell population of the umbilical cord can, for example, be obtained by removing the arteries and veins from the umbilical cord tissue, cutting the remaining tissue and the Wharton’s jelly into piece and and cultivating the umbilical cord tissue (by tissue explant) in the culture medium of the present invention.
  • a mixed mesenchymal stem cell population of the umbilical cord may also be obtained by culturing entire umbilical cord tissue with intact umbilical vessels as tissue explant under the conditions (cultivation in serum-supplemented DMEM with 10% fetal bovine serum, 10% horse serum, and 1% Penicillin/Streptomycin) as described by Schugar et al.“High harvest yield, high expansion, and phenotype stability of CD 146 mesenchymal stromal cells from whole primitive human umbilical cord tissue. Journal of biomedicine & biotechnology. 2009; 2009:789526”.
  • a mesenchymal stem cell population of the cord-placenta junction can be isolated as described by Beeravolu et al.“Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.” J Vis Exp. 2017; (122): 55224.
  • the mesenchymal stem cell population that is cultivated in the present invention in a culture medium comprising DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum) to induce or improve its wound healing properties can be isolated from its natural environment prior to cultivation in the culture medium of the present invention.
  • DMEM Denbecco’s modified eagle medium
  • F12 Ham’s F12 Medium
  • M171 Medium 171
  • FBS Fetal Bovine Serum
  • a mesenchymal stem cell population of the umbilical cord a mesenchymal stem cell population of the placenta or an adipose-tissue derived mesenchymal stem cell population.
  • a stem cell population say a mesenchymal stem cell population of Wharton’s jelly may first be isolated as described above 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” and then be subjected to cultivation of the isolated mesenchymal stem cell population in the culture medium of the present invention that comprises DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum).
  • DMEM Disbecco’s modified eagle medium
  • F12 Ham’s F12 Medium
  • M171 Medium 171
  • a placental mesenchymal stem cell population may be isolated from placenta as described in European patent application EP1 288 293, Talwadekar et al,“Cultivation and Cryopreservation of Cord Tissue MSCs with Cord Blood AB Plasma” Biomed Res J 2014;1(2): 126-136, Talwadekar et al, "Placenta-derived mesenchymal stem cells possess better immunoregulatory properties compared to their cord-derived counterparts - a paired sample study” Scientific Reports 5: 15784 (2015), or Beeravolu et al.“Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.” J Vis Exp.
  • an adipose-tissue derived mesenchymal stem cell population may be isolated as described by Schneider et al,“Adipose-derived mesenchymal stem cells from liposuction and resected fat are feasible sources for regenerative medicine” Eur J Med Res. 2017; 22: 17 as the references cited therein and subsequently cultivated in the culture medium of the present invention (cf, also the Experimental Section).
  • a mesenchymal stem cell population of the cord-placenta junction can first be isolated as described by Beeravolu et al.“Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.” J Vis Exp. 2017; (122): 55224 and subsequently cultivated in the culture medium of the present invention.
  • the mesenchymal stem cell population can be isolated directly from its natural tissue environment by cultivating the natural tissue in the cell culture medium of the invention.
  • Such a methodology is particularly suited for cultivation of mesenchymal stem cell populations from umbilical cord tissue, placental tissue (the placental tissue may, for example, comprise or be the amniotic membrane of placenta) or from the cord-placenta junction.
  • the culture medium of the present invention therefore also allows the isolation of a mesenchymal stem cell population (also referred hereas as“mesenchymal stem cells”) from its natural environment. Accordingly, the culture medium of the present invention also isolation of a mesenchymal stem cell population under conditions that allow cell proliferation of the mesenchymal stem/progenitor cells without differentiation of the mesenchymal stem/progenitor cells.
  • the culture medium of the present invention allows the isolation of mesenchymal stem cell population from the amniotic membrane under conditions that allow cell proliferation of the mesenchymal stem/progenitor cells without differentiation of the mesenchymal stem/progenitor cells.
  • the isolated mesenchymal stem/progenitor cell population has the capacity to differentiate into multiple cell types as described in US patent application 2006/0078993, US patent 9,085,755, International patent application W02006/019357, US patent 8,287,854 or W02007/046775, for instance.
  • the mesenchymal stem cells of the amniotic membrane of the umbilical cord have a spindle shape, express the following genes: POU5fl, Bmi-l, leukemia inhibitory factor (LIF), and secrete Activin A and Follistatin.
  • the mesenchymal stem cells isolated in the present invention can, for example, be differentiated into any type of mesenchymal cell 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 (for example, b-islet cells) or neurectodermal cells.
  • the stem cells isolated in the present invention can be differentiated in vitro in order to subsequently use the differentiated cell for medical purposes.
  • mesenchymal stem cells of the invention can be used in their undifferentiated state for cell-based therapy, for example, for wound healing purposes such as treatment of burns or chronic diabetic wounds.
  • the mesenchymal stem cells of the invention can either serve to promote wound healing by interacting with the surrounding diseased tissue or can also differentiate into a respective skin cell (cf., again W02007/046775, for example).
  • such a mesenchymal stem cell population described herein can be isolated and cultivated (i.e. are derived) from any umbilical cord tissue as long as the umbilical cord tissue contains the amniotic membrane (which is also referred to as “cord lining”). Accordingly, the mesenchymal stem cell population can be isolated from (pieces of) the entire umbilical cord as described in the Experimental section of the present application. This umbical cord tissue may thus contain, in addition to the amniotic membrane, any other tissue/component of the umbilical cord.
  • the amniotic membrane of the umbilical cord is the outmost part of the umbilical cord, covering the cord.
  • 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).
  • these three blood vessels are embedded in the Wharton's jelly, a gelatinous substance made largely from mucopolysaccharides.
  • the umbilical cord tissue used in the present invention can also comprise this one vein, the two arteries and the Wharton's jelly.
  • the use of such an entire (intact) section of the umbilical cord has the advantage that the amniotic membrane does not need to be separated from the other components of the umbilical cord. This reduces the isolation steps and thus makes the method of the present invention, simpler, faster, less error prone and more economical - which are all important aspects for the GMP production that is necessary for therapeutic application of the mesenchymal stem cells.
  • the isolation of the mesenchymal stem cells can thus start by tissue explant, which may be followed by subsequent subculturing (cultivation) of the isolated mesenchymal stem cells if greater amounts of the mesenchymal stem cells are desired, for example, for use in clinical trials.
  • tissue explant which may be followed by subsequent subculturing (cultivation) of the isolated mesenchymal stem cells if greater amounts of the mesenchymal stem cells are desired, for example, for use in clinical trials.
  • tissue explant or“tissue explant method” is used in its regular meaning in the art to refer a method in which a tissue (for example, placental tissue or umbilical cord tissue), once being harvested, or a piece of the tissue is being placed in a cell culture dish containing culture (growth) medium and by which over time, the stem cells migrate out of the tissue onto the surface of the dish. These primary stem cells can then be further expanded and transferred into fresh dishes through micropropagation (subculturing) as also described here.
  • tissue for example, placental tissue or umbilical cord tissue
  • a mesenchymal stem cell population of the present invention for example, 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, while in the subsequent subculturing a working cell bank can be obtained.
  • a mesenchymal stem cell population of the invention in particular a population of the mesenchymal stem cells of which at least about 97 % or more, 98% or more or 99 % or more of the cells express each of the markers CD73, CD90 and CD105 and lack expression of each of the markers: CD34, CD45 and HLA-DR
  • a cell population of the working cell bank will be typically used for this purpose.
  • Both the stem cell population of the isolation step (which may make up the master cell bank) and the stem cell population of the subculturing step (which may make up the working cell bank) can, for example, be stored in cryo- preserved form.
  • the present method of inducing or improving the wound healing properties of the mesenchymal cell population (and optionally at the same time of isolating mesenchymal stem cells from a tissue such as Wharton’s jelly or the amniotic membrane of umbilical cord) has the advantage that all components used in the culture medium of the invention are available in GMP quality and thus provide the possibility to isolate the mesenchymal stem cells under GMP conditions for subsequent therapeutic administration.
  • inducing or improving wound healing properties of a mesenchymal stem cell population is meant herein the ability of the culture medium to increase or start (induce) the expression and/or secretion of at least one of the proteins Ang-l, TGF-b I , VEGF, and HGF by the mesenchymal stem cell population.
