Methods for assessing the wound-healing efficacy of mesenchymal stem cell populations, and related methods for selecting mesenchymal stem cells and identifying tissues as starting materials for generating mesenchymal stem cell populations.

Abstract

This invention relates to a method for evaluating the wound-healing efficacy of mesenchymal stem cell (MSC) populations. Furthermore, this invention relates to a method for selecting MSCs for generating stem cell populations under cGMP conditions, and a method for selecting MSC populations for generating stem cell populations for subsequent drug administration. Additionally, this invention relates to a method for selecting MSC populations for generating a master cell bank, and a method for identifying tissues suitable as starting materials for generating MSC populations for drug use. The method includes measuring the levels of at least two proteins selected from angiopoietin-1 (Ang-1), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted by MSCs into the culture medium.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 912,374, filed October 8, 2019, the contents of which are incorporated herein by reference for all purposes.

[0003] sequence list

[0004] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Technical Field

[0005] This invention relates to a method for evaluating the wound-healing efficacy of mesenchymal stem cell populations. Furthermore, this invention relates to a method for selecting mesenchymal stem cell populations for generating stem cell populations under cGMP conditions, and a method for selecting mesenchymal stem cell populations for generating stem cell populations for subsequent drug administration. Additionally, this invention relates to a method for selecting mesenchymal stem cell populations for generating a master cell bank, and a method for identifying tissues suitable as starting materials for generating mesenchymal stem cell populations for drug use. This invention also relates to the use of at least one protein for evaluating the wound-healing efficacy of mesenchymal stem cell populations. This invention also relates to the use of at least one protein for selecting mesenchymal stem cell populations for generating stem cell populations under cGMP conditions. Furthermore, this invention relates to the use of at least one protein for selecting mesenchymal stem cell populations for generating stem cell populations for subsequent drug administration. This invention also relates to the use of at least one protein for selecting mesenchymal stem cell populations for generating a master cell bank, and to the use of at least one protein for selecting mesenchymal stem cell populations for selecting mesenchymal stem cell populations from tissues suitable as starting materials for generating mesenchymal stem cell populations for drug use. Furthermore, the present invention relates to a method for identifying a culture medium suitable for inducing or improving the wound healing properties of mesenchymal stem cell populations. Background Technology

[0006] Mesenchymal stem cells (MSCs) are capable of self-renewal and multi-lineage differentiation. Therefore, these cells are an attractive and promising tool for regenerative medicine. MSCs can be isolated from a variety of tissues, such as bone marrow stroma, adipose tissue, dermis, placenta, cord blood, or various umbilical cord tissues, including Wharton's glial, the subendothelial layer of the umbilical vein, or amnion tissue (Mitchell, Ke.E. et al. (2003) Stem Cells 21, 50-60; US Patent 5,919,702; US Patent Application 2004 / 0136967; Romanov, YA et al. (2003) Stem Cells 21, 105-110; Covas, DT et al. (2003) Braz. J. Med. Biol. Res. 36, 1179-1183; US2006 / 0078993). Mesenchymal stem cells isolated from the umbilical cord amnion have been first reported in U.S. Patent Application 2006 / 0078993 (which became granted U.S. Patents 9,085,755, 9,737,568, and 9,844,571) and the corresponding International Patent Application WO2006 / 019357. Furthermore, such populations of mesenchymal stem cells from the umbilical cord amnion have recently been described in U.S. Application 20181 / 27721 or the corresponding International Application WO2018 / 067071.

[0007] The mesenchymal stem cell population described in U.S. Application 20181 / 27721 or the corresponding international application WO2018 / 067071 has the advantage that 99% or more of the stem cells in the population express the three MSC markers CD73 and CD90, but lack the expression of CD34, CD45 and HLA-DR. Therefore, this highly homologous and well-defined cell population is an ideal candidate for clinical trials and cell-based therapies because, for example, it fully meets the criteria commonly accepted for human MSCs used in cell therapy, such as those defined by Dominii et al., “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement”, Cytotherapy (2006) Vol.8, No.4, 315-317; Sensebe et al., “Production of mesenchymalstromal / stem cells according to good manufacturing practices: a review”, Stem Cell Research & Therapy 2013, 4:66; Vonk et al., Stem Cell Research & Therapy (2015) 6:94; or Kundrotas Acta Medica Lituanica. 2012. Vol.19. No.2. P.75–79. As described in International Application WO2018 / 067071, this population of mesenchymal stem cells can, for example, be used in its undifferentiated state for wound healing purposes, such as the treatment of burns or chronic diabetic wounds. Alternatively, this population of mesenchymal stem cells can differentiate into, for example, insulin-producing β-islet cells, which can then be administered (e.g., by implantation) to patients with insulin deficiency such as diabetes (see also International Application WO2007 / 046775 in this regard).

[0008] International application WO2018 / 067071 also describes a method for producing such a population of mesenchymal stem cells, suitable for use under Good Manufacturing Practice (GMP) conditions, such as those required for allogeneic cell-based therapies. However, GMP production requires quality control of the prepared pharmaceutical product, regardless of whether the product is a small organic molecule, biomolecule, or even a cell population, as in the case of the mesenchymal stem cell population in international application WO2018 / 067071. Therefore, it is anticipated that a quality control assay for the GMP production of the mesenchymal stem cell population in international application WO2018 / 067071 will soon be available.

[0009] In this context, mesenchymal stem cells (MSCs), like any other biological material, are known to possess intrinsic variability. For example, cell biology studies of MSCs have identified a variety of factors influencing their lifespan and expansion potential. Issues affecting the overall quality of MSCs have been described, such as donor tissue origin, donor age, environmental context, and isolation methods (see Paladino et al., “Comparison between isolation protocols highlights intrinsic variability of human umbilical cord mesenchymal cells,” Cell and Tissue Banking, vol. 17, no. 1, pp. 123–136, (2016), https: / / doi.org / 10.1007 / s10561-015-9525-6). Furthermore, Paladino et al., ibid., 2016 addressed individual variability in MSCs by comparing three different methods of isolating MSCs from the umbilical cord for cell bank construction and identifying advantages in cell viability, culture lifespan, expansion potential, and differentiation efficiency. The authors reported that, because the same samples were treated using at least two of the three study protocols under highly controlled experimental conditions, their results revealed that a portion of the observed variability was clearly intrinsic to each donor, with readings in doubling time and lifespan. In a further study, Paladino et al. (2017) “Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy”, Hindawi, Stem Cells International Volume 2017, Article ID 8492797, 12 pages, https: / / doi.org / 10.1155 / 2017 / 8492797 showed that gene expression of immunomodulatory molecules differed in Wharton's glial mesenchymal stem cell (WJ-MSC) samples without a specific pattern. In co-culture, all WJ-MSCs were able to inhibit mitogen-activated CD3+ T cell proliferation, albeit to varying degrees, and each PBMC responded with different levels of inhibition. The authors propose that each WJ-MSC exhibits unique behavior, differing in cytokine mRNA expression patterns and immunomodulatory potency. They also hypothesize that variability between samples plays a role in the effectiveness of WJ-MSCs used in therapy.

[0010] Based on these results, MSCs derived from the umbilical cord amnion may also exhibit inherent variability in the production of specific molecules, which in turn could affect their suitability for therapeutic applications such as wound healing or diabetes. Therefore, for example, it would be advantageous to have a method for identifying MSC populations or MSC-containing donor tissues suitable for wound healing purposes. Ideally, this method could also be used to generate master cell banks (essential for the production of cell therapy drug products) or to generate stem cell populations for subsequent drug administration under cGMP conditions.

[0011] Therefore, the object of the present invention is to provide a method, for example, for identifying donors of tissue containing MSCs or subsequently identifying donors of MSC populations suitable for therapeutic applications such as wound healing. Summary of the Invention

[0012] This objective is achieved by methods and uses that have the features of the independent claims.

[0013] In a first aspect, the present invention provides a method for evaluating the wound healing efficacy of a mesenchymal stem cell population, wherein the method includes measuring the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

[0014] In a second aspect, the present invention provides a method for selecting a mesenchymal stem cell population for generating a stem cell population under cGMP conditions, wherein the method includes measuring the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

[0015] In a third aspect, the present invention provides a method for selecting a mesenchymal stem cell population for generating a stem cell population for subsequent drug administration, wherein the method includes measuring the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

[0016] In a fourth aspect, the present invention provides a method for selecting a population of mesenchymal stem cells for generating a master cell bank, wherein the method includes measuring the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the population of mesenchymal stem cells.

[0017] In a fifth aspect, the present invention provides a method for identifying tissue suitable as starting material for producing a population of mesenchymal stem cells for pharmaceutical purposes, wherein the method includes determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in a culture medium from a sample of the tissue or from cells isolated from the tissue.

[0018] In a sixth aspect, the present invention provides the use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for evaluating the wound healing efficacy of mesenchymal stem cell populations.

[0019] In a seventh aspect, the present invention provides the use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for selecting a mesenchymal stem cell population for generating a stem cell population under cGMP conditions.

[0020] In an eighth aspect, the present invention provides the use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for selecting a mesenchymal stem cell population for generating a stem cell population for subsequent drug administration.

[0021] In a ninth aspect, the present invention provides the use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for selecting a population of mesenchymal stem cells for generating a master cell bank.

[0022] In a tenth aspect, the present invention provides the use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for identifying tissues suitable as starting materials for generating populations of mesenchymal stem cells for pharmaceutical use.

[0023] In an eleventh aspect, the present invention provides a method for identifying a culture medium suitable for inducing or improving wound healing properties of a mesenchymal stem cell population, wherein the method includes determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

[0024] In a twelfth aspect, the present invention provides the use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for identifying a culture medium suitable for inducing or improving wound healing properties of mesenchymal stem cell populations. Attached Figure Description

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

[0026] Figure 1This diagram illustrates a flowchart of experimental steps representing an exemplary example of a method for generating a therapeutic composition comprising a population of MSCs with suitable wound-healing efficacy. The method includes identifying tissues suitable as starting material for generating a population of MSCs, assessing the wound-healing efficacy of the MSC population, selecting a population of MSCs for generating a master cell bank, and selecting a population of MSCs for drug administration. The stem cells used in this embodiment are isolated from the umbilical cord amnion, also referred to herein as umbilical cord lining membrane stem cells (CLSCs). This embodiment begins by establishing a tissue bank containing umbilical cord tissue, which can be used as starting material for MSC culture. For this purpose, donor consent for tissue donation can be obtained (Phase 1). Furthermore, blood samples from the mother and newborn are screened for infectious diseases, and the umbilical cord is tested for microbial contamination. For example, up to 100 umbilical cord samples can be collected in such a tissue bank. It has been found that tissues not critical to contamination or the donor's infectious disease are used to develop cultures for generating pure MSC lines (Phase 2). For this purpose, products growing from approximately 10 individual amniotic membranes of the umbilical cord are cultured for passages 0 to 2 for propagation. MSC markers from passage 2 (P2) cells were assessed by flow cytometry, and cytokine production in the supernatant was evaluated. In-process release criteria for MSCs in Phase 2 included >95% positivity for CD73, CD90, and CD105, and <5% positivity for CD34, CD45, and HLA-DR, with in vitro production of angiopoietin-1 and TGF-β >500 pg / ml, VEGF and HGF >100 pg / ml, and negative sterility. Cell lines meeting the Phase 2 release criteria were then further propagated in a Terumo Quantum bioreactor for Phase 3 of the manufacturing process. In Phase 3, MSC release criteria included >95% positivity for CD73, CD90, and CD105, and <5% positivity for CD34, CD45, and HLA-DR, and testing for sterility, endotoxins, mycoplasma, human pathogen viruses, and foreign viruses. The tested mesenchymal cell lines (population) were used as the master cell bank for construction (Phase 3). Thaw the master cell bank and inoculate it into cultures. Release criteria are sterility, mycoplasma, and endotoxin. Finally, test 1×, 3×, and 5× 10⁻⁶ cells. 6 MSCs are loaded into the carrier culture medium, such as 1 ml. Store in a bottle at 2-8°C before drug administration (Stage 4).