  • the mesenchymal stem cell population will secrete a bigger amount (corresponding to a higher secretion level or a higher concentration) of at least one of the four marker proteins Ang- 1 , TGF-b 1 , VEGF, and HGF into the supematant/culture medium, when cultivated in a culture medium of the invention compared to cultivation of the mesenchymal stem cell population in the reference medium, then the wound healing properties of the mesenchymal stem cell population are increased.
  • the wound healing properties of the stem cell population are induced.
  • the wound healing properties of the mesenchymal stem cell population are also improved when the expression or secretion of least two or of least three or of all of the four marker proteins Ang-l, TGE-b I , VEGF, and HGF is increased relative to cultivation of the stem cell population in the reference medium.
  • the secretion of the four marker proteins into the culture medium can be measured/determined with any suitable method, for example, by measuring the amount of protein by means of commercially available antibodies/immunoassays (cf, the Experimental Section). Such measurements can be made in an automated fashion, using, for example a system such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).
  • DMEM Dulbecco’s modified eagle medium which was developed in 1969 and is a modification of basal medium eagle (BME) (cf. Fig.1 showing the data sheet of DMEM available from Lonza).
  • BME basal medium eagle
  • the original DMEM formula contains 1000 mg/L of glucose and was first reported for culturing embryonic mouse cells.
  • DMEM has since then become a standard medium for cell culture that is commercially available from various sources such as ThermoFisher Scientific (catalogue number 11965-084), Sigma Aldrich (catalogue number D5546) or Lonza, to name only a few suppliers.
  • any commercially available DMEM can be used in the present invention.
  • the DMEM used herein is the DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g/L glucose and L- glutamine). In another preferred embodiment the DMEM used herein is the DMEM medium of Sigma Aldrich catalogue number D5546 that contains 1000 mg/L glucose, and sodium bicarbonate but is without L-glutamine.
  • Ham Ham
  • F12 medium is meant Ham’s F12 medium.
  • This medium is also a standard cell culture medium and is a nutrient mixture initially designed to cultivate a wide variety of mammalian and hybridoma cells when used with serum in combination with hormones and transferrin (cf. Fig. 2, showing the data sheet of Ham’s F12 medium from Lonza).
  • Any commercially available Ham’s F12 medium for example, from ThermoFisher Scientific (catalogue number 11765-054), Sigma Aldrich (catalogue number N4888) or Lonza, to new only a few suppliers
  • Ham’s F12 medium from Lonza is used.
  • DMEM/F12 or“DMEM:Fl2” is meant a 1 : 1 mixture of DMEM with Ham’s F12 culture medium (cf. Fig. 3 showing the data sheet for DMEM: F12 (1: 1) medium from Lonza).
  • DMEM/F12 (1: 1) medium is a widely used basal medium for supporting the growth of many different mammalian cells and is commercially available from various supplier such as ThermoFisher Scientific (catalogue number 11330057), Sigma Aldrich (catalogue number D6421) or Lonza. Any commercially available DMEM:Fl2 medium can be used in the present invention.
  • the DMEM:Fl2 medium used herein is the DMEM/F12 (1: 1) medium available from Lonza under catalog number 12-719F (which is DMEM: F12 with L-glutamine, 15 mM HEPES, and 3.151 g/L glucose).
  • M171 is meant culture medium 171, which has been developed as basal medium for the culture of for the growth of normal human mammary epithelial cells (cf. Fig. 4 showing the data sheet for M171 medium from Life Technologies Corporation). Also this basal medium is widely used and is commercially available from supplier such as ThermoFisher Scientific or Life Technologies Corporation (catalogue number M 171500), for example. Any commercially available M 171 medium can be used in the present invention. In preferred embodiments, the M171 medium used herein is the M171 medium available from Life Technologies Corporation under catalogue number M171500.
  • FBS fetal bovine serum
  • fetal calf serum i.e. the blood fraction that remains after the natural coagulation of blood, followed by centrifugation to remove any remaining red blood cells.
  • Fetal bovine serum is the most widely used serum- supplement for in vitro cell culture of eukaryotic cells because it has a very low level of antibodies and contains more growth factors, allowing for versatility in many different cell culture applications.
  • the FBS is preferably obtained from a member of the International Serum Industry Association (ISIA) whose primary focus is the safety and safe use of serum and animal derived products through proper origin traceability, truth in labeling, and appropriate standardization and oversight.
  • ISIA International Serum Industry Association
  • FBS FBS
  • ISIA members include Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific and Life Science Production, to mention only a few.
  • the FBS is obtained from GE Healthcare under catalogue number A15-151.
  • the culture medium may comprise for inducing or improving the wound healing properties or for the isolation or cultivation of the mesenchymal stem cells DMEM in a final concentration of about 55 to 65 % (v/v), F12 in a final concentration of about 5 to 15 % (v/v), M171 in a final concentration of about 15 to 30 % (v/v) and FBS in a final concentration of about 1 to 8 % (v/v).
  • the value (v/v)” as used herein refers to the volume of the indivual component relative to the final volume of the culture medium. This means, if DMEM is, for example, present in the culture medium a final concentration of about 55 to 65 % (v/v), 1 liter of culture medium contains about 550 to 650 ml DMEM.
  • the culture medium may comprise DMEM in a final concentration of about 57.5 to 62.5 % (v/v), F12 in a final concentration of about 7.5 to 12.5 % (v/v), M171 in a final concentration of about 17.5 to 25.0 % (v/v) and FBS in a final concentration of about 1.75 to 3.5 % (v/v) .
  • the culture medium may comprise DMEM in a final concentration of about 61.8 % (v/v), F12 in a final concentration of about 11.8 % (v/v), M171 in a final concentration of about 23.6 % (v/v) and FBS in a final concentration of about 2.5 % (v/v).
  • the culture medium may comprise supplements that are advantageous for cultivation of the mesenchymal cord lining stem cells.
  • the culture medium of the present invention may, for example, comprises Epidermal Growth Factor (EGF). If present, EGF may be present in the culture medium in a final concentration of about 1 ng/ml to about 20 ng/ml. In some of these embodiments, the culture medium may comprise EGF in a final concentration of about lOng/ml.
  • EGF Epidermal Growth Factor
  • the culture medium of the present invention may also comprise insulin. If present, insulin may be present in a final concentration of about 1 pg/ml to 10 pg/ml. In some of these embodiments, the culture medium may comprise Insulin in a final concentration of about 5 pg/ml.
  • the culture medium may further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-Triiodo-L-thyronine sodium salt (T3).
  • the culture medium may comprise all three of adenine, hydrocortisone, and 3,3',5-Triiodo-L-thyronine sodium salt (T3).
  • the culture medium may comprise adenine in a final concentration of about 0.05 to about 0.1 pg/ml adenine, hydrocortisone in a final concentration of about 1 to about 10 pg/ml hydrocortisone and/or 3,3',5-Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • adenine in a final concentration of about 0.05 to about 0.1 pg/ml adenine
  • hydrocortisone in a final concentration of about 1 to about 10 pg/ml hydrocortisone
  • T3 3,3',5-Triiodo-L-thyronine sodium salt
  • tissue such as umbilical cord tissue or placental may be cultured till a suitable number of (primary) mesenchymal stem cells such as cord lining stem cells, Wharton’s Jelly or placental stem cells cells have outgrown from the tissue.
  • the umbilical cord tissue is cultivated until cell outgrowth of the mesenchymal stem cells of the respective tissue reaches about 70 to about 80% confluency.
  • the term“confluency” or“confluence” is used in its regular meaning in the art of cell culture and is meant as an estimate/indicator of the number of adherent cells in a culture dish or a flask, referring to the proportion of the surface which is covered by cells. For example, 50 percent confluence means roughly half of the surface is covered and there is still room for cells to grow. 100 percent confluence means the surface is completely covered by the cells, and no more room is left for the cells to grow as a monolayer. [0079] Once a suitable number of primary cells (mesenchymal stem cells) have been obtained from the respective tissue by tissue explant, the mesenchymal stem cells are removed from the cultivation container used for the cultivation.
  • a master cell bank containing the (primary) isolated mesenchymal stem cells of for example, the umbilical cord or the placeta can be obtained.
  • the enzymatic treatment may comprise trypsination as described in International US patent application 2006/0078993, International patent application W02006/019357 or International patent application W02007/046775, meaning outgrowing cells can be harvested by trypsinization (0.125% trypsin/0.05% EDTA) for further expansion.
  • the harvested mesenchymal stem cells are, for example, used for generating a master cell bank, the cells can also be cryo-preserved and stored for further use as explained herein below.
  • the mesenchymal stem cells can be transferred to a cultivation container for subculturing.
  • Subculturing or culturing will be also be carried out if a mesenchymal stem cell population is employed that has been isolated from its natural environment earlier (as explained above, such isolated stem cells used in the method of the invention may be from cord blood, bone marrow or adipose tissue but also from cord tissue or placental tissue).
  • the subculturing can also be started from frozen primary cells, i.e. from the master cell bank. For subculturing any suitable amount of cells can be seeded in a cultivation container such as cell culture plate.
  • the mesenchymal cells can, for this purpose, be suspended in a suitable medium (most conveniently, the culture medium of the present invention) for subculturing at a concentration of, for example, about 0.5 x 10 6 cells/ml to about 5.0 x 10 6 cells/ml. In one embodiment the cells are suspended for subcultivation at a concentration of about 1.0 x 10 6 cells/ml.
  • the subculturing can be carried by cultivation either in simple culture flasks but also, for example, in a multilayer system such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA) that can be stacked in incubators.
  • the subculturing can also be carried out in a closed self- contained system such as a bioreactor.
  • a bioreactor Different designs of bioreactors are known to the person skilled in the art, for example, parallel-plate, hollow-fiber, or micro-fluidic bioreactors. See, for example, Sensebe et al.’’Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra.
  • An illustrative example of a commercially available hollow-fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT, Inc) that has, for example, been used for the expansion of bone marrow mesenchymal stem cells for clinical trials (cf., Hanley et al, Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August ; 16(8): 1048-1058).
  • Another example of a commercially available bioreactors that can be used for the subculturing of the mesenchymal stem cell population of the present invention is the Xuri Cell Expansion System available from GE Heathcare.
  • the cultivation of the mesenchymal stem cell population in an automated system such as the Quantum® Cell Expansion System is of particular benefit if a working cell bank for therapeutic application is to be produced under GMP conditions and a high number of cells is wanted.
  • the culture medium of the present invention can be used both for the isolation of the mesenchymal stem cell population, for example, from the amniotic membrane of placenta, or from the amniotic membrane or from Wharton’ s jelly of umbilical cord and the subsequent cultivation of the isolated primay cells by subcultivation.
  • the mesenchymal stem cells can be cultured till a suitable amount of cells have grown. In illustrative embodiments the mesenchymal stem cells are subcultured till the mesenchymal stem cells reach about 70 to about 80% confluency.
  • the isolation/cultivation of the population of mesenchymal stem cell population can be carried out under standard condition for the cultivation of mammalian cells.
  • the method of the invention of isolating the population of the mesenchymal stem cells is typically carried out under conditions (temperature, atmosphere) that are normally used for cultivation of cells of the species of which the cells are derived.
  • conditions temperature, atmosphere
  • human umbilical cord tissue and the mesenchymal cord lining stem cells, respectively, are usually cultivated at 37°C in normal atmosphere with 5%C0 2 .
  • the mesenchymal cell population may be derived of any mammalian species, such as mouse, rat, guinea pig, pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, with mesenchymal stem cells of human origin being preferred in one embodiment.
  • the mesenchymal stem cells are harvested by removing them from the cultivation container used for the subcultivation.
  • the harvesting of the mesenchymal stem cells is typically again carried out by enzymatic treatment, including comprises trypsination of the cells.
  • the isolated mesenchymal stem cells are subsequently collected and are either be directedly used or preserved for further use. Typically, preserving is carried out by cryo-preservation.
  • cryo-preservation is used herein in its regular meaning to describe a process where the mesenchymal stem cells are preserved by cooling to low sub-zero temperatures, such as (typically) -80°C or -l96°C (the boiling point of liquid nitrogen). Cryo-preservation can be carried out as known to the person skilled in the art and can include the use of cryo-protectors such as dimethylsulfoxide (DMSO) or glycerol, which slow down the formation of ice-crystals in the cells of the umbilical cord.
  • DMSO dimethylsulfoxide
  • glycerol glycerol
  • the isolated population of the mesenchymal stem cells that is obtained by the cultivation and/or isolation method of the present invention is highly defined and homogenous. In typical embodiments of the method at least about 90 % or more, about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90 and CD105.
  • 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 lack expression of the following markers: CD34, CD45 and HLA-DR.
  • about 97 % or more, about 98 % or more, or about 99 % or more of the isolated mesenchymal stem cell population express CD73, CD90 and CD 105 while lacking expression of CD34, CD45 and HLA-DR.
  • the present invention is also directed to a mesenchymal stem population such as a placental mesenchymal stem cell population, or an umbilical cord mesenchymal stem cell population (for example, isolated from Wharton’s jelly or the amniotic membrane of the umbilical cord), wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD 105.
  • a mesenchymal stem population such as a placental mesenchymal stem cell population, or an umbilical cord mesenchymal stem cell population (for example, isolated from Wharton’s jelly or the amniotic membrane of the umbilical cord), wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD 105.
  • 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 cells of the isolated mesenchymal stem cell population are CD73+, CD90+ and CD105+, meaning that this percentage of the isolate cell population express each of CD73, CD90 and CD 105 (cf. the Experimental Section of the present application).
  • 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.
  • about 97 % or more, about 98 % or more, or about 99 % or more of the isolated mesenchymal stem cell population express CD73, CD90 and CD 105 while lacking expression of CD34, CD45 and HLA-DR.
  • mesenchymal stem cells derived from the amniotic membrane of the umbilical cord has been reported here for the first time and meets the critiera for mesenchymal stem cells to be used for cellular therapy (also cf. the Experimental Section and, for example, Sensebe et al.’’Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra). It is noted in this context that this mesenchymal stem cell population can be obtained by either the isolating method of the present invention but also by a different method such as cell sorting, if wanted.
  • a mesenchymal stem cell population of the umbilical cord of the present invention wherein at least about 91 % or more cells of the stem cell population express each CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord is excluded.
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a mesenchymal stem population as described herein, wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD105 and optionally, lack expression of CD34, CD45 and HLA-DR.
  • the pharmaceutical composition may comprise any pharmaceutically acceptable excipient and may be formulated for any desired pharmaceutical way of administration.
  • the pharmaceutical composition may, for example, be adapted for systemic or topical application.
  • the present invention also provides a pharmaceutical composition that contains three or four of Ang- 1 , TGF-b 1 , VEGF, or HGF as the only wound healing proteins.
  • Such a pharmaceutical composition may be formulated as a liquid, for example, by using a pharmaceutically suitable buffer such 0.9 % saline, Ringer’ s solution or phosphate buffered saline (PBS) or a lyophilisate/freeze-dried formulation.
  • a pharmaceutically suitable buffer such 0.9 % saline, Ringer’ s solution or phosphate buffered saline (PBS) or a lyophilisate/freeze-dried formulation.
  • the invention is directed to a method of making a culture medium for inducing or improving wound healing properties and/or for isolating the mesenchymal stem cell population, wherein the method comprises mixing to obtain a final volume of 500 ml culture medium:
  • Fetal Bovine Serum iv. 12.5 ml Fetal Bovine Serum (FBS) to reach a final concentration of 2.5% (v/v).
  • DMEM/F 12 medium is a 1 : 1 mixture of DMEM and Ham’ s
  • 118 ml DMEM/F12 medium contain 59 ml DMEM and 59 ml F12. Accordingly, when using this method of making a culture medium, the final concentrations (v/v) mit 500 ml total volume are as follows:
  • M171: 118 ml, corresponds to 118/500 23.6 % (v/v)
  • Embodiments of this method of making a culture medium further comprise adding
  • EGF stock solution 5 pg/ml
  • Insulin 0.175 ml stock solution 14.28 mg/ml
  • the above-mentioned volumes of these components i. to vi when mixed result in a final volume of 499.675 ml culture medium.
  • the remaining 0.325 ml can, for example, be any of components i. to iv, that means either DMEM, M 171 , DMEM/F 12 or FB S .
  • the concentration of the stock solution of EGF or Insulin can of course be adjusted such that the total volume of the culture medium is 500 ml.