[0027] Figure 2 This paper presents the results of an analysis of the secretion levels of Ang-1, VEGF, HGF, and TGF-β (here, TGF-β1) in 10 individual MSC populations obtained from 10 different umbilical cord donors. Figure 1 The cultivation described after stage 2, Figure 2 A shows the secretion levels of transforming growth factor β (TGF-β) in individual MSC populations 8049356, 8049358, 8049359, 8049364, 8049365, 8049369, 8049370, 8049372, 8049373, and 8049384. All 10 populations had secretion levels exceeding the TGF-β threshold of approximately 500 pg / ml, indicated by the red line. Figure 2 B shows the hepatocyte growth factor (HGF) secretion levels of individual MSC populations 8049356, 8049358, 8049359, 8049364, 8049365, 8049369, 8049370, 8049372, 8049373, and 8049384. The secretion levels of 8049365, 8049369, 8049372, 8049373, and 8049384 exceeded the HGF threshold of approximately 100 pg / ml, and are indicated by the red line. Figure 2 C shows the angiopoietin-1 (Ang-1) secretion levels of individual MSC populations 8049356, 8049358, 8049359, 8049364, 8049365, 8049369, 8049370, 8049372, 8049373, and 8049384. All 10 populations had Ang-1 secretion levels exceeding the threshold of approximately 500 pg / ml, indicated by the red line. Figure 2 D shows the vascular endothelial growth factor (VEGF) secretion levels in individual MSC populations 8049356, 8049358, 8049359, 8049364, 8049365, 8049369, 8049370, 8049372, 8049373, and 8049384. Except for 8049358, 8049359, and 8049370, the secretion levels of all populations exceeded the VEGF threshold of approximately 100 pg / ml, as indicated by the red line.

[0028] Figure 3 This study assesses the stability of cytokine secretion in an MSC population (8049372). For this purpose, the secretion levels of TGF-β, HGF, Ang-1, and VEGF in two independent samples from the same population after phase 4 were measured and compared. Figure 3 A shows the secretion levels of TGF-β in two independent samples, indicating stable protein secretion of approximately 2230 pg / ml and 2419 pg / ml after stage 4, respectively. Figure 3 B shows the HGF secretion levels in two independent samples, indicating stable protein secretion of approximately 933 pg / ml and 985 pg / ml after stage 4, respectively. Figure 3C shows the secretion levels of Ang-1 in the two separate samples, indicating stable protein secretion of approximately 1800 pg / ml and 1854 pg / ml, respectively, after stage 4. Figure 3 D shows the VEGF secretion levels in two independent samples, indicating stable protein secretion of approximately 210 pg / ml and 219 pg / ml after stage 4, respectively.

[0029] Figure 4 Results of assays used to identify suitable culture media for inducing or improving wound healing properties of mesenchymal stem cell populations are presented. MSCs obtained from the placenta, Wharton's glial (WJ-MSC), and umbilical cord amnion (also known as umbilical cord liner MSCs (CL-MSC)) were cultured in different media suitable for MSC culture (PTT4, PTT6, and DMEM / F12). Protein detection was performed in the MSC supernatant, and analysis was performed using Luminex 200 and Xponent software. Figure 4 A summarizes the measurements of Ang-1. S1 represents the highest standard used in this assay. Any sample falling below this standard is considered extrapolated (overly concentrated). The figure illustrates that CL-MSCs, WJ-MSCs, and placental MSCs all produced significantly higher Ang-1 levels when grown in PTT6 compared to when MSCs were grown in PTT4 or DMEM / F12. Figure 4 B summarizes the measurements of VEGF in the analytical supernatants of CL-MSCs, WJ-MSCs, and placental MSCs cultured in PTT6, PTT4, or DMEM / F12. S1 represents the highest standard used in this assay. Any sample falling on this standard is considered extrapolated (overly concentrated). As can be seen from the figure, CL-MSCs, WJ-MSCs, and placental MSCs all produced significantly higher VEGF levels when grown in PTT6 compared to when MSCs were grown in PTT4 or DMEM / F12. Figure 4 C summarizes the HGF measurements. The figure illustrates that CLMSCs, WJMSCs, and placental MSCs all produced significantly higher Ang-1 levels when grown in PTT6 compared to when MSCs were grown in PTT4 or DMEM / F12. Figure 4 Figure D shows the single-weight measurement of TGF-β1. As can be seen from the figure, CL-MSCs, WJ-MSCs, and placental MSCs all produced more TGF-β1 when grown in PTT6 than when grown in DMEM / F12. Detailed Implementation

[0030] This invention relates to several methods suitable for quality control at various stages of a GMP production process for mesenchymal stem cell populations intended for therapeutic use, both for validation and / or for the treatment of these populations. All of these methods utilize the determination of the level of at least one protein selected from Ang-1, TGF-β, VEGF, and HGF secreted into the culture medium by the mesenchymal stem cell population.

[0031] Therefore, it has been surprisingly found that measuring the secretion levels of Ang-1, TGF-β, VEGF, and HGF in the culture medium of MSC populations is a suitable standard for several aspects of GMP production of MSC populations. Measuring the secretion levels of Ang-1, TGF-β, VEGF, and HGF in the culture medium of cultured or stored MSC populations can be used to assess the wound-healing efficacy of MSC populations, to identify suitable (donor) tissues as starting material for producing MSC populations with appropriate wound-healing efficacy, to select MSC populations for cGMP production, or to select MSC populations for subsequent drug administration or for generating master cell banks. Furthermore, measuring the secretion levels of Ang-1, TGF-β, VEGF, and HGF can be used to identify suitable culture media for inducing or improving the wound-healing properties of mesenchymal stem cell populations.

[0032] It is noted here that the involvement of Ang-1, TGF-β1, VEGF, and HGF in the wound healing process is known to those skilled in the art. For the involvement of Ang-1 (SEQ ID NO:1) in wound healing, see, for example, Li et al., Stem Cell Research & Therapy 2013, 4:113, “Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing” or Bitto et al., “Angiopoietin-1 gene transfer improves the impaired wound healing of the genetically diabetic mice without increasing VEGF expression”, Clinical Science, May 14, 2008, 114(12)707-718. In the study by Li et al., the Ang-1 gene was inserted into bone marrow mesenchymal stem cells, and the results showed that “Ang-1-MSCs significantly promoted wound healing, increased epidermal and dermal regeneration, and enhanced angiogenesis compared with MSCs, Ad-Ang-1, or sham surgery.” It is worth noting that Li et al. pointed out that MSCs alone cannot produce enough Ang-1. Therefore, the authors inserted the Ang-1 gene into MSCs to provide genetically modified cells.

[0033] For information on the role of transforming growth factor β, including TGF-β1 (SEQ ID NO:2), TGF-β2, and TGF-β3, in wound healing, particularly in the healing of chronic / non-healing wounds, see, for example, Ramirez et al., “The Role of TGFβ 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 Betain Different Phases of Wound Healing,” Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.

[0034] For the involvement of VEGF (SEQ ID NO:3) in wound healing, particularly in the healing of chronic / non-healing wounds, see, for example, Froget et al., Eur. Cytokine Netw., Vol. 14, March 2003, 60–64 or Bao et al., “The Role of Vascular Endothelial Growth Factor in Wound Healing” J Surg Res. 2009 May 15; 153(2): 347–358.

[0035] Returning to the role of HGF (SEQ ID NO:4) in wound healing, particularly chronic / non-healing wound healing, see, for example, Yoshida et al., “Neutralization of Hepatocyte Growth Factor Leads to Retarded Cutaneous Wound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation” J. Invest. Dermatol. 120:335-343, 2003; Li, Jin-Feng et al., “HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through β1-Integrin / ILK Pathway” BioMed Research International 2013(2013):470418; or Conway et al., “Hepatocyte growth factor regulation: An integral part of why wounds become chronic” Wound Rep Reg (2007) 15 683–692.

[0036] Wound healing potency can describe the potential, ability, efficacy, or effectiveness of promoting or accelerating wound healing. In this invention, for example, a population of MSCs is considered to have sufficient wound healing potency, or the tissue is considered suitable as starting material for producing a pharmaceutically suitable population of MSCs, if the secretion levels (also referred to as concentrations) of one, two, three, or all four of Ang-1, TGF-β, VEGF, and HGF are equal to or higher than a specific threshold for each of these proteins as defined herein (see also Example 2). Therefore, assessing wound healing involves determining the secretion levels of Ang-1, TGF-β, VEGF, and HGF, and subsequently determining whether a specific threshold is reached or exceeded. The assessment of the wound healing potency of a population of MSCs can be performed at different stages of MSC culture. Wound healing potency can be assessed directly on the tissue prior to MSC culture to identify tissues suitable as starting material for producing a population of MSCs with wound healing potency, and thus suitable for subsequent pharmaceutical use. For suitability for pharmaceutical application, the population of MSCs may have to be produced under current pharmaceutical manufacturing practices (cGMP). Therefore, measuring the secretion levels of Ang-1, TGF-β, VEGF, and HGF before generating an MSC population can be suitable for selecting MSC populations that will generate MSC populations under cGMP conditions. Furthermore, the methods described herein can be used to select MSC populations for subsequent drug administration.

[0037] The MSC population selected according to the present invention can also be used to generate a master cell bank. In this case, the integrity and contaminants, such as bacteria, fungi, mycoplasma, and viruses, of the selected MSC population can be further characterized and tested before cryopreservation. Once established, the MSC master cell bank allows for the expansion of specific MSC populations to form cultures for further research or production processes when needed. For example, an MSC master cell bank containing MSCs with wound-healing properties allows for the expansion of specific MSC populations that secrete amounts of Ang-1, TGF-β, VEGF, and HGF ideal for wound healing.

[0038] In the methods described herein, the secretion levels of Ang-1, TGF-β, VEGF, and / or HGF are used as selection criteria. Secretion levels equal to or exceeding a threshold can, for example, indicate (i) the wound-healing efficacy of the MSC population or (ii) a tissue or isolated cell population suitable as starting material for generating an MSC population. In this invention, the Ang-1 threshold may be about 100 pg / ml, about 200 pg / ml, about 300 pg / ml, about 400 pg / ml, about 500 pg / ml, about 600 pg / ml, about 700 pg / ml, about 800 pg / ml, about 900 pg / ml, or about 1000 pg / ml. Preferably, the Ang-1 threshold is about 500 pg / ml. The threshold for TGF-β can be approximately 100 pg / ml, approximately 200 pg / ml, approximately 300 pg / ml, approximately 400 pg / ml, approximately 500 pg / ml, approximately 600 pg / ml, approximately 700 pg / ml, approximately 800 pg / ml, approximately 900 pg / ml, or approximately 1000 pg / ml. Preferably, the threshold for TGF-β is approximately 500 pg / ml. The threshold for VEGF can be approximately 80 pg / ml, approximately 100 pg / ml, approximately 120 pg / ml, approximately 140 pg / ml, approximately 160 pg / ml, approximately 180 pg / ml, or approximately 200 pg / ml, wherein the threshold for VEGF is preferably approximately 100 pg / ml. The threshold for HGF can be approximately 80 pg / ml, approximately 100 pg / ml, approximately 120 pg / ml, approximately 140 pg / ml, approximately 160 pg / ml, approximately 180 pg / ml, or approximately 200 pg / ml. Preferably, the threshold for HGF is approximately 100 pg / ml.

[0039] In one embodiment of the invention, the following threshold level (value) is used:

[0040] The threshold for angiopoietin-1 (Ang-1) is approximately 400 pg / ml.

[0041] The threshold for transforming growth factor β (TGF-β) is approximately 400 pg / ml.

[0042] - The threshold for vascular endothelial growth factor (VEGF) is approximately 80 pg / ml.

[0043] The threshold for hepatocyte growth factor (HGF) is approximately 80 pg / ml.

[0044] In another embodiment of the invention, the following threshold level (value) is used:

[0045] The threshold for angiopoietin-1 (Ang-1) is approximately 500 pg / ml.

[0046] The threshold for transforming growth factor β (TGF-β) is approximately 500 pg / ml.

[0047] - The threshold for vascular endothelial growth factor (VEGF) is approximately 100 pg / ml.

[0048] The threshold for hepatocyte growth factor (HGF) is approximately 100 pg / ml.

[0049] In both examples, the secretion levels of all four proteins were equal to or exceeded their respective secretion level / concentration thresholds (see Example 2) to, for example, consider the MSC population to have suitable wound healing properties or to consider the tissue as a suitable starting material for generating a pharmaceutically suitable MSC population. It should be noted here that the concentrations and therefore threshold levels determined in this invention are preferably absolute concentrations.

[0050] Any pharmaceutically suitable MSC population can be used in this invention. Therefore, the MSC population can be derived from any mammalian tissue or compartment / body part known to contain MSCs. In exemplary instances, the MSC population can be an umbilical cord MSC population, a placental MSC population, an umbilical cord-placental junction MSC population, an umbilical cord blood MSC population, a bone marrow MSC population, or an adipose tissue-derived MSC population. The umbilical cord MSC population can be derived from any compartment of umbilical cord tissue containing MSCs, such as the amnion, perivascular MSC population, Wharton's glial MSC population, umbilical cord-amniotic membrane MSC population, and a mixed umbilical cord MSC population, meaning an MSC population comprising stem cells from two or more of these compartments. The MSCs in these compartments and their isolation are known to those skilled in the art and are described, for example, in Subramanian et al., “Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton's Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells”, PLoS ONE 10(6):e0127992, 2015, and in reference cited therein, Van Pham et al., “Isolation and proliferation of umbilical cord tissue-derived mesenchymal stem cells for clinical applications”, Cell Tissue Bank (2016) 17:289-302, 2016. A mixed population of MSCs from the umbilical cord can be obtained, for example, by removing arteries and veins from the umbilical cord tissue, cutting the remaining tissue and Wharton's glial into fragments, and culturing the umbilical cord tissue in the medium of the present invention (via tissue explants).Mixed MSC populations from the umbilical cord can also be obtained as tissue explants by culturing whole umbilical cord tissue with intact umbilical vessels under the conditions described in Schugar et al., “High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue. Journal of biomedicine & biotechnology. 2009; 2009: 789526” (cultured in DMEM supplemented with serum containing 10% fetal bovine serum, 10% horse serum, and 1% penicillin / streptomycin). In this case, it is noted that MSC populations at the cord-placental junction can be isolated as described in Beeravolu et al., “Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta.” J Vis Exp. 2017; (122): 55224. In embodiments of the invention, if the MSC population is derived from the umbilical cord or umbilical cord-amniotic membrane (see Examples 1-4), the umbilical cord-amniotic MSC population can be highly defined and homologous. Therefore, in one embodiment of the invention, the mesenchymal stem cell population described in International Application WO2018 / 067071 is used. Thus, in a typical example of this 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 MSCs express the following markers: CD73 (SEQ ID NO. 5), CD90 (SEQ ID NO. 6), and CD105 (SEQ ID NO. 7). Furthermore, in these instances, at least approximately 90% or more, approximately 91% or more, approximately 92% or more, approximately 93% or more, approximately 94% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, and approximately 99% or more of MSCs may lack the expression of the following biomarkers: CD34 (SEQ ID NO. 8), CD45 (SEQ ID NO. 9), and HLA-DR (SEQ ID NO. 10). In specific instances, approximately 97% or more, approximately 98% or more, or approximately 99% or more of the MSC population express CD73, CD90, and CD105, while lacking the expression of CD34, CD45, and HLA-DR.In preferred embodiments, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, and about 99% or more of the MSC population express each of CD73, CD90, and CD105, while at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, and about 99% or more of the MSC population may lack expression of CD34, CD45, and HLA-DR. In specific embodiments, about 97% or more, about 98% or more, or about 99% or more of the MSC population express CD73, CD90, and CD105, while lacking expression of CD34, CD45, and HLA-DR.

[0051] In this invention, the levels of Ang-1, TGF-β, VEGF, and HGF are typically determined in the supernatant of the culture medium, and the MSC population is stored, transported, or cultured in the medium. The MSC population can be stored long-term or short-term. Examples of long-term storage media include, but are not limited to, glycerol and trehalose, which allow storage at approximately -80°C, or cryoprotectants such as dimethyl sulfoxide (DMSO), which allow storage at approximately -195°C. Short-term storage may include transport to the administration location (e.g., a physician's office or hospital) and / or storage for a period of time until the MSC population is administered to a subject. Excipients This is an exemplary example of a short-term storage medium. Such a preservation medium is suitable for transport, allowing MSCs to be stored at approximately 2-8°C. Another example of a transportable medium is Plasmalyte. Examples of media suitable for MSC culture may include, but are not limited to, commercially available media such as CTS StemPro MSC SFM, MesenPRO RS medium, and StemPro MSC SFMXenoFree. In one embodiment of the invention, the MSC cell culture medium may be PTT6, the medium described in International Application WO2018 / 067071. According to the disclosure of International Application WO2018 / 067071, the MSC cell culture medium may therefore comprise Dulbecco Modified Eagle Medium (DMEM), Ham's F12 Medium (F12), serum-free basal media such as M171, and fetal bovine serum (FBS). Therefore, in one example, the culture medium may contain DMEM at a final concentration of approximately 55-65% (v / v), F12 at a final concentration of approximately 5-15% (v / v), M171 at a final concentration of approximately 15-30% (v / v), and FBS at a final concentration of approximately 1-8% (v / v). The value “% (v / v)” used herein refers to the volume of a single component relative to the final volume of the culture medium. This means that if DMEM is present in the culture medium, for example, at a final concentration of approximately 55-65% (v / v), then 1 liter of culture medium contains approximately 550-650 ml of DMEM. In other examples, the culture medium may contain DMEM at a final concentration of approximately 57.5-62.5% (v / v), F12 at a final concentration of approximately 7.5-12.5% ​​(v / v), M171 at a final concentration of approximately 17.5-25.0% (v / v), and FBS at a final concentration of approximately 1.75-3.5% (v / v). In another example, the culture medium may contain DMEM at a final concentration of about 61.8% (v / v), F12 at a final concentration of about 11.8% (v / v), M171 at a final concentration of about 23.6% (v / v), and FBS at a final concentration of about 2.5% (v / v). In addition to the above components, the culture medium may contain supplements that are beneficial to MSC culture. In this invention, the MSC culture medium may, for example, contain epidermal growth factor (EGF). If EGF is present, its final concentration in the culture medium is from about 1 ng / ml to about 20 ng / ml. In some of these examples, the culture medium may contain EGF at a final concentration of about 10 ng / ml. The culture medium of this invention may also contain insulin. If present, insulin may be present at a final concentration of from about 1 μg / ml to 10 μg / ml. In some of these examples, the culture medium may contain insulin at a final concentration of about 5 μg / ml. The culture medium may further contain at least one of the following supplements: adenine, hydrocortisone, and sodium 3,3',5-triiodo-L-thyroxine (T3).In such examples, the culture medium may contain all three of adenine, hydrocortisone, and sodium 3,3',5-triiodo-L-thyroxine (T3). In these embodiments, the culture medium may contain adenine at a final concentration of about 0.05 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 to about 10 μg / ml, and / or sodium 3,3',5-triiodo-L-thyroxine (T3) at a final concentration of about 0.5 to about 5 ng / ml. In this case, it is noted that by culturing MSC populations in the culture medium described herein, the expression and / or secretion of at least one protein selected from Ang-1, TGF-β, VEGF, and HGF can be increased.

[0052] In this method, centrifugation of the culture medium may be necessary to determine the concentrations of Ang-1, TGF-β, VEGF, and HGF in the medium containing MSCs, typically after a suitable time period. In this case, a suitable time period can be any incubation period (if the cell population is stored or transported, for example, in a storage or transport medium such as Hypothermosol) or culture period (if the cell population is cultured in a medium) suitable for the secretion of detectable amounts of protein by the MSC population. In an exemplary instance, a suitable time period could be an incubation or culture period of approximately 6 hours, approximately 12 hours, approximately 18 hours, approximately 24 hours, approximately 30 hours, approximately 36 hours, approximately 42 hours, approximately 46 hours, approximately 48 hours, approximately 50 hours, or approximately 54 hours. Any immunoassay suitable for detecting one or more secreted proteins in a culture medium can be used in this invention. Exemplary examples of suitable immunoassays for detecting proteins in a culture medium are enzyme-linked immunosorbent assays (ELISA) or singlet assays. Single assays (e.g., available from BioVendor, Brno, Czech Republic under the trade name Q-Plex, or from R&D Systems Inc, Minneapolis, USA) can be performed by placing two spots consisting of capture antibodies at the bottom of each well of a 96-well ELISA plate (the second spot being a positive control in addition to the assay spot to ensure proper assay procedure). An example of a suitable assay for detecting multiple proteins in a culture medium is a multiple assay. In such a multiple assay, multiple analytes can be immobilized on a solid surface, such as an ELISA plate, which spatially separates the analytes. Alternatively, multiple assays can be performed using analytes immobilized on beads or particles. In this case, different beads / particles are used for the assay of each analyte. In exemplary examples, bead-based multiple assays can be used to detect Ang-1, TGF-β, VEGF, and HGF secreted in a culture medium (see Examples 1 and 4). Such multiple assay systems for the simultaneous detection and quantification of multiple target analytes in complex samples such as cell culture media are commercially available, for example from R&D Systems Inc, Minneapolis, USA. Measurement and Highly efficient measurement.

[0053] This invention also relates to the use of at least one protein selected from Ang-1, TGF-β, VEGF, and HGF for evaluating the wound healing efficacy of MSC populations. Furthermore, this invention relates to the use of at least one protein selected from Ang-1, TGF-β, VEGF, and HGF for selecting MSC populations for generating stem cell populations under cGMP conditions. Therefore, this invention relates to the use of at least one protein selected from Ang-1, TGF-β, VEGF, and HGF for selecting MSC populations for generating stem cell populations for subsequent drug administration. This invention also relates to the use of at least one protein selected from Ang-1, TGF-β, VEGF, and HGF for selecting MSC populations for generating master cell banks.

[0054] Additionally, this invention relates to the use of at least one protein selected from Ang-1, TGF-β, VEGF, and HGF for selecting a population of MSCs from a tissue suitable as a starting material for generating a population of MSCs for pharmaceutical use. In this invention, such tissue can be any mammalian tissue or compartment / body part known to contain MSCs. Examples of such tissues include, but are not limited to, bone marrow, adipose tissue, placental tissue, tissue from the cord-placental junction, umbilical cord tissue such as Wharton's glial, and umbilical cord-amniotic membrane, to name just a few known tissue sources of MSCs. In one example, the tissue is the umbilical cord or umbilical cord-amniotic membrane, and the population of MSCs generated therefrom can be a population of MSCs from the umbilical cord-amniotic membrane.

[0055] To determine whether tissue, for example, from a specific donor, is suitable as starting material for generating a population of MSCs for pharmaceutical use, tissue can be directly cultured as tissue explants, for example. For such tissue explants, samples of various tissues (e.g., Wharton's colloid, placental amnion, or umbilical cord amnion) can be placed in tissue culture dishes and cultured in suitable culture / growth media as described herein (see also U.S. Patents 9,085,755 or 9,737,568 in this regard). Then, after a suitable culture period, cells grow from the tissue (MSCs migrate from the tissue to the surface of the culture dish), and the secretion of Ang-1, TGF-β, VEGF, and HGF is analyzed on the culture medium. Alternatively, a population of MSCs can first be isolated from selected tissues using known isolation methods, and then the isolated population of MSCs can be cultured in suitable media and the secretion of Ang-1, TGF-β, VEGF, and HGF examined. Tissue-independent, the use of Ang-1, TGF-β, VEGF, and HGF in the methods described herein may include determining at least one, at least two, at least three, or all four of the proteins secreted into the culture medium by a population of MSCs or by a sample of tissue or by cells isolated from a sample of tissue.