  • M171 and DMEM/F12 can be mixed together and then combined with DMEM and FBS to reach final concentrations as described here, i.e. a final concentration of DMEM of about 55 to 65 % (v/v), a final concentration of F12 of about 5 to 15 % (v/v), a final concentration of M171 of about 15 to 30 % (v/v) and a final concentration of FBS of about 1 to 8 % (v/v).
  • the method further comprises adding to DMEM a volume of 0.325 ml of one or more of the following supplements: adenine, hydrocortisone, 3,3 ',5- Triiodo-L-thyronine sodium salt (T3), thereby reaching a total volume of 500 ml culture medium.
  • the final concentration of these supplements in DMEM may be as follows:
  • T3 3,3',5-Triiodo-L-thyronine sodium salt
  • T3 1,3',5-Triiodo-L-thyronine sodium salt
  • the invention is also directed to a cell culture medium that is obtainable or that is obtained by the method of making the medium as described here.
  • the invention also concerns a method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, wherein this method comprises cultivating amniotic membrane tissue in the culture medium prepared by the method as described here.
  • the present invention is also directed to a cell culture medium comprising:
  • the medium comprises DMEM in the final concentration of about 57.5 to 62.5 % (v/v), F12 in a final concentration of about 7.5 to 12.5 % (v/v), M171 in a final concentration of about 17.5 to 25.0 % (v/v) and FBS in a final concentration of about 1.75 to 3.5 % (v/v).
  • the culture medium may comprise DMEM in a final concentration of about 61.8 % (v/v), F12 in a final concentration of about 11.8 % (v/v), M171 in a final concentration of about 23.6 % (v/v) and FBS in a final concentration of about 2.5 % (v/v).
  • the culture medium may further comprise Epidermal Growth Factor (EGF) in a final concentration of about 1 ng/ml to about 20 ng/ml.
  • EGF Epidermal Growth Factor
  • the culture medium comprise EGF in a final concentration of about lOng/ml.
  • the culture medium described herein may further comprise Insulin in a final concentration of about 1 pg/ml to 10 pg/ml. In such embodiments the culture medium may comprise Insulin in a final concentration of about 5 pg/ml.
  • the cell culture medium of the invention may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-Triiodo-F-thyronine sodium salt (T3).
  • the culture medium comprises all three of adenine, hydrocortisone, and 3,3',5-Triiodo-F-thyronine sodium salt (T3).
  • the culture medium may comprise adenine in a final concentration of about 0.01 to about 0.1 pg/ml adenine or of about 0.05 to about 0.1 pg/ml adenine, hydrocortisone in a final concentration of about 0.1 to about 10 pg/ml hydrocortisone or of about 1 to about 10 pg/ml hydrocortisone and/or 3,3',5-Triiodo-F-thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • adenine in a final concentration of about 0.01 to about 0.1 pg/ml adenine or of about 0.05 to about 0.1 pg/ml adenine
  • hydrocortisone in a final concentration of about 0.1 to about 10 pg/ml hydrocortisone or of about 1 to about 10 pg/ml hydrocortisone and/or 3,3',5-Triiodo-
  • 500 ml of the cell culture medium of the present invention comprise:
  • Fetal Bovine Serum (FBS) (final concentration of 2.5%)
  • the cell culture medium may further comprise v. EGF in a final concentration of lOng/ml, and
  • Both, insulin and and EGF can be added to to the culture medium using a stock solution of choice, such that the total volume of the culture medium does not exceed 500 ml.
  • the components i. to vi. of the culture medium of the present invention are the components indicated in Figure 5, meaning they are obtained from the respective manufacturers using the catalogue number indicated in Figure 5.
  • the medium that is obtained from mixing the components i. to vi. as indicated in Figure 5 is also referred herein as“PTT-6”. It is again noted in this context that the constituents i. to vi. as well as any other ingredient such as an antibiotic of any other commercial supplier can be used in making the medium of the present invention.
  • the cell culture medium of the invention may comprise adenine in a final concentration of about 0.01 to about 0.1 pg/ml adenine or of about 0.05 to about 0.1 pg/ml adenine, hydrocortisone in a final concentration of about 0.1 to 10 pg/ml, of about 0.5 to about lOpg/ml, or of about 1 to about 10 pg/ml hydrocortisone and/or 3,3',5-Triiodo-L- thyronine sodium salt (T3) in a final concentration of about 0.1 to about 5 ng/ml or of about 0.5 to about 5 ng/ml.
  • adenine in a final concentration of about 0.01 to about 0.1 pg/ml adenine or of about 0.05 to about 0.1 pg/ml adenine
  • hydrocortisone in a final concentration of about 0.1 to 10 pg/ml, of about 0.5 to about lOp
  • the invention also provides a method of treating a non-human mammal (such as cats, dogs, horses, to name only a few) or a human patient having a disease or suffering from a condition, the method comprising administering to the non-human mammal or human patient a mesenchymal stem cell population or a pharmaceutical composition containing a stem cell population as disclosed herein.
  • the disease can be any disease or condition, in particular any disease or condition in which healing of wound is wanted/required.
  • the subject patient or non-human mammal
  • the patient may, for example, be afflicted with Type I or Type II diabetes and suffers from chronic foot ulcers.
  • the mesenchymal stem cell population of the invention may be administered in any suitable way, for example, including but not limited to, topical administration, by implantation or by injection. In principle any way of topical administration is meant herein.
  • the administering the mesenchymal stem cell population may be performed by means of a syringe. It is however also possible, to contact the mesenchymal stem cells within a cream, ointment, gel, suspension or any other suitable substance before applying the mesenchymal stem cells to the subject.
  • the stem cell population may, for example, then be placed directly onto a wound such as a burn or a diabetic wound (see International patent application W02007/046775). After its application to the subject the mesenchymal stem cell population may be held in place e.g. by a dressing such as Tegaderm® dressing and a crepe bandage to cover the Tegaderm® dressing. Alternatively, the stem cell population may also be implanted subcutaneously, for example, directly under the skin, in body fat or in the peritoneum.
  • the present invention also relates to a unit dosage comprising about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells of a mesenchymal stem cell population as described herein.
  • the unit dosage 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.
  • the unit dosage comprises about 10 million cells.
  • the unit dosage comprises about 1000 cells to about 5 million cells.
  • the unit dosage can be applied in a dosage of about 100,000 cells, 300,000 cells or 500,000 cells. As described herein the unit dosage may be applied topically, in particular if used for wound healing. For example, the unit dosage may be applied topically per cm 2 .
  • the unit dosage may be applied once, twice, three times or more a week.
  • the unit dosage can be applied for one, two, three, four, five, six, seven, eight, nine, ten, elven weeks or more.
  • the unit dosage comprising of about 100,000 cells, about 300,000 cells or about 500,000 cells can be applied two times a week for 8 weeks, preferably onto 1 cm 2 .
  • the unit dosage can be contained in any suitable container.
  • the unit dosage can be contained in a 1 ml vial. In such cases, for example 0.1 ml of the vial can be applied onto the subject, preferably per cm 2 .
  • the unit dosage may alternatively be contained in a syringe.
  • the unit dosage of the present invention the cells can be in contact with a pharmaceutically acceptable carrier, for example a liquid carrier.
  • the carrier may be any known carrier such as HypoThermosolTM , HypothermosolTM-FRS or PlasmaLyte.
  • the culture medium of the present invention may also be used as carrier for a (unit dosage) of the mesenchymal stem cell population of the present invention.
  • the mesenchymal stem cells may be separated from the carrier before administration.
  • the cells can be centrifuged and isolated before administration to a subject.
  • the method of treatment and the unit dosage of the present invention can comprise utilization of viable cells.
  • the viability of the mesenchymal stem cell population can be tested with known methods, for example, staining with Tryphan Blue as described in the Experimental Section.
  • Umbilical cord tissue (the umbilical cords were donated with informed consent of the mother) was processed for the subsequent isolation of the mesenchymal stel cells from the amniotic membrane of the umbilical cord as follows.
  • d Use the cover of the 150 mm culture dish as a resting place for forceps and scalpel.
  • e. Remove 25ml Plasmalyte A (Baxter, Catalog # 2B2543Q) with a 30 ml syringe. Hold the syringe at a 45° angle using one hand and dispense the Plasmalyte A directly onto the umbilical cord tissue.
  • tissue i. Place the tissue into a new labeled tissue culture dish to continue cutting the tissue. Place 20 ml of Plasmalyte A into the dish so the tissue does not dry out while cutting it.