[0056] The present invention also relates to a method for identifying a culture medium suitable for inducing or improving wound healing properties of mesenchymal stem cell populations. This method also includes determining the level of at least one protein selected from Ang-1, TGF-β, VEGF, and HGF secreted into the cell culture medium by the MSC population. Therefore, the present invention also relates to the use of at least one protein for identifying a culture medium suitable for inducing or improving wound healing properties of mesenchymal stem cells. This use may include determining the level of at least two, at least three, or all four proteins selected from Ang-1, TGF-β, VEGF, and HGF secreted into the cell culture medium by the mesenchymal stem cell population.

[0057] The present invention will be further illustrated by the following non-limiting experimental examples.

[0058] The polypeptide sequences disclosed in this paper are described in Table 1.

[0059]

[0060]

[0061]

[0062] Table 1

[0063] Experimental Example

[0064] Example 1: Identifying suitable umbilical cords containing MSCs, evaluating and selecting those obtained from the umbilical cord for generating suitable wounds. MSC populations of oral healing drug compositions.

[0065] MSCs are derived from fresh umbilical cord tissue collected by Colorado Hospital University.

[0066] Phase 1: Organization Library

[0067] In the first step, typically after obtaining Institutional Review Board (IRB) approval from the tissue donor, the umbilical cord is collected. The freshly collected umbilical cord is then cut into 1-2 mm pieces. 3 Slices were frozen at a controlled rate in vials containing 4-5 fragments and stored in isolation in the vapor phase of liquid nitrogen (LN2) at -196°C (see [link to documentation]). Figure 1 Blood samples collected from mothers within seven days of delivery are screened for infectious diseases, and tissues are tested for microbial contamination. Release criteria during Phase 1 include negative results for all infectious diseases except CMV, negative sterility, and an acceptable maternal questionnaire. Although umbilical cords are naturally contaminated by vaginal flora during delivery, collection under antibiotic conditions will ensure some of them are sterile. Up to 100 umbilical cord samples can be collected in such a tissue bank. Sterile tissue will be used in Phase 2.

[0068] Stage 2: Developmental Culture

[0069] The developmental culture, derived from umbilical cord tissue, was used to generate a pure MSC line. Ten tissue fragments were placed in 6-well plates and proliferated for approximately 10-20 days to produce passage 0 (P0) cells. P0 cells were then seeded in 175 cm² wells. 2 The first generation (P1) cells were obtained by culturing in a flask. The P1 cells were then cultured at a rate of 1–3 × 10⁻⁶ cells / mL. 6 Cells / vials are frozen in CryoStor 5 or seeded for culture. First-generation cells are cultured at 2-3 × 10⁶ cells / vial. 5 Cells / 175cm 2 Flask inoculation. Cell morphology was recorded during propagation. MSC markers from passage 2 (P2) cells were assessed by flow cytometry, and cytokine production in the supernatant was evaluated. More specifically, P2 cells were subjected to multiplex analysis with the following analytes: Ang-1, VEGF, and HGF (R&D Systems / Bio-techne catalog number LXSAHM), and singlex assay with TGF-β. The assays were performed as follows:

[0070] Multiple determination:

[0071] (i) Standards were prepared by combining 100 μl of each standard into a single microcentrifuge tube containing an appropriate volume of complete PTT6 medium to make a total volume of 1000 μl. Standard S1 contained all the multiplex standards combined in a single vial. To prepare a 3-fold serial dilution of S1, 200 μl of complete PTT6 medium was pipetted into each of five 1.5 ml polypropylene tubes labeled S2-S6. Then, 100 μl was transferred from S1 to S2. After vortexing, 100 μl was transferred from S2 to S3. This process continued until S6. The complete PTT6 medium served as a blank.

[0072] (ii) Prepare beads by gently vortexing the vial to resuspend it. It is important to be careful not to invert the vial. If using an entire plate, mix 500 μl of beads with 5.0 ml of diluent RD2-1; adjust the volume accordingly if using fewer wells. Protect the vial or wells from light.

[0073] (iii) If required: Sample preparation. All samples are used undiluted unless the sample concentration exceeds the highest standard value (S1). In this case, repeat the determination with an appropriately diluted sample. For dilution, use PTT6. All samples are measured in triplicate.

[0074] (iv) Before adding 50 μl to each well using a multichannel pipette and reservoir, gently vortex the bead.

[0075] (v) Add 50 μl of standard or sample to each well. Then, cover the plate with plate sealant and incubate on a fixed-track shaker at 800 rpm in the dark at room temperature (RT) for 2 hours.

[0076] (vi) Prepare the biotin-antibody mixture by gently vortexing the vial to resuspend it during sample incubation. It is important to be careful not to invert the vial. Then mix 500 μl of the biotin-antibody mixture with 5.0 ml of diluent RD2-1 to thoroughly mix the solution.

[0077] (vii) Prepare streptavidin phycoerythrin (PE) by gently vortexing the vial to resuspend it (carefully do not invert the vial). Then, mix 200 μl of streptavidin-PE concentrate with 5.35 mL of wash buffer. Mix the solution thoroughly and protect it from light.

[0078] (viii) Wash the plate as follows: Attach the plate to the magnet and let it stand for at least one minute. While the plate is attached to the magnet, quickly invert the plate to pour it into the water bath, then move it downwards relatively forcefully (1-2 times) to empty the wells. Complete removal of liquid by inversion is necessary, but do not aspirate the plate. Disconnect the magnet and fill the wells with 100 μl of washing buffer using a multichannel pipette. Then, reattach the magnet and let it stand for one minute before pouring as described above. Repeat the washing process for a total of 3 washes.

[0079] (ix) Using a multichannel pipette, add 50 μl of diluted biotinylate antibody to each well. Cover the plate and incubate at room temperature for 1 hour on a shaker set to 800 rpm. Then, wash the plate three times as previously described.

[0080] (x) Add 50 μl of diluted streptoantibiotic-PE to each well. Cover the plate and incubate on a shaker at room temperature for 30 minutes as previously described. Then, wash the plate three times as previously described.

[0081] (xi) Add 100 μl of wash buffer to each well and incubate the plate on a shaker at room temperature for 2 minutes as previously described. Then, immediately transfer the well contents to a Costa 6509 96-well 4-plate using a multichannel pipette set to 120 μl. Then place the plate into the assembly mold in a Luminex 3D scanner.

[0082] (xii) The board was read and analyzed using Luminex 3D and Xponent software.

[0083] TGF-β1 single weight:

[0084] (i) The standard was prepared by using a 1.5 ml polypropylene tube to prepare the diluent. For this purpose, 500 μl of standard S1 was pipetted into tube S1. 200 μl of complete PTT6 medium was added to tubes S2-S6. The standard was diluted 1:3 by transferring 100 μl from one S1 to S7, ensuring thorough mixing.

[0085] (ii) Prepare beads by gently vortexing the vial to resuspend it. It is important to be careful not to invert the vial. If using an entire plate, mix 50 μl of beads with 5.0 ml of particulate diluent RD2-1; adjust the volume accordingly if using fewer wells. Protect the vial or wells from light.

[0086] (iii) To make TGF-β1 immunoreactive (for samples only, not standards), add 30 μl of activating reagent to 150 μl of supernatant. Mix the solution thoroughly and incubate at room temperature for 10 minutes. All samples are used undiluted unless the sample concentration exceeds the highest standard value (S1). In this case, repeat the assay with an appropriately diluted sample. For dilution, use PTT6. All samples are measured in triplicate.

[0087] (iv) Before adding 50 μl to each well using a multichannel pipette and reservoir, gently vortex the bead.

[0088] (v) Add 50 μl of standard or sample to each well. Then, cover the plate with plate sealant and incubate on a fixed-track shaker at 800 rpm in the dark at room temperature for 2 hours.

[0089] (vi) Prepare the biotin-antibody mixture by gently vortexing the vial to resuspend it during sample incubation. It is important to be careful not to invert the vial. Then mix 50 μl of biotin-antibody concentrate with 5.0 ml of biotin-antibody diluent. Mix the solution thoroughly.

[0090] (vii) Prepare streptavidin phycoerythrin (PE) by gently vortexing the vial to resuspend it (carefully do not invert the vial). Then, mix 55 μl of 100× streptavidin-PE concentrate with 5.35 ml of wash buffer. Mix the solution thoroughly and protect it from light.

[0091] (viii) Wash the plate as follows: Attach the plate to the magnet and let it stand for at least one minute. While the plate is attached to the magnet, quickly invert the plate to pour it into the water bath, then move it downwards relatively forcefully (1-2 times) to empty the wells. Complete removal of liquid by inversion is necessary, but do not aspirate the plate. Disconnect the magnet and fill the wells with 100 μl of washing buffer using a multichannel pipette. Then, reattach the magnet and let it stand for one minute before pouring as described above. Repeat the washing process for a total of 3 washes.

[0092] (ix) Using a multichannel pipette, add 50 μl of diluted biotinylate antibody to each well. Cover the plate and incubate at room temperature for 1 hour on a shaker set to 800 rpm. Then, wash the plate three times as previously described.

[0093] (x) Add 50 μl of diluted streptoantibiotic-PE to each well. Cover the plate and incubate on a shaker at room temperature for 30 minutes as previously described. Then, wash the plate three times as previously described.

[0094] (xi) Add 100 μl of wash buffer to each well and incubate the plate on a shaker at room temperature for 2 minutes as previously described. Then, immediately transfer the well contents to a Costa 6509 96-well 4-plate using a multichannel pipette set to 120 μl. Then place the plate into the assembly mold in a Luminex 3D scanner.

[0095] (xii) The board was read and analyzed using Luminex 3D and Xponent software.

[0096] Phase 3: Master Cell Bank

[0097] Then, the three cell lines that met the release criteria during Phase 2 were fed at 20-40 × 10⁻⁶. 6 Live cells were seeded into the Terumo Quantum bioreactor and multiplied for approximately 7 to 14 days. After Quantum, the cells were tested for sterility, endotoxins, mycoplasma, human pathogens, and foreign viruses. The cells were cultured at 9 to 10 × 10⁶ cells / day. 6 Cells were cryopreserved at 50-70 vials per batch. MSCs were propagated in flasks and Terumo Quantum in PTT6 medium, prepared as follows: 1000 ml (500 ml PTT6 basal medium, 236 ml M171, 236 ml DMEM F12, 25 ml fetal bovine serum, 0.1 ml 0.1 mg / ml epidermal growth factor [10 ng / ml final concentration]), 0.35 ml insulin (5 μg / ml final concentration), and incubated at 37°C in 5% CO2.

[0098] Phase 4: Therapeutic Cultures

[0099] The 1×, 3× and 5×10⁻⁶ samples tested positive for Ang-1, TGF-β, VEGF and HGF 6 MSC bottles were packaged at It should be stored at 2-8℃ before drug application.

[0100] Example 2: Protein secretion level analysis for identifying suitable donor umbilical cords

[0101] For analysis, 10 umbilical cords were collected from different donors. These cords were used to generate 10 individual MSC populations derived from the umbilical amnion.

[0102] The purpose of this experiment was to determine the secretion levels of Ang-1, VEGF, HGF, and TGF-β1 in order to draw conclusions about the changes in secretion distribution in individual MSC populations and to identify MSC populations with sufficient secretion of Ang-1, VEGF, HGF, and TGF-β1.

[0103] Therefore, according to the present invention, individual MSC populations were cultured, and after stage 2, the secretion levels of Ang-1, VEGF, HGF, and TGF-β (here, TGF-β1) in each MSC population were measured as described in Example 1. The results of the secretion level analysis are shown in... Figure 2 The specific protein thresholds (Ang-1 and TGF-β are 500 pg / ml, and VEGF and HGF are 100 pg / ml) are represented by horizontal black lines.

[0104] The results show that TGF-β1 secretion levels were more than twice the threshold in all 10 individual MSC populations. Therefore, for all individual MSC populations, TGF-β1 exceeded the threshold of 500 pg / ml. HGF secretion level plotting showed that 5 out of 10 samples exceeded the threshold of 100 pg / ml: MSC population 8049365 showed approximately 600 pg / ml, MSC population 8049369 showed approximately 300 pg / ml, MSC population 8049372 showed approximately 1450 pg / ml, MSC population 8049373 showed approximately 380 pg / ml, and MSC population 8049384 showed approximately 190 pg / ml. Ang-1 secretion level plotting showed that all individual MSC populations exceeded the threshold of 500 pg / ml. VEGF secretion levels showed that all samples, except for 8049358, 8049359, and 8049370, exceeded the threshold of 100 pg / ml. In this case, the secretion level of 8049359 was only about 50 pg / ml, while the levels of 8049358 and 8049359 were close to zero.

[0105] The results showed that the protein secretion levels varied among different MSC populations, confirming that MSCs possess individual secretion characteristics. To identify MSC populations with sufficient secretion of Ang-1, VEGF, HGF, and TGF-β1, the secretion levels of these proteins were analyzed.

[0106] In this context, MSCs exhibited sufficient secretion of Ang-1, TGF-β, VEGF, and HGF if the levels of these proteins exceeded their respective thresholds. Therefore, MSC populations 8049365, 8049369, 8049372, 8049373, and 8049384 (each derived from different donor umbilical cords) showed sufficient secretion of Ang-1, TGF-β, VEGF, and HGF because these were the only MSC populations exceeding the given threshold for each of the four selected proteins. For subsequent experiments (establishing a master cell bank and for cell production for drug purposes), MSC population 8049372 was selected.

[0107] Example 3: Analysis of the stability of secretion levels

[0108] In Example 2, a population of MSCs 8049372 demonstrating sufficient secretion of proteins Ang-1, TGF-β, VEGF, and HGF was used to analyze the stability of protein secretion levels. Therefore, the MSC population was further cultured until the fourth passage. Then, as described in Example 1, the secretion levels of Ang-1, VEGF, HGF, and TGF-β (here, TGF-β1) in two samples from the MSC population (MSCs in well 1 and MSCs in well 2) after stage 4 were analyzed. The protein secretion levels of MSC population 8049372 measured in this experiment (after stage 4) were then compared with the secretion levels of MSC population 8049372 measured in Example 2 (after stage 2). This reveals changes in endocrine levels at different time points. Therefore, conclusions can be drawn regarding the stability of protein secretion levels, and thus regarding the sufficiency of protein secretion over time. The results are shown in... Figure 3 .

[0109] After phase 2, MSC population 8049372 showed a TGF-β1 secretion level of approximately 2200 pg / ml. After two cell passages, after phase 4, MSC population 8049372 showed an average TGF-β1 secretion level of approximately 2345 pg / ml. Therefore, after more than two cell passages, the secretion level of MSC population 8049372 increased by approximately 7%. HGF secretion levels decreased by approximately 33% after two cell passages, from approximately 1450 pg / ml after phase 2 to an average of approximately 959 pg / ml after phase 4; MSC population 8049372 showed an average Ang-1 secretion level of approximately 1827 pg / ml after phase 4, a decrease of approximately 9% from the 2000 pg / ml Ang-1 level after phase 2; VEGF secretion levels increased by approximately 43% after two cell passages, from approximately 150 pg / ml after phase 2 to an average of approximately 215 pg / ml after phase 4. The results showed that the secretion levels of proteins Ang-1, VEGF, HGF, and TGF-β (here, TGF-β1) changed after more than two cell passages. However, after more than two cell passages, the levels of all analyzed proteins still exceeded the corresponding thresholds selected in this study. Therefore, the results of this experiment indicate that the MSC population 8049372 maintained relative protein secretion stability, thus preserving its wound-healing efficacy. Thus, the results suggest that the wound-healing efficacy of the MSC population can be stable over a certain period. Based on these results, the MSC population 8049372 is a suitable candidate as a starting material for generating a master cell bank or for producing pharmaceutical compositions subsequently administered to subjects.

[0110] Example 4: Identification of culture media suitable for inducing or improving the wound healing properties of mesenchymal stem cells

[0111] For this experiment, as described in International Application WO2018 / 067071, various MSC populations isolated from umbilical cord amniotic membrane were cultured in PTT4, PTT6, or DMEM / F12, and the secretion of wound healing marker proteins was then analyzed for comparison.

[0112] Culture protocols for culturing isolated MSCs

[0113] • Plate 5 million MSCs in 100 mm tissue culture dishes in DMEM / F12 / 10% FCS for 24 hours.

[0114] • Discard the culture medium and add PTT4, PTT6 / DMEM / F12 and incubate for 24 hours.

[0115] • Discard the culture medium and wash the cells with PBS.

[0116] Add 10ml of DMEM and incubate for 24 hours.

[0117] • Discard the culture medium and add 5 ml of DMEM for further culture.

[0118] • After 24 hours of culture, the conditioned medium was harvested, cell debris was removed by centrifugation, and the supernatant was divided into equal portions into test tubes and stored at -80°C. Subsequently, the secretion of marker proteins was analyzed by cytokine assay.

[0119] Secretion levels of PTT4 and PTT6 were analyzed in MSC supernatant, unlike the analysis of secretion levels in DMEM / F12 medium supernatant. Measurements and analyses were performed using Luminex 200 and Xponent software.

[0120] In addition to placental supernatant samples, each sample was tested three times. The aim of this experiment was to generate cytokine signatures of MSCs cultured in PTT4 or PTT6 and to compare the signatures of MSCs from different tissue sources (umbilical cord lining membrane vs. Wharton's glial vs. placental MSCs). Cytokine assays were performed as described below. This signature will elucidate which stem cell populations, grown in which culture medium, will secrete more of the target cytokines, indicating that the medium is suitable for inducing or promoting wound-healing properties of MSCs.

[0121] Multiple analysis

[0122] Multiple information:

[0123] R&D Systems / Bio-techne catalog number LXSAHM. This kit is lot number L123680, expired on 08 / 28 / 18, and contains the following analytes:

[0124] Ang-1, angiopoietin

[0125] VEGF, vascular endothelial growth factor

[0126] HGF, hepatocyte growth factor

[0127] TGF-β1 single-weight information: R&D Systems / Bio-techne:

[0128] • Base reagent kit, catalog number LTGM00, batch number P156217, received on 02 / 27 / 18, expires on 08 / 30 / 18.

[0129] • TGF-β1 component, catalog number LTGM100, batch number P161760, received on 02 / 27 / 18, expired on 11 / 27 / 19.

[0130] Multiple information:

[0131] R&D Systems / Bio-techne catalog number LXSAHM. This kit, lot number L123999, expires on 09 / 25 / 18 and contains the following analytes:

[0132] Ang-1, angiopoietin

[0133] VEGF, vascular endothelial growth factor

[0134] HGF, hepatocyte growth factor

[0135] Data registration

[0136] The original data output is in PDF and Excel formats. We will use the Excel format data for data processing.

[0137] program

[0138] Protein detection in MSC supernatant was performed according to the detailed protocol information. As part of this experiment, the protocol had a single modification: Standard 8 in the multiplex kit was no longer used. Standard 8 was discontinued because the R&DSystems protocol itself only uses standards 1 through 6. Furthermore, the sequence of Standard 8 was only validated for two of the six analytes constituting the multiplex analytes, including HGF in the multiplex. In the case of HGF, the analyte fell in the middle region of the standard curve. Since the standards were reconstructed using growth media, the standard curves were constructed using PTT4 and PTT6. Test samples grown in PTT4 or PTT6 were extrapolated from their respective standard curves. Results were extrapolated from analyte-specific standard curves using Luminex software, which was generated by the same software: the analysis algorithm was set to utilize weighted Logistic 5P with 1 / y² weighting.

[0139] sample

[0140] 1. DMEM / F12, PTT6, and PTT4 media (not exposed to MSCs)

[0141] 2. Supernatant of the MSC to be tested

[0142] 3. Optional: supernatant from different donors; CR001A, C, D and G.

[0143] The results for Ang-1 are shown in Figure 4 In A, it was shown that MSCs from the umbilical cord amnion produced more Ang-1 when grown in PTT6 than when grown in DMEM / F12 or PTT4. VEGF results were shown in... Figure 4In B, it was shown that MSCs from the umbilical amnion produced more VEGF when grown in PTT6 than when grown in DMEM / F12 or PTT4. Results for HGF were shown in... Figure 4 In C, it was shown that MSCs from the umbilical cord amnion produced more HGF when grown in PTT6 than when grown in DMEM / F12 or PTT4. Finally, the results for TGF-β1 were shown in... Figure 4 In D, it was shown that MSCs from the umbilical amnion produced more TGF-β1 when grown in PTT6 than when grown in DMEM / F12 or PTT4.

[0144] The following conclusions can be drawn from the above experiments. When MSCs are cultured in PTT6 medium, the secretion of Ang-1, TGF-β1, VEGF, and HGF by the MSC population is significantly increased compared to their secretion levels in PTT4 or commercially available media such as DMEM / F12. PTT6 medium has the highest potency in inducing or improving the wound healing properties of MSC populations by promoting the secretion of all Ang-1, TGF-β1, VEGF, and HGF (which, as described herein, are known to be involved in wound healing) in the MSC population. Therefore, measuring the secretion levels of Ang-1, TGF-β1, VEGF, and HGF can be used to identify suitable media for inducing or improving the wound healing properties of mesenchymal stem cell populations.

[0145] It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0146] All patents and publications mentioned in this specification represent the level of skill of one ordinary person in the art to which this invention pertains. All patents and publications are incorporated herein by reference to the extent that each individual publication is specifically and individually indicated as being incorporated herein by reference.

[0147] The invention described herein by way of example may be practiced without any elements or limitations specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and not restrictively. Furthermore, the terminology and expressions used herein are used as descriptive rather than restrictive terms, and in using these terms and expressions, no equivalents of the shown and described features or portions thereof are intended to be excluded; however, it should be recognized that various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed by way of preferred embodiments and optional features, modifications and variations of the invention as disclosed herein can be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention. The invention has been described broadly and generally herein. Each narrower group of species and subgenus falling within the general disclosure also forms part of the invention. This includes the general description of the invention, with any subject matter removed from that genus by incidental conditions or negative limitations, regardless of whether the removed material is specifically described herein. Furthermore, where features or aspects of the invention are described in the form of the Markush group, those skilled in the art will recognize that the invention is also described in the form of any single member or subgroup of the Markush group. Other embodiments of the invention will become apparent from the following claims.

[0148] The invention is further characterized by the following items.

[0149] 1. A method for evaluating the wound healing efficacy of a mesenchymal stem cell population, wherein the method comprises determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

[0150] 2. A method for identifying tissues suitable as starting material for generating populations of mesenchymal stem cells for pharmaceutical use, wherein the method comprises determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in a culture medium from a sample of the tissue or from cells isolated from the tissue.

[0151] 3. A method for selecting a mesenchymal stem cell population for generating a stem cell population under cGMP conditions, wherein the method includes determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

[0152] 4. A method for selecting a population of mesenchymal stem cells for generating a population of stem cells for subsequent drug administration, wherein the method includes determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the population of mesenchymal stem cells.