  • i Prepare 50 ml freezing solution consisting of 60% Plasmalyte A, 30% of 5% Human Serum Albumin, and 10% dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • Preparing media for processing MSCs from umbilical cord tissue a. To make 500 ml PTT-6 (culture/growth media) add the following in the order listed: i. DMEM, 250 ml
  • an antibiotic such as Penicillin-Streptomycin- Amphotericin can be added to result in a final volume of 500 ml.
  • c. Prepare 10 mL of rinse medium consisting of 70% to 90% PlasmaFyte A in the clean room under a biosafety cabinet. Sterile filter the solution with a 0.2-pm syringe filter attached to a 10 ml syringe and keep the solution refrigerated until use. d. Place a processing label on a 50 ml conical tube.
  • Umbilical cord tissue processing should be performed in an environmentally monitored (EM) clean room:. At the end of each shift, full room and hood cleaning are performed b. Prepare/clean the biosafety cabinet.
  • EM environmentally monitored
  • Initiating MSC outgrowth from tissue a. Label the bottom of a 6-well plate“Outgrowth 1” with MSC lot number or umbilical cord tissue ID and the date outgrowth is initiated. If 60 mm tissue culture dish is used, divide the plate into 4 quadrants by drawing a grid on the bottom of the dish.
  • in“Outgrowth 3” plate discard the tissue. If the tissues are very small and do not seem to interfere with cell growth, dispose of the tissue when subculturing.
  • the average number of mesenchymal stem cells harvested from an explant is typically about 4,000 - 6,000 cells/explant. Accordingly, when the mesenchymal stem cells are simultaneously grown out of 48 explants about 300,000 cells can be obtained at harvest. These 300,000 mesenchymal stem cells collected from explants can then be used for subculturing by seeding a l75cm 2 cell culture flask with such 300,000 cells as described in the following Example 2.5 (this can be referred to as Passage 1).
  • the mesenchymal stem cells obtained from this passage 1 can then be used to seed again l75cm 2 flasks (Passage 2) and expand the cells as described in the following Example 2.5.
  • the cells obtained from both Passage 1 and Passage 2 can be “banked” by cryo-preservation, with the mesenchymal stem cells obtained after Passage 2 being considered to represent the Master Cell Bank which will be for further expansion of the mesenchymal stem cells, for example, in a bioreactor as explained below in Example 2.7.
  • Count cells using a hemocytometer Expect to count 20-100 cells/square. If the count higher than 100, dilute the original sample 1:5 and repeat Trypan Blue method using a hemocytometer.
  • Viable cells/ml viable cell count x dilution factor x 10 4
  • Total viable cells viable cell count x dilution factor x total volume x 10 4
  • % viability viable cell count x 100 /(viable cell count + dead cell count)
  • total viable cell number is 1.0 x 10 7 ;
  • cell suspension is less than l06/ml, determine the volume required to seed 2 x 106 cells for each 150 mm petri dish or 175 cm2 flask.
  • viable cells/ml is 8 x 10 5 cells/ml
  • viii Seed 2 x 10 6 cells to each 150 mm petri dish or 175 cm 2 flask with 30 ml PTT-6. ix. Observe cells for attachment, colony formation, and confluence every three days. When cells reach 40-50% confluence, observe cells every one-two days to prevent over-expansion. DO NOT allow cells to expand beyond 80% confluence. A real time cell culturing monitoring system can be used in place of the light microscope.
  • Quantum Bioreactor can be used to expand the MSC.
  • the starting cell number for the expansion in the Quantum Bioreactor should range between 20 to 30 million cells per run.
  • the typical yield per run is 300 to 700 million MSC at harvest.
  • the Bioreactor is operated following the protocol of the manufacturer.
  • the so obtained mesenchymal stem cells are typically cryo-preserved (see below) and serve as Working Cell Bank.
  • PROCEDURE [00131] PROCEDURE:
  • the actual maximal tolerable lactate concentration will be defined by a flask culture from which the cells originate. Determine if adequate PTT-6 media is in the media bag. If necessary, replace the PTT-6 media bag with a fresh PTT-6 media bag. 4) When the flow rate has reached the desired value, measure lactate level every 8- 12 hours. If the lactate level does not decrease or if the lactate level continues to increase, harvest the cells.
  • mesenchymal stem cells were isolated from umbilical cord tissue by cultivation of the umbilical cord tissue in three different cultivation media, followed by subculturing of the mesenchymal stem cells in the respective medium as set forth in Example 2.
  • Total cell count is given as (Average number of cells/quadrant) x 10 4 cells/ml.
  • Fig. 6a shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and cultivation in DMEM/10% FBS
  • Fig. 6b shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD 105 after isolation from umbilical cord tissue and cultivation in PTT-4
  • Fig. 6c shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD 105 after isolation from umbilical cord tissue and cultivation in PTT-6.
  • Fig. 6a shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and cultivation in PTT-6.
  • the population isolated using DMEM/10 % FBS as culture medium cultivation has about 75% CD73+ cells, 78 % 90+ cells and 80 % CD105+ cells (average of two experiments), while after isolation/cultivation of umbilical cord tissue using PTT-4 culture medium (see Fig. 6b) the number of mesenchymal stem cells that are CD73-positive, CD90-positive and CD 105-positive are about 87 % (CD73+ cells), 93 % /CD90+ cells) and 86 % (CD 105+ cells) average of two experiments.
  • the purity of the mesenchymal stem cell population that was obtained by means of cultivation in the PTT-6 medium of the present invention is at least 99.0 % with respect to all three markers (CD73, CD90, CD105), meaning the purity of this cell population is significant higher than for cultivation using PTT-4 medium or DMEM/10 % FBS.
  • the mesenchymal stem cell population obtained by means of cultivation in PTT-6 is essentially a 100% pure and defined stem cell population. This makes the stem cell population of the present invention the ideal candidate for stem cell-based therapies. Thus, this population of mesenchymal cord lining stem cells may become the gold standard for such stem cell-based therapeutic approaches.
  • Fig. 7a shows the percentage of isolated mesenchymal cord lining stem cells (mesenchymal stem cells of the amniotic membrane of umbilical cord) that express the stem cell markers CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR after isolation from umbilical cord tissue and cultivation in PTT-6 medium.
  • stem cells mesenchymal stem cells of the amniotic membrane of umbilical cord
  • the mesenchymal stem cell population contained 97.5 % viable cells of which 100 % expressed each of CD73, CD90 and CD105 (see the rows“CD73+CD90+” and“CD73+CD105+”) while 99.2 % of the stem cell population did not express CD45 and 100 % of the stem cell population did not express CD34 and HLA-DR (see the rows “CD34-CD45- and“CD34-HLA-DR-).
  • the mesenchymal stem cells population obtained by cultivation in PTT-6 medium is essentially a 100% pure and defined stem cell population that meets the criteria that mesenchymal stem cells are to fulfill to be used for cell therapy (95% or more of the stem cell population express CD73, CD90 and CD105, while 98 % or more of the stem cell population lack expression of CD34, CD45 and HLA-DR, see Sensebe et al.’’Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra).
  • mesenchymal stem cells of the amniotic membrane are adhere to plastic in standard culture conditions and differentiate in vitro into osteoblasts, adipocytes and chondroblasts, see US patents 9,085,755, US patent 8,287,854 or W02007/046775 and thus meet the criteria generally accepted for use of mesenchymal stem cells in cellular therapy.
  • Fig. 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR.
  • the bone marrow mesenchymal stem cell population contained 94.3 % viable cells of which 100 % expressed each of CD73, CD90 and CD105 (see the rows “CD73+CD90+” and“CD73+CD105+”) while only 62.8 % of the bone marrow stem cell population lacked expression of CD45 and 99.9 % of the stem cell population lacked expression CD34 and HLA-DR (see the rows“CD34-CD45- and“CD34-HLA-DR-).
  • the bone marrow mesenchymal stem cells that are considered to be the gold standard of mesenchymal stem cells are by far less homogenous/pure in terms of stem cell marker than the mesenchymal stem cells population (of the amniotic membrane of the umbilical cord) of the present application.
  • This finding also shows that the stem cell population of the present invention may be the ideal candidate for stem cell-based therapies and may become the gold standard for stem cell-based therapeutic approaches.