[0153] 5. A method for selecting a population of mesenchymal stem cells for generating a master cell bank, wherein the method includes determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the population of mesenchymal stem cells.

[0154] 6. The method of any one of items 1 to 5, wherein the method includes determining the level of at least two, at least three, or all four proteins selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by a population of mesenchymal stem cells.

[0155] 7. As in any of items 1 to 6, where a secretion level equal to or exceeding a threshold indicates:

[0156] (i) The wound healing efficacy of mesenchymal stem cell populations; or

[0157] (ii) Tissues or isolated cells suitable as starting material for generating a population of mesenchymal stem cells.

[0158] 8. The method of Project 7, wherein the threshold for angiopoietin-1 (Ang-1) is approximately 400 pg / ml or approximately 500 pg / ml.

[0159] 9. The method as described in item 7 or 8, wherein the threshold for transforming growth factor β (TGF-β) is approximately 400 pg / ml or approximately 500 pg / ml.

[0160] 10. The method of any one of items 7 to 9, wherein the threshold for vascular endothelial growth factor (VEGF) is approximately 80 mg / ml or approximately 100 pg / ml.

[0161] 11. The method of any one of items 7 to 10, wherein the threshold for hepatocyte growth factor (HGF) is approximately 80 pg / ml or approximately 100 pg / ml.

[0162] 12. The method described in any of items 8 to 11, wherein the secretion levels of all four proteins are equal to or exceed their respective thresholds, and wherein the threshold is...

[0163] The threshold for angiopoietin-1 (Ang-1) is approximately 400 pg / ml.

[0164] The threshold for transforming growth factor β (TGF-β) is approximately 400 pg / ml.

[0165] - The threshold for vascular endothelial growth factor (VEGF) is approximately 80 pg / ml, and

[0166] The threshold for hepatocyte growth factor (HGF) is approximately 80 pg / ml.

[0167] 13. The method described in any of items 8 to 11, wherein the secretion levels of all four proteins are equal to or exceed their respective thresholds, and wherein the threshold is...

[0168] The threshold for angiopoietin-1 (Ang-1) is approximately 500 pg / ml.

[0169] The threshold for transforming growth factor β (TGF-β) is approximately 500 pg / ml.

[0170] - The threshold for vascular endothelial growth factor (VEGF) is approximately 100 pg / ml, and

[0171] The threshold for hepatocyte growth factor (HGF) is approximately 100 pg / ml.

[0172] 14. The method of any one of items 1 to 13, wherein the mesenchymal stem cell population is selected from umbilical cord mesenchymal stem cell populations, placental mesenchymal stem cell populations, umbilical cord-placental junction mesenchymal stem cell populations, umbilical cord blood mesenchymal stem cell populations, bone marrow mesenchymal stem cell populations, and adipose tissue-derived mesenchymal stem cell populations.

[0173] 15. The method of Project 14, wherein the mesenchymal stem cell population of the umbilical cord is selected from the mesenchymal stem cell population of the amnion (AM), the perivascular (PV) mesenchymal stem cell population, the Wharton's glial (WJ) mesenchymal stem cell population, the mesenchymal stem cell population of the umbilical cord amnion, and the mixed mesenchymal stem cell population (MC) of the umbilical cord.

[0174] 16. The method of Project 2, wherein the tissue is the umbilical cord or umbilical cord amnion, and the mesenchymal stem cell population is the stem cell population of the umbilical cord amnion.

[0175] 17. The method of Item 15 or 16, wherein the mesenchymal stem cell population of the umbilical cord amnion is a mesenchymal stem cell population, wherein at least about 90% or more of the cells in the stem cell population express each of the following markers: CD73, CD90, and CD105.

[0176] 18. The method of Project 17, wherein at least about 90% or more of the cells in the mesenchymal stem cell population lack the expression of the following markers: CD34, CD45 and HLA-DR.

[0177] 19. The method of Item 17 or 18, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the mesenchymal stem cell population express each of CD73, CD90 and CD105 and lack expression of each of CD34, CD45 and HLA-DR.

[0178] 20. The method of any of the preceding items, wherein the culture medium is a cell culture medium or a storage culture medium.

[0179] 21. The method of Project 20, wherein the storage medium is Hypothermosol or Plasmalyte.

[0180] 22. The method of any one of items 1 to 19, wherein the culture medium comprises Dulbecco modified Eagle medium (DMEM) at a final concentration of about 55-65% (v / v), Ham's F12 medium (F12) at a final concentration of about 5-15% (v / v), serum-free basal medium at a final concentration of about 15-30% (v / v), and fetal bovine serum (FBS) at a final concentration of about 1-8% (v / v).

[0181] 23. The method of Project 22, wherein the culture medium comprises Dulbecco modified Eagle medium (DMEM) at a final concentration of about 57.5-62.5% (v / v), Ham's F12 medium (F12) at a final concentration of about 7.5-12.5% ​​(v / v), serum-free basal medium at a final concentration of about 17.5-25.0% (v / v), and fetal bovine serum (FBS) at a final concentration of about 1.75-3.5% (v / v).

[0182] 24. The method of Item 23, wherein the culture medium comprises Dulbecco modified Eagle medium (DMEM) at a final concentration of about 61.8% (v / v), Ham's F12 medium (F12) at a final concentration of about 11.8% (v / v), serum-free basal medium at a final concentration of about 23.6% (v / v), and fetal bovine serum (FBS) at a final concentration of about 2.5% (v / v).

[0183] 25. As in Project 23 or 24, wherein the serum-free basal medium is M171.

[0184] 26. The method of any one of items 22 to 25, wherein the culture medium further comprises epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml.

[0185] 27. The method of Project 26, wherein the culture medium contains epidermal growth factor (EGF) at a final concentration of approximately 10 ng / ml.

[0186] 28. The method of any of items 22 to 27, wherein the culture medium contains insulin at a final concentration of about 1 μg / mL to 10 μg / mL.

[0187] 29. The method of Project 28, wherein the culture medium contains insulin at a final concentration of approximately 5 μg / ml.

[0188] 30. The method of any one of items 22 to 29, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone and sodium 3,3',5-triiodo-L-thyroxine (T3).

[0189] 31. The method of any one of items 22 to 30, wherein the culture medium contains all three of adenine, hydrocortisone and sodium 3,3',5-triiodo-L-thyroxine (T3).

[0190] 32. The method of Item 30 or 31, wherein the culture medium comprises adenine at a final concentration of about 0.01 to about 0.1 μg / ml, hydrocortisone at a final concentration of about 0.1 to about 10 μg / ml, and / or sodium 3,3',5-triiodo-L-thyroxine (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0191] 33. The method of any of items 1 to 32, wherein culturing a population of mesenchymal stem cells in a medium as defined in any of items 22 to 32 above results in an increase in the expression and / or secretion of at least one protein selected from angiopoietin 1 (Ang-1), transforming growth factor β (TGF-β; particularly TGF-β1), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF), relative to a reference medium that does not contain all of DMEM (Dulbecco modified Eagle medium), F12 (Ham's F12 medium), M171 (medium 171), and FBS (fetal bovine serum).

[0192] 34. The method of any one of items 1 to 33, wherein the cell culture medium is centrifuged after a suitable culture period.

[0193] 35. The method of item 34, wherein a suitable incubation period includes about 12 hours, about 24 hours, about 36 hours, about 46 hours, about 48 hours or about 50 hours, preferably about 48 hours.

[0194] 36. The method of item 34 or 35, wherein multiple determinations are performed on the supernatant of centrifuged cell culture medium.

[0195] 37. The method of item 36, in which multiple determinations are based on beads.

[0196] 38. Use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for evaluating the wound healing efficacy of mesenchymal stem cell populations.

[0197] 39. Use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for selecting a mesenchymal stem cell population for generating a stem cell population under cGMP conditions.

[0198] 40. Use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for selecting a population of mesenchymal stem cells for generating a population of stem cells for subsequent drug administration.

[0199] 41. The use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for selecting a population of mesenchymal stem cells for generating a master cell bank.

[0200] 42. The use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for identifying tissues suitable as starting material for generating mesenchymal stem cell populations for pharmaceutical use.

[0201] 43. As in the use of item 42, wherein the tissue is the umbilical cord or umbilical cord amnion, and the mesenchymal stem cell population is a stem cell population of the umbilical cord amnion.

[0202] 44. The use of any of items 38 to 41, wherein the use includes determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in a cell culture medium secreted by a population of mesenchymal stem cells into the cell culture medium.

[0203] 45. As described in item 42 or 43, wherein the use includes determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in a cell culture medium from a sample of tissue or from cells isolated from tissue.

[0204] 46. ​​The use of any of items 38 to 44, wherein the use includes determining the level of at least two, at least three, or all four proteins selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in a cell culture medium secreted by a population of mesenchymal stem cells into the cell culture medium.

[0205] 47. A method for identifying a culture medium suitable for inducing or improving wound healing properties of a mesenchymal stem cell population, wherein the method comprises determining the level of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

[0206] 48. Use of at least one protein selected from angiopoietin-1 (Ang-1), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for identifying a culture medium suitable for inducing or improving wound healing properties of mesenchymal stem cell populations.

[0207] 49. As described in item 48, wherein the use includes determining the levels of at least two, at least three, or all four proteins secreted into the culture medium by a population of mesenchymal stem cells. sequence list <110> Cell Research Private Limited <120> Methods for assessing the wound-healing efficacy of mesenchymal stem cell populations, and related methods for selecting mesenchymal stem cells and identifying tissues as starting materials for generating mesenchymal stem cell populations. <130> LC22310006P <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 498 <212> PRT <213> people() <400> 1 Met Thr Val Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His 1 5 10 15 Ile Gly Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30 Tyr Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45 Glu His Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60 Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser 65 70 75 80 Gln Lys Leu Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp 85 90 95 Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met 100 105 110 Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu 115 120 125 Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140 Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu 145 150 155 160 Ile Gln Leu Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165 170 175 Leu Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser 180 185 190 Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu 195 200 205 Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr 210 215 220 Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala 225 230 235 240 Thr Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp 245 250 255 Thr Val His Asn Leu Val Asn Leu Cys Thr Lys Glu Gly Val Leu Leu 260 265 270 Lys Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp 275 280 285 Val Tyr Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile 290 295 300 Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn 305 310 315 320 Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp 325 330 335 Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser 340 345 350 Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln 355 360 365 Arg Gln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg 370 375 380 Ala Tyr Ser Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn 385 390 395 400 Tyr Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser 405 410 415 Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn 420 425 430 Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp 435 440 445 Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala 450 455 460 Gly Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys 465 470 475 480 Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu 485 490 495 Asp Phe <210> 2 <211> 503 <212> PRT <213> Human() <400> 2 Met Glu Ala Ala Val Ala Ala Pro Arg Pro Arg Leu Leu Leu Leu Val 1 5 10 15 Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr 20 25 30 Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys 35 40 45 Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys 50 55 60 Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg 65 70 75 80 Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr 85 90 95 Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro 100 105 110 Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Ala Ala 115 120 125 Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser Leu Met Leu Met 130 135 140 Val Tyr Ile Cys His Asn Arg Thr Val Ile His His Arg Val Pro Asn 145 150 155 160 Glu Glu Asp Pro Ser Leu Asp Arg Pro Phe Ile Ser Glu Gly Thr Thr 165 170 175 Leu Lys Asp Leu Ile Tyr Asp Met Thr Thr Ser Gly Ser Gly Ser Gly 180 185 190 Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Arg Thr Ile Val Leu Gln 195 200 205 Glu Ser Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg Gly Lys Trp 210 215 220 Arg Gly Glu Glu Val Ala Val Lys Ile Phe Ser Ser Arg Glu Glu Arg 225 230 235 240 Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met Leu Arg His 245 250 255 Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn Lys Asp Asn Gly Thr 260 265 270 Trp Thr Gln Leu Trp Leu Val Ser Asp Tyr His Glu His Gly Ser Leu 275 280 285 Phe Asp Tyr Leu Asn Arg Tyr Thr Val Thr Val Glu Gly Met Ile Lys 290 295 300 Leu Ala Leu Ser Thr Ala Ser Gly Leu Ala His Leu His Met Glu Ile 305 310 315 320 Val Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser 325 330 335 Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu 340 345 350 Gly Leu Ala Val Arg His Asp Ser Ala Thr Asp Thr Ile Asp Ile Ala 355 360 365 Pro Asn His Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu 370 375 380 Asp Asp Ser Ile Asn Met Lys His Phe Glu Ser Phe Lys Arg Ala Asp 385 390 395 400 Ile Tyr Ala Met Gly Leu Val Phe Trp Glu Ile Ala Arg Arg Cys Ser 405 410 415 Ile Gly Gly Ile His Glu Asp Tyr Gln Leu Pro Tyr Tyr Asp Leu Val 420 425 430 Pro Ser Asp Pro Ser Val Glu Glu Met Arg Lys Val Val Cys Glu Gln 435 440 445 Lys Leu Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys Glu Ala Leu 450 455 460 Arg Val Met Ala Lys Ile Met Arg Glu Cys Trp Tyr Ala Asn Gly Ala 465 470 475 480 Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ser Gln Leu Ser 485 490 495 Gln Gln Glu Gly Ile Lys Met 500 <210> 3 <211> 232 <212> PRT <213> Human() <400> 3 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135 140 Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr 145 150 155 160 Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp 165 170 175 Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 180 185 190 His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn 195 200 205 Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr 210 215 220 Cys Arg Cys Asp Lys Pro Arg Arg 225 230 <210> 4 <211> 728 <212> PRT <213> Human() <400> 4 Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu 1 5 10 15 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 145 150 155 160 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170 175 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205 Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220 His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 225 230 235 240 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250 255 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365 Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380 Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met 385 390 395 400 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp 405 410 415 Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His 435 440 445 Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu 465 470 475 480 Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val 485 490 495 Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg 500 505 510 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp 515 520 525 Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr 530 535 540 Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys 545 550 555 560 Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575 Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605 Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615 620 Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu 625 630 635 640 Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu 645 650 655 Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp 660 665 670 Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu 675 680 685 Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 700 Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile 705 710 715 720 Leu Thr Tyr Lys Val Pro Gln Ser 725 <210> 5 <211> 574 <212> PRT <213> Human() <400> 5 Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu 1 5 10 15 Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu 20 25 30 His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser 35 40 45 Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu '50 55 60 Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu 65 70 75 80 Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr 85 90 95 Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala 100 105 110 Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile 115 120 125 Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro Ile Leu Ser Ala Asn Ile 130 135 140 Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro 145 150 155 160 Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr 165 170 175 Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val 180 185 190 Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu Val Asp Lys Leu Lys 195 200 205 Thr Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu 210 215 220 Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val 225 230 235 240 Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys 245 250 255 Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile Val Thr Ser Asp Asp Gly 260 265 270 Arg Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly 275 280 285 Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His 290 295 300 Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile 305 310 315 320 Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr 325 330 335 Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser 340 345 350 Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met 355 360 365 Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val 370 375 380 Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu 385 390 395 400 Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro 405 410 415 Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys 420 425 430 Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu 435 440 445 Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys 450 455 460 Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg 465 470 475 480 Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile 485 490 495 Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys 500 505 510 Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val 515 520 525 Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly 530 535 540 Arg Ile Lys Phe Ser Thr Gly Ser His Cys His Gly Ser Phe Ser Leu 545 550 555 560 Ile Phe Leu Ser Leu Trp Ala Val Ile Phe Val Leu Tyr Gln 565 570 <210> 6 <211> 161 <212> PRT <213> Human() <400> 6 Met Asn Leu Ala Ile Ser Ile Ala Leu Leu Leu Thr Val Leu Gln Val 1 5 10 15 Ser Arg Gly Gln Lys Val Thr Ser Leu Thr Ala Cys Leu Val Asp Gln 20 25 30 Ser Leu Arg Leu Asp Cys Arg His Glu Asn Thr Ser Ser Ser Pro Ile 35 40 45 Gln Tyr Glu Phe Ser Leu Thr Arg Glu Thr Lys Lys His Val Leu Phe 50 55 60 Gly Thr Val Gly Val Pro Glu His Thr Tyr Arg Ser Arg Thr Asn Phe 65 70 75 80 Thr Ser Lys Tyr Asn Met Lys Val Leu Tyr Leu Ser Ala Phe Thr Ser 85 90 95 Lys Asp Glu Gly Thr Tyr Thr Cys Ala Leu His His Ser Gly His Ser 100 105 110 Pro Pro Ile Ser Ser Gln Asn Val Thr Val Leu Arg Asp Lys Leu Val 115 120 125 Lys Cys Glu Gly Ile Ser Leu Leu Ala Gln Asn Thr Ser Trp Leu Leu 130 135 140 Leu Leu Leu Leu Ser Leu Ser Leu Leu Gln Ala Thr Asp Phe Met Ser 145 150 155 160 Leu <210> 7 <211> 658 <212> PRT <213> Human() <400> 7 Met Asp Arg Gly Thr Leu Pro Leu Ala