  • WJ-MSC - mesenchymal stem cells of the Wharton’s jelly
  • This population of WJ-MSC was isolated by tissue explant (cultivation in DMEM with 4,500 mg/mL glucose and 2 mM L- glutamine, supplemented with 10% human serum/FBS and antibiotic solution) of Wharton’s jelly of human umbilical cord as described by Beeravolu et al.“Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.” J Vis Exp. 2017; (122): 55224.
  • AT-MSC Adipose-tissue derived mesenchymal stem cells
  • BM-MSC Bone Marrow mesenchymal stem cells
  • PT-MSC placental mesenchymal stem cells
  • Cytokine Detection was performed in MSC Supernatants. Measurements and analysis has been conducted using Luminex 200 and Xponent software.
  • the goal of this experiment was to measure relative levels of Multiplex (PDGF- AA, PDGF-BB, IL-10, VEGF, Ang-l, and HGF), TGFpl Singleplex, andbFGF2 Singleplex cytokines on cell culture supernatants.
  • the supernatants are (MSC, mesenchymal stem cell; CL, cord lining; WJ, Wharton’s Jelly; AT, adipose tissue; BM, bone marrow):
  • samples CR001A, CR001C, CR001D, and CR001G were included as a positive control to validate the cytokine assay (the conditioned media from CR001A, CR001C, CR001D and CR001G were not prepared by cultivation of cells in PTT-6 or PTT-4)
  • the aim of this experiment was to generate cytokine profiles of MSCs cultured either in PTT-4 or PTT-6 and to compare the profiles of MSCs from different tissue origins (umbilical cord lining vs. Wharton’s Jelly vs. adipose tissue vs. bone marrow).
  • the profile will shed light onto which stem cell population grown in which medium would secrete more of the cytokines of interest in order to promote wound healing.
  • MSC mesenchymal stem cell
  • CL cord lining
  • WJ Wharton’s Jelly
  • AT adipose tissue
  • BM bone marrow.
  • VEGF vascular endothelial growth factor
  • PDGF-AA platelet-derived growth factor
  • PDGF-BB platelet-derived growth factor
  • TGFp 1 component cat. # FTGM100, lot # P161760, received 02/27/18, expires 11/27/19.
  • VEGF vascular endothelial growth factor
  • PTT-4 and PTT-6 media (not exposed to MSCs)
  • FIG. 9 Singleplex measurement of TGFP 1. As can be seen cultures CL-MSC and WJ- MSC produce more TGFp i when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced more or less equal amounts of TGFP 1 when grown in PTT-6 or PTT-4. All error bars are standard deviation from triplicate measurements.
  • Figure 10 Fig.lOA Multiplex measurement of PDGF-AA. As can be seen cultures CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produce more PDGF-AA when grown in PTT-4 than when grown in PTT-6. All error bars are standard deviation from triplicate measurements. Fig. 10B Multiplex measurement of VEGF. As can be seen cultures CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviation from triplicate measurements. Fig.lOC Multiplex measurement of Ang- 1.
  • Figure 11 Multiplex measurement of HGF. As can be seen cultures CL-MSC and WJ-MSC cultures produce much more HGF when grown in PTT-6 than when grown in PTT-4. Cultures AT-MSC and BM-MSC essentially did not produce any HGF. All error bars are standard deviation from triplicate measurements.
  • Figure 12 Multiplex measurement of PDGF-AA. As can be seen cultures CL- MSC and WJ-MSC cultures produce 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 culture media. All error bars are standard deviation from triplicate measurements.
  • FIG.l3A Multiplex measurement of VEGF.
  • cultures CL-MSC, WJ-MSC, AT-MSC and BM-MSC cultures produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviation from triplicate measurements.
  • Fig. 13B Multiplex measurement of Ang-l Multiplex Assay.
  • cultures CL-MSC and WJ-MSC cultures produce much more Ang-lwhen grown in PTT-6 than when grown in PTT-4.
  • Cultures AT-MSC and BM-MSC essentially did not produce any Ang-l. All error bars are standard deviation from triplicate measurements.
  • Fig.l3C Multiplex measurement of HGF.
  • Figure 14 Multiplex measurement of bFGF. As can be seen cultures CL-MSC and WJ-MSC cultures produce 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 deviation from triplicate measurements.
  • Figures 15 to Figure 21 depict a summary of data obtained over different experiments.
  • Figure 15 Summarizes measurement of TGFpl over 5 different experiments (170328, 170804, 170814, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the TGFB standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the TGFB standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that cultures CL-MSC and WJ- MSC produce more TGFP 1 when grown in PTT-6 than when grown in PTT-4.
  • AT-MSC and BM-MSC cultures produced equal amounts of TGFpl when grown in PTT-6 or PTT-4. All error bars are standard deviation from different measurements for the experiments 170328, 170804, 170814, 180105, 180226.
  • FIG. 16 Summarizes measurement of Ang-l over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the Ang-l standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the Ang-l standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that cultures CL- MSC and WJ- MSC produce more Ang- 1 when grown in PTT-6 than when grown in PTT-4. Only AT-MSC and BM-MSC cultures produced essentially equal amounts of Ang-l when grown in PTT-6 or PTT-4. All error bars are standard deviation from different measurements for the experiments 170602, 170511, 170414, 170224, 180105, 180226.
  • MFI Mean fluorescent intensity measured for the PDGF-BB standard curves across experiments is depicted in the graph below on the left-hand side. MFI for the PDGF-BB standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs. Notably, in none of the experiments PDGF-BB has been detected.
  • FIG. 18 Summarizes measurement of PDGF-AA over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the PDGF-AA standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the PDGF-AA standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that 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 deviation from measurements of experiments 170602, 170511, 170414, 170224, 180105, 180226.
  • FIG. 19 Summarizes measurement of IL-10 over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the IL-10 standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the IL-10 standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs. Notably, in none of the experiments IL-10 has been detected.
  • Figure 20 Summarizes measurement of VEGF over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the VEGF standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the VEGF standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that cultures CL- MSC, AT-MSC and BM-MSC and WJ-MSC produce more VEGF when grown in PTT-6 than when grown in PTT-4. All error bars are standard deviation from different measurements for the experiments 170602, 170511, 170414, 170224, 180105, 180226.
  • FIG. 21 Summarizes measurement of HGF over 6 different experiments (170602, 170511, 170414, 170224, 180105, 180226).
  • Mean fluorescent intensity (MFI) measured for the HGF standard curves across experiments is depicted in the graph below on the left-hand side.
  • MFI for the HGF standard curves obtained in PTT-4 and PTT-6 medium are shown in above graphs.
  • the graph below on the right-hand side depicts that cultures CL- MSC, and WJ- MSC produce more HGF when grown in PTT-6 than when grown in PTT-4.
  • cultures AT-MSC and BM-MSC did not produce as much HGF as the other cultures. All error bars are standard deviation from different measurements for the experiments 170602, 170511, 170414, 170224, 180105, 180226.
  • the cytokine detection was performed in MSC Supernatants. Measurements and analysis were conducted as described above.
  • the supernatants are obtained from mesenchymal stem cells from cord lining (CL), from Wharton’s Jelly (WJ) and from placenta.
  • the mesenchymal stem cells were cultivated in PTT-6, PPT-4 or DMEM/F12 medium.
  • FIG. 22 Singleplex measurement of TGFP 1 .
  • Mean fluorescent intensity (MFI) measured for the standard TGFP 1 curves across experiments is depicted in the graph on the left-hand side As can be seen the graph on the right-hand sidall of CL-MSC, WJ- MSC and placental MSC produce more TGFP 1 when grown in PTT-6 than when grown in PTT-4 or DMEM/F12 (referred to only as DMEM in Fig. 22).
  • MFI Mean fluorescent intensity
  • Figure 23 Summarizes measurement of PDGF-BB in the analysed supernatants of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM/F12. Mean fluorescent intensity (MFI) measured for the PDGF-BB standard curves across experiments is depicted in the graph on the left-hand side. Notably, in none of the experiments PDGF-BB has been detected.
  • MFI Mean fluorescent intensity
  • Figure 24 Summarizes measurement of IL-10 in the analysed supernatants of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM/F12.
  • Mean fluorescent intensity (MFI) measured for the VEGF standard curves across experiments is depicted in the graph on the left-hand side.
  • S6 denotes the lowest standard used in the assay. Any samples that fall below are considered below detection.
  • all of CL-MSC, WJ- MSC and placental MSC produce detectable levels of IL- 10 when grown in PTT-6 while little or no IL- 10 were detected when the MSC’ s were grown in PTT-4 or DMEM/F12
  • FIG. 25 Summarizes measurement of VEGF in the analysed supernatants of CL-MSC, WJ-MSC and placental MSC cultured in PTT-6, PTT-4 or DMEM/F12.
  • Mean fluorescent intensity (MFI) measured for the VEGF standard curves across experiments is depicted in the graph on the left-hand side.
  • S 1 denotes the highest standard used in the assay. Any samples that fall above are considered extrapolated (too concentrated).
  • all of CL-MSC, WJ- MSC and placental MSC produce much higher levels of VEGF when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • Figure 26 Summarizes multiplex measurement of bFGF.
  • Mean fluorescent intensity (MFI) measured for the PDGF-AA standard curves across experiments is depicted in the graph on the left-hand side.
  • CL-MSC and WJ-MSC produce more bFGF when grown in PTT-6 than when grown in PTT-4.
  • all of CF-MSC, WJ- MSC and placental MSC produce much lower levels of bFGF when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • Figure 27 Summarizes measurement of PDGF-AA. Mean fluorescent intensity
  • MFI measured for the PDGF-AA standard curves across experiments is depicted in the graph on the left-hand side.
  • S6 denotes the lowest standard used in the assay. Any samples that fall below are considered below detection.
  • all of CF-MSC, WJ- MSC and placental MSC produce higher levels of PDGF-AS when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • FIG. 28 Summarizes measurement of Ang-l.
  • Mean fluorescent intensity (MFI) measured for the Ang- 1 standard curves across experiments is depicted in the graph on the left-hand side.
  • S 1 denotes the highest standard used in the assay. Any samples that fall above are considered extrapolated (too concentrated).
  • the graph on the right-hand side depicts that all of CF-MSC, WJ- MSC and placental MSC produce much higher levels of Ang-l when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • FIG. 29 Summarizes measurement of HGF.
  • Mean fluorescent intensity (MFI) measured for the HGF standard curves across experiments is depicted in the graph on the left-hand side.
  • the graph on the right-hand side depicts that all of CF-MSC, WJ- MSC and placental MSC produce much higher levels of Ang-l when grown in PTT-6 compared to when the MSC’s were grown in PTT-4 or DMEM/F12.
  • mesenchymal stem cells in particular mesenchymal stem cells isolated from a compartment of the umbilical cord or isolated from the placenta, are cultured in PTT-6 medium
  • the secretion of the factors Angiopoietin 1 (Ang-l), TGF-b I , VEGF, and HGF by the mesenchymal stem cell population is significantly increased when compared to their production level in PTT-4 medium or a commercially available culture medium such as DMEM/F12.
  • PTT-6 medium is able to increase the production/secretion of these factors irrespective of the natural environment/ compartment of the mesenchymal stem population.
  • the PTT-6 medium causes secretion of all of Ang-l, TGF-b I , VEGF, and HGF (the involvement of which in wound healing is known, as discussed herein) in mesenchymal stem cell populations, it is clear that the PTT-6 medium has the effect of inducing or improving wound healing properties of a wide range of mesenchymal stem cell population, irrespective of the natural environment/ compartment of the mesenchymal stem population from which the mesenchymal stem cells have been originally derived - it is noted here again that Experiment 4 was carried out with cell populations that had been isolated from their natural environment prior to cultivation in PTT-6.
  • mesenchymal stem cells in PTT-6 by tissue explant provides a highly homogenous mesenchymal stem cell population (that contained 97.5 % viable cells of which 100 % expressed each of CD73, CD90 and CD105 while 99.2 % of the stem cell population did not express CD45 and 100 % of the stem cell population did not express CD34 and HLA-DR (see the rows“CD34-CD45- and“CD34-HLA-DR-) of the amniotic membrane of the umbilical.
  • tissue explant of placental tissue including the amniotic membrane of placenta by cultivation in PTT-6 can be expect to yield a placental mesenchymal stem cell population of similar homogeneity.
  • the present provides a generally applicable methodology to obtain a mesenchymal stem cell population, 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 cells of the isolated mesenchymal stem cell population express each of CD73, CD90 and CD105 and lack expression of each of CD34, CD45 and HLA-DR.
  • the invention is also characterized by the following items.
  • a method of inducing or improving wound healing properties of a mesenchymal stem cell population comprising cultivating the mesenchymal stem cell population in a culture medium comprising DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum).
  • DMEM Denbecco’s modified eagle medium
  • F12 Haam’s F12 Medium
  • M171 Medium 171
  • FBS Fetal Bovine Serum
  • the mesenchymal stem cell population is selected from the group consisting of a mesenchymal stem cell population of the umbilical cord, a placental mesenchymal stem cell population, a mesenchymal stem cell population of the cord-placenta junction, a mesenchymal stem cell population of the cord blood, a mesenchymal stem cell population of the bone marrow, and an adipose-tissue derived mesenchymal stem cell population.
  • the mesenchymal stem cell population of the umbilical cord is selected from the group consisting of a mesenchymal stem cell population of the amnion (AM), a perivascular (PV) mesenchymal stem cell population, a mesenchymal stem cell population of Wharton’s jelly (WJ), a mesenchymal stem cell population of the amniotic membrane of umbilical cord and a mixed mesenchymal stem cell population of the umbilical cord (MC).
  • AM mesenchymal stem cell population of the amnion
  • PV perivascular
  • WJ mesenchymal stem cell population of Wharton’s jelly
  • MC mixed mesenchymal stem cell population of the umbilical cord
  • the culture medium comprises DMEM in a final concentration of about 55 to 65 % (v/v), F12 in a final concentration of about 5 to 15 % (v/v), M171 in a final concentration of about 15 to 30 % (v/v) and FBS in a final concentration of about 1 to 8 % (v/v).
  • the culture medium comprises DMEM in a final concentration of about 57.5 to 62.5 % (v/v), F12 in a final concentration of about 7.5 to 12.5 % (v/v), M171 in a final concentration of about 17.5 to 25.0 % (v/v) and FBS in a final concentration of about 1.75 to 3.5 % (v/v).
  • the culture medium comprises DMEM in a final concentration of about 61.8 % (v/v), F12 in a final concentration of about 11.8 % (v/v) , M171 in a final concentration of about 23.6 % (v/v) and FBS in a final concentration of about 2.5 % (v/v).
  • the culture medium further comprises Epidermal Growth Factor (EGF) in a final concentration of about 1 ng/ml to about 20 ng/ml.
  • EGF Epidermal Growth Factor
  • the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-Triiodo-L- thyronine sodium salt (T3).
  • the culture medium comprises adenine in a final concentration of about 0.01 to about 0.1 pg/ml adenine, hydrocortisone in a final concentration of about 0.1 to about 10 pg/ml hydrocortisone and/or 3, 3', 5-Triiodo-L- thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • the umbilical cord tissue is selected from the group consisting of tissue of the entire umbilical cord, tissue comprising the amniotic membrane of umbilical cord, tissue comprising Wharton’s jelly, tissue comprising the amniotic membrane, the amnion and Wharton’s jelly, the isolated umbilical cord blood vessels, Wharton’s jelly separated from the other components of umbilical cord tissue, and isolated amniotic membrane of the umbilical cord.
  • tissue comprises or is amniotic membrane tissue of placenta.
  • the umbilical cord tissue is a piece of the entire umbilical cord, a piece of the amniotic membrane of the umbilical cord or a piece of the amniotic membrane of placenta.
  • bioreactor is selected from the group consisting of a parallel-plate bioreactor, a hollow-fiber bioreactor and and a micro-fluidic bioreactor.
  • An isolated mesenchymal stem population wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD105.
  • the mesenchymal stem cell population of item 43 wherein least about 90 % or more cells of the stem cell population lack expression of the following markers: CD34, CD45 and HLA-DR.
  • 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 cells of the isolated mesenchymal stem cell population express each of CD73, CD90 and
  • CD 105 and lack expression of each of CD34, CD45 and HLA-DR. 46.
  • mesenchymal stem cell population of the umbilical cord a placental mesenchymal stem cell population, a mesenchymal stem cell population of the cord blood, a mesenchymal stem cell population of the bone marrow, and an adipose-tissue derived mesenchymal stem cell population.
  • mesenchymal stem cell population of any of items 43 to 46 wherein the mesenchymal stem cell population of the umbilical cord is seleced from the group consisting of a mesenchymal stem cell population of the amnion (AM), a perivascular (PV) mesenchymal stem cell population, a mesenchymal stem cell population of Wharton’s jelly (WJ), a mesenchymal stem cell population of the amniotic membrane of umbilical cord and a mixed mesenchymal stem cell population of the umbilical cord (MC).
  • AM mesenchymal stem cell population of the amnion
  • PV perivascular mesenchymal stem cell population
  • WJ mesenchymal stem cell population of Wharton’s jelly
  • MC mixed mesenchymal stem cell population of the umbilical cord
  • a pharmaceutical composition comprising an isolated mesenchymal stem cell population as defined in any of items 43 to 47, wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD 105 and lack expression of each of the following markers: CD34, CD45 and HLA- DR.
  • composition 51 The pharmaceutical composition of item 50, wherein the pharmaceutical composition is adapted for systemic or topical application.
  • a method of making a culture medium suitable of inducing or improving wound healing properties of a mesenchymal stem cell population comprising, mixing to obtain a final volume of 500 ml culture medium:
  • Fetal Bovine Serum (FBS) (final concentration of 2.5%)
  • EGF stock solution 5 pg/ml
  • Insulin 0.175 ml stock solution (14.28 mg/ml) to achieve a final concentration of 5 pg/ml.
  • T3 3,3',5-Triiodo-L-thyronine sodium salt
  • T3 1,3',5-Triiodo-L-thyronine sodium salt
  • a cell culture medium obtainable by the method of any of items 53 to 56.
  • a method of inducing or improving wound healing properties of a mesenchymal stem cell population comprising cultivating amniotic membrane tissue in the culture medium prepared by the method as defined in any of items 53 to 56.
  • the mesenchymal stem cell population is selected from the group consisting of a mesenchymal stem cell population of the umbilical cord, a placental mesenchymal stem cell population, a mesenchymal stem cell population of the cord blood, a mesenchymal stem cell population of the bone marrow, and an adipose- tissue derived mesenchymal stem cell population. 60.
  • the mesenchymal stem cell population of the umbilical cord is seleced from the group consisting of a mesenchymal stem cell population of the amnion (AM), a perivascular (PV) mesenchymal stem cell population, a mesenchymal stem cell population of Wharton’s jelly (WJ), a mesenchymal stem cell population of the amniotic membrane of umbilical cord and a mixed mesenchymal stem cell population of the umbilical cord (MC).
  • AM mesenchymal stem cell population of the amnion
  • PV perivascular
  • WJ mesenchymal stem cell population of Wharton’s jelly
  • MC mixed mesenchymal stem cell population of the umbilical cord
  • a cell culture medium comprising:
  • the cell culture medium of item 61 wherein the culture medium comprises DMEM in the final concentration of about 57.5 to 62.5 % (v/v), F12 in a final concentration of about 7.5 to 12.5 % (v/v), M171 in a final concentration of about 17.5 to 25.0 % (v/v) and FBS in a final concentration of about 1.75 to 3.5 % (v/v).
  • the cell culture medium of item 62 wherein the culture medium comprises DMEM in a final concentration of about 61.8 % (v/v), F12 in a final concentration of about 11.8 % (v/v) , M 171 in a final concentration of about 23.6 % (v/v) and FBS in a final
  • EGF Epidermal Growth Factor
  • the cell culture medium of item 68 wherein the culture medium comprises all three of adenine, hydrocortisone, and 3, 3', 5-Triiodo-L-thyronine sodium salt (T3).
  • the cell culture medium of item 68 or 69 wherein the culture medium comprises adenine in a final concentration of about 0.05 to about 0.1 pg/ml adenine, hydrocortisone in a final concentration of about 1 to about 10 pg/ml hydrocortisone and/or 3, 3', 5- Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • T3 5- Triiodo-L-thyronine sodium salt
  • the cell culture medium of any of items 61 to 70, wherein 500 ml of the cell culture medium comprise:
  • Fetal Bovine Serum (FBS) (final concentration of 2.5%)
  • the cell culture medium of item 71 or 72 further comprising adenine in a final concentration of about 0.05 to about 0.1 pg/ml adenine, hydrocortisone in a final concentration of about 1 to about 10 pg/ml hydrocortisone and/or 3,3', 5-Triiodo-L- thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • 74 The use of a cell culture medium as defined in any of items 61 to 73 for inducing or improving wound healing properties of a mesenchymal stem cell population. 75. The use of a cell culture medium as defined in any of items 61 to 73 for isolation of a mesenchymal stem cell population.
  • mesenchymal stem cell population is selected from the group consisting of a mesenchymal stem cell population of the umbilical cord, a placental mesenchymal stem cell population, a mesenchymal stem cell population of the cord blood, a mesenchymal stem cell population of the bone marrow, and an adipose- tissue derived mesenchymal stem cell population.
  • the mesenchymal stem cell population of the umbilical cord is selected from the group consisting of a mesenchymal stem cell population of the amnion (AM), a perivascular (PV) mesenchymal stem cell population, a mesenchymal stem cell population of Wharton’s jelly (WJ), a mesenchymal stem cell population of the amniotic membrane of umbilical cord and a mixed mesenchymal stem cell population of the umbilical cord (MC).
  • AM mesenchymal stem cell population of the amnion
  • PV perivascular
  • WJ mesenchymal stem cell population of Wharton’s jelly
  • MC mixed mesenchymal stem cell population of the umbilical cord

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Abstract

La présente invention concerne un procédé d'induction ou d'amélioration des propriétés de cicatrisation d'une population de cellules souches mésenchymateuses, le procédé comprenant la culture de la population de cellules souches mésenchymateuses dans un milieu de culture comprenant du DMEM (milieu Eagle modifié de Dulbecco), F12 (milieu F12 de Ham), M171 (milieu 171) et FBS (sérum bovin foetal). L'invention concerne également une population de cellules souches mésenchymateuses, au moins environ 90% ou plus des cellules de la population de cellules souches exprimant chacune les marqueurs suivants : CD73, CD90 et CD105, et n'exprimant pas les marqueurs suivants : CD34, CD45 et HLA-DR. L'invention concerne également une composition pharmaceutique de cette population de cellules souches mésenchymateuses.
PCT/SG2019/050204 2018-04-12 2019-04-12 Procédé d'induction ou d'amélioration des propriétés de cicatrisation de cellules souches mésenchymateuses WO2019199234A1 (fr)

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AU2019250653A AU2019250653A1 (en) 2018-04-12 2019-04-12 A method of inducing or improving wound healing properties of mesenchymal stem cells
KR1020207030469A KR20200143399A (ko) 2018-04-12 2019-04-12 중간엽 줄기 세포의 창상 치유 특성을 유도 또는 개선시키는 방법
SG11202009687PA SG11202009687PA (en) 2018-04-12 2019-04-12 A method of inducing or improving wound healing properties of mesenchymal stem cells
BR112020020092-1A BR112020020092A2 (pt) 2018-04-12 2019-04-12 método de induzir ou melhorar propriedades de cicatrização de feridas de células tronco mesenquimais
EP19784508.4A EP3775163A4 (fr) 2018-04-12 2019-04-12 Procédé d'induction ou d'amélioration des propriétés de cicatrisation de cellules souches mésenchymateuses
JP2020555404A JP2021526125A (ja) 2018-04-12 2019-04-12 間葉系幹細胞の創傷治癒特性を誘導するまたは改善する方法
CN201980039206.6A CN112262211A (zh) 2018-04-12 2019-04-12 一种诱导或改善间充质干细胞的创伤愈合性质的方法
CA3095490A CA3095490A1 (fr) 2018-04-12 2019-04-12 Procede d'induction ou d'amelioration des proprietes de cicatrisation de cellules souches mesenchymateuses
ZA2020/05967A ZA202005967B (en) 2018-04-12 2020-09-28 A method of inducing or improving wound healing properties of mesenchymal stem cells
PH12020551658A PH12020551658A1 (en) 2018-04-12 2020-10-07 A method of inducing or improving wound healing properties of mesenchymal stem cells
JP2024035680A JP2024069365A (ja) 2018-04-12 2024-03-08 間葉系幹細胞の創傷治癒特性を誘導するまたは改善する方法

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