Val Ala Leu Leu Leu Ala Ser 1 5 10 15 Cys Ser Leu Ser Pro Thr Ser Leu Ala Glu Thr Val His Cys Asp Leu 20 25 30 Gln Pro Val Gly Pro Glu Arg Gly Glu Val Thr Tyr Thr Thr Ser Gln 35 40 45 Val Ser Lys Gly Cys Val Ala Gln Ala Pro Asn Ala Ile Leu Glu Val 50 55 60 His Val Leu Phe Leu Glu Phe Pro Thr Gly Pro Ser Gln Leu Glu Leu 65 70 75 80 Thr Leu Gln Ala Ser Lys Gln Asn Gly Thr Trp Pro Arg Glu Val Leu 85 90 95 Leu Val Leu Ser Val Asn Ser Ser Val Phe Leu His Leu Gln Ala Leu 100 105 110 Gly Ile Pro Leu His Leu Ala Tyr Asn Ser Ser Leu Val Thr Phe Gln 115 120 125 Glu Pro Pro Gly Val Asn Thr Thr Glu Leu Pro Ser Phe Pro Lys Thr 130 135 140 Gln Ile Leu Glu Trp Ala Ala Glu Arg Gly Pro Ile Thr Ser Ala Ala 145 150 155 160 Glu Leu Asn Asp Pro Gln Ser Ile Leu Leu Arg Leu Gly Gln Ala Gln 165 170 175 Gly Ser Leu Ser Phe Cys Met Leu Glu Ala Ser Gln Asp Met Gly Arg 180 185 190 Thr Leu Glu Trp Arg Pro Arg Thr Pro Ala Leu Val Arg Gly Cys His 195 200 205 Leu Glu Gly Val Ala Gly His Lys Glu Ala His Ile Leu Arg Val Leu 210 215 220 Pro Gly His Ser Ala Gly Pro Arg Thr Val Thr Val Lys Val Glu Leu 225 230 235 240 Ser Cys Ala Pro Gly Asp Leu Asp Ala Val Leu Ile Leu Gln Gly Pro 245 250 255 Pro Tyr Val Ser Trp Leu Ile Asp Ala Asn His Asn Met Gln Ile Trp 260 265 270 Thr Thr Gly Glu Tyr Ser Phe Lys Ile Phe Pro Glu Lys Asn Ile Arg 275 280 285 Gly Phe Lys Leu Pro Asp Thr Pro Gln Gly Leu Leu Gly Glu Ala Arg 290 295 300 Met Leu Asn Ala Ser Ile Val Ala Ser Phe Val Glu Leu Pro Leu Ala 305 310 315 320 Ser Ile Val Ser Leu His Ala Ser Ser Cys Gly Gly Arg Leu Gln Thr 325 330 335 Ser Pro Ala Pro Ile Gln Thr Thr Pro Pro Lys Asp Thr Cys Ser Pro 340 345 350 Glu Leu Leu Met Ser Leu Ile Gln Thr Lys Cys Ala Asp Asp Ala Met 355 360 365 Thr Leu Val Leu Lys Lys Glu Leu Val Ala His Leu Lys Cys Thr Ile 370 375 380 Thr Gly Leu Thr Phe Trp Asp Pro Ser Cys Glu Ala Glu Asp Arg Gly 385 390 395 400 Asp Lys Phe Val Leu Arg Ser Ala Tyr Ser Ser Cys Gly Met Gln Val 405 410 415 Ser Ala Ser Met Ile Ser Asn Glu Ala Val Val Asn Ile Leu Ser Ser 420 425 430 Ser Ser Pro Gln Arg Lys Lys Val His Cys Leu Asn Met Asp Ser Leu 435 440 445 Ser Phe Gln Leu Gly Leu Tyr Leu Ser Pro His Phe Leu Gln Ala Ser 450 455 460 Asn Thr Ile Glu Pro Gly Gln Gln Ser Phe Val Gln Val Arg Val Ser 465 470 475 480 Pro Ser Val Ser Glu Phe Leu Leu Gln Leu Asp Ser Cys His Leu Asp 485 490 495 Leu Gly Pro Glu Gly Gly Thr Val Glu Leu Ile Gln Gly Arg Ala Ala 500 505 510 Lys Gly Asn Cys Val Ser Leu Leu Ser Pro Ser Pro Glu Gly Asp Pro 515 520 525 Arg Phe Ser Phe Leu Leu His Phe Tyr Thr Val Pro Ile Pro Lys Thr 530 535 540 Gly Thr Leu Ser Cys Thr Val Ala Leu Arg Pro Lys Thr Gly Ser Gln 545 550 555 560 Asp Gln Glu Val His Arg Thr Val Phe Met Arg Leu Asn Ile Ile Ser 565 570 575 Pro Asp Leu Ser Gly Cys Thr Ser Lys Gly Leu Val Leu Pro Ala Val 580 585 590 Leu Gly Ile Thr Phe Gly Ala Phe Leu Ile Gly Ala Leu Leu Thr Ala 595 600 605 Ala Leu Trp Tyr Ile Tyr Ser His Thr Arg Ser Pro Ser Lys Arg Glu 610 615 620 Pro Val Val Ala Val Ala Ala Pro Ala Ser Ser Glu Ser Ser Ser Thr 625 630 635 640 Asn His Ser Ile Gly Ser Thr Gln Ser Thr Pro Cys Ser Thr Ser Ser 645 650 655 Met Ala <210> 8 <211> 385 <212> PRT <213> Human() <400> 8 Met Leu Val Arg Arg Gly Ala Arg Ala Gly Pro Arg Met Pro Arg Gly 1 5 10 15<00009�9>Trp Thr Ala Leu Cys Leu Leu Ser Leu Leu Pro Ser Gly Phe Met Ser 20 25 30 Leu Asp Asn Asn Gly Thr Ala Thr Pro Glu Leu Pro Thr Gln Gly Thr 35 40 45 Phe Ser Asn Val Ser Thr Asn Val Ser Tyr Gln Glu Thr Thr Thr Pro 50 55 60<00009۵۵>Ser Thr Leu Gly Ser Thr Ser Leu His Pro Val Ser Gln His Gly Asn 65 70 75 80 It should be noted that there seems to be an error in the tag where it should likely be instead of <00009�9> and where it should likely be instead of <00009۵۵> . This translation is done based on the provided text with these potential errors.Glu Ala Thr Thr Asn Ile Thr Glu Thr Thr Val Lys Phe Thr Ser Thr 85 90 95 Ser Val Ile Thr Ser Val Tyr Gly Asn Thr Asn Ser Ser Val Gln Ser 100 105 110 Gln Thr Ser Val Ile Ser Thr Val Phe Thr Thr Pro Ala Asn Val Ser 115 120 125 Thr Pro Glu Thr Thr Leu Lys Pro Ser Leu Ser Pro Gly Asn Val Ser 130 135 140 Asp Leu Ser Thr Thr Ser Thr Ser Leu Ala Thr Ser Pro Thr Lys Pro 145 150 155 160 Tyr Thr Ser Ser Ser Pro Ile Leu Ser Asp Ile Lys Ala Glu Ile Lys 165 170 175 Cys Ser Gly Ile Arg Glu Val Lys Leu Thr Gln Gly Ile Cys Leu Glu 180 185 190 Gln Asn Lys Thr Ser Ser Cys Ala Glu Phe Lys Lys Asp Arg Gly Glu 195 200 205 Gly Leu Ala Arg Val Leu Cys Gly Glu Glu Gln Ala Asp Ala Asp Ala 210 215 220 Gly Ala Gln Val Cys Ser Leu Leu Leu Ala Gln Ser Glu Val Arg Pro 225 230 235 240 Gln Cys Leu Leu Leu Val Leu Ala Asn Arg Thr Glu Ile Ser Ser Lys 245 250 255 Leu Gln Leu Met Lys Lys His Gln Ser Asp Leu Lys Lys Leu Gly Ile 260 265 270 Leu Asp Phe Thr Glu Gln Asp Val Ala Ser His Gln Ser Tyr Ser Gln 275 280 285 Lys Thr Leu Ile Ala Leu Val Thr Ser Gly Ala Leu Leu Ala Val Leu 290 295 300 Gly Ile Thr Gly Tyr Phe Leu Met Asn Arg Arg Ser Trp Ser Pro Thr 305 310 315 320 Gly Glu Arg Leu Gly Glu Asp Pro Tyr Tyr Thr Glu Asn Gly Gly Gly 325 330 335 Gln Gly Tyr Ser Ser Gly Pro Gly Thr Ser Pro Glu Ala Gln Gly Lys 340 345 350 Ala Ser Val Asn Arg Gly Ala Gln Glu Asn Gly Thr Gly Gln Ala Thr 355 360 365 Ser Arg Asn Gly His Ser Ala Arg Gln His Val Val Ala Asp Thr Glu 370 375 380 Leu 385 <210> 9 <211> 1304 <212> PRT <213> Person() <400> 9 Met Tyr Leu Trp Leu Lys Leu Leu Ala Phe Gly Phe Ala Phe Leu Asp 1 5 10 15 Thr Glu Val Phe Val Thr Gly Gln Ser Pro Thr Pro Ser Pro Thr Gly 20 25 30 Leu Thr Thr Ala Lys Met Pro Ser Val Pro Leu Ser Ser Asp Pro Leu 35 40 45 Pro Thr His Thr Thr Ala Phe Ser Pro Ala Ser Thr Phe Glu Arg Glu 50 55 60 Asn Asp Phe Ser Glu Thr Thr Thr Ser Leu Ser Pro Asp Asn Thr Ser 65 70 75 80 Thr Gln Val Ser Pro Asp Ser Leu Asp Asn Ala Ser Ala Phe Asn Thr 85 90 95 Thr Gly Val Ser Ser Val Gln Thr Pro His Leu Pro Thr His Ala Asp 100 105 110 Ser Gln Thr Pro Ser Ala Gly Thr Asp Thr Gln Thr Phe Ser Gly Ser 115 120 125 Ala Ala Asn Ala Lys Leu Asn Pro Thr Pro Gly Ser Asn Ala Ile Ser 130 135 140 Asp Val Pro Gly Glu Arg Ser Thr Ala Ser Thr Phe Pro Thr Asp Pro 145 150 155 160 Val Ser Pro Leu Thr Thr Thr Leu Ser Leu Ala His His Ser Ser Ala 165 170 175 Ala Leu Pro Ala Arg Thr Ser Asn Thr Thr Ile Thr Ala Asn Thr Ser 180 185 190 Asp Ala Tyr Leu Asn Ala Ser Glu Thr Thr Thr Leu Ser Pro Ser Gly 195 200 205 Ser Ala Val Ile Ser Thr Thr Thr Ile Ala Thr Thr Pro Ser Lys Pro 210 215 220 Thr Cys Asp Glu Lys Tyr Ala Asn Ile Thr Val Asp Tyr Leu Tyr Asn 225 230 235 240 Lys Glu Thr Lys Leu Phe Thr Ala Lys Leu Asn Val Asn Glu Asn Val 245 250 255 Glu Cys Gly Asn Asn Thr Cys Thr Asn Asn Glu Val His Asn Leu Thr 260 265 270 Glu Cys Lys Asn Ala Ser Val Ser Ile Ser His Asn Ser Cys Thr Ala 275 280 285 Pro Asp Lys Thr Leu Ile Leu Asp Val Pro Pro Gly Val Glu Lys Phe 290 295 300 Gln Leu His Asp Cys Thr Gln Val Glu Lys Ala Asp Thr Thr Ile Cys 305 310 315 320 Leu Lys Trp Lys Asn Ile Glu Thr Phe Thr Cys Asp Thr Gln Asn Ile 325 330 335 Thr Tyr Arg Phe Gln Cys Gly Asn Met Ile Phe Asp Asn Lys Glu Ile 340 345 350 Lys Leu Glu Asn Leu Glu Pro Glu His Glu Tyr Lys Cys Asp Ser Glu 355 360 365 Ile Leu Tyr Asn Asn His Lys Phe Thr Asn Ala Ser Lys Ile Ile Lys 370 375 380 Thr Asp Phe Gly Ser Pro Gly Glu Pro Gln Ile Ile Phe Cys Arg Ser 385 390 395 400 Glu Ala Ala His Gln Gly Val Ile Thr Trp Asn Pro Pro Gln Arg Ser 405 410 415 Phe His Asn Phe Thr Leu Cys Tyr Ile Lys Glu Thr Glu Lys Asp Cys 420 425 430 Leu Asn Leu Asp Lys Asn Leu Ile Lys Tyr Asp Leu Gln Asn Leu Lys 435 440 445 Pro Tyr Thr Lys Tyr Val Leu Ser Leu His Ala Tyr Ile Ile Ala Lys 450 455 460 Val Gln Arg Asn Gly Ser Ala Ala Met Cys His Phe Thr Thr Lys Ser 465 470 475 480 Ala Pro Pro Ser Gln Val Trp Asn Met Thr Val Ser Met Thr Ser Asp 485 490 495 Asn Ser Met His Val Lys Cys Arg Pro Pro Arg Asp Arg Asn Gly Pro 500 505 510 His Glu Arg Tyr His Leu Glu Val Glu Ala Gly Asn Thr Leu Val Arg 515 520 525 Asn Glu Ser His Lys Asn Cys Asp Phe Arg Val Lys Asp Leu Gln Tyr 530 535 540 Ser Thr Asp Tyr Thr Phe Lys Ala Tyr Phe His Asn Gly Asp Tyr Pro 545 550 555 560 Gly Glu Pro Phe Ile Leu His His Ser Thr Ser Tyr Asn Ser Lys Ala 565 570 575 Leu Ile Ala Phe Leu Ala Phe Leu Ile Ile Val Thr Ser Ile Ala Leu 580 585 590 Leu Val Val Leu Tyr Lys Ile Tyr Asp Leu His Lys Lys Arg Ser Cys 595 600 605 Asn Leu Asp Glu Gln Gln Glu Leu Val Glu Arg Asp Asp Glu Lys Gln 610 615 620 Leu Met Asn Val Glu Pro Ile His Ala Asp Ile Leu Leu Glu Thr Tyr 625 630 635 640 Lys Arg Lys Ile Ala Asp Glu Gly Arg Leu Phe Leu Ala Glu Phe Gln 645 650 655 Ser Ile Pro Arg Val Phe Ser Lys Phe Pro Ile Lys Glu Ala Arg Lys 660 665 670 Pro Phe Asn Gln Asn Lys Asn Arg Tyr Val Asp Ile Leu Pro Tyr Asp 675 680 685 Tyr Asn Arg Val Glu Leu Ser Glu Ile Asn Gly Asp Ala Gly Ser Asn 690 695 700 Tyr Ile Asn Ala Ser Tyr Ile Asp Gly Phe Lys Glu Pro Arg Lys Tyr 705 710 715 720 Ile Ala Ala Gln Gly Pro Arg Asp Glu Thr Val Asp Asp Phe Trp Arg 725 730 735 Met Ile Trp Glu Gln Lys Ala Thr Val Ile Val Met Val Thr Arg Cys 740 745 750 Glu Glu Gly Asn Arg Asn Lys Cys Ala Glu Tyr Trp Pro Ser Met Glu 755 760 765 Glu Gly Thr Arg Ala Phe Gly Asp Val Val Val Lys Ile Asn Gln His 770 775 780 Lys Arg Cys Pro Asp Tyr Ile Ile Gln Lys Leu Asn Ile Val Asn Lys 785 790 795 800 Lys Glu Lys Ala Thr Gly Arg Glu Val Thr His Ile Gln Phe Thr Ser 805 810 815 Trp Pro Asp His Gly Val Pro Glu Asp Pro His Leu Leu Leu Lys Leu 820 825 830 Arg Arg Arg Val Asn Ala Phe Ser Asn Phe Phe Ser Gly Pro Ile Val 835 840 845 Val His Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Tyr Ile Gly Ile 850 855 860 Asp Ala Met Leu Glu Gly Leu Glu Ala Glu Asn Lys Val Asp Val Tyr 865 870 875 880 Gly Tyr Val Val Lys Leu Arg Arg Gln Arg Cys Leu Met Val Gln Val 885 890 895 Glu Ala Gln Tyr Ile Leu Ile His Gln Ala Leu Val Glu Tyr Asn Gln 900 905 910 Phe Gly Glu Thr Glu Val Asn Leu Ser Glu Leu His Pro Tyr Leu His 915 920 925 Asn Met Lys Lys Arg Asp Pro Pro Ser Glu Pro Ser Pro Leu Glu Ala 930 935 940 Glu Phe Gln Arg Leu Pro Ser Tyr Arg Ser Trp Arg Thr Gln His Ile 945 950 955 960 Gly Asn Gln Glu Glu Asn Lys Ser Lys Asn Arg Asn Ser Asn Val Ile 965 970 975 Pro Tyr Asp Tyr Asn Arg Val Pro Leu Lys His Glu Leu Glu Met Ser 980 985 990 Lys Glu Ser Glu His Asp Ser Asp Glu Ser Ser Asp Asp Asp Ser Asp 995 1000 1005 Ser Glu Glu Pro Ser Lys Tyr Ile Asn Ala Ser Phe Ile Met Ser Tyr 1010 1015 1020 Trp Lys Pro Glu Val Met Ile Ala Ala Gln Gly Pro Leu Lys Glu Thr 1025 1030 1035 1040 Ile Gly Asp Phe Trp Gln Met Ile Phe Gln Arg Lys Val Lys Val Ile 1045 1050 1055 Val Met Leu Thr Glu Leu Lys His Gly Asp Gln Glu Ile Cys Ala Gln 1060 1065 1070 Tyr Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp Ile Glu Val Asp Leu 1075 1080 1085 Lys Asp Thr Asp Lys Ser Ser Thr Tyr Thr Leu Arg Val Phe Glu Leu 1090 1095 1100 Arg His Ser Lys Arg Lys Asp Ser Arg Thr Val Tyr Gln Tyr Gln Tyr 1105 1110 1115 1120 Thr Asn Trp Ser Val Glu Gln Leu Pro Ala Glu Pro Lys Glu Leu Ile 1125 1130 1135 Ser Met Ile Gln Val Val Lys Gln Lys Leu Pro Gln Lys Asn Ser Ser 1140 1145 1150 Glu Gly Asn Lys His His Lys Ser Thr Pro Leu Leu Ile His Cys Arg 1155 1160 1165 Asp Gly Ser Gln Gln Thr Gly Ile Phe Cys Ala Leu Leu Asn Leu Leu 1170 1175 1180 Glu Ser Ala Glu Thr Glu Glu Val Val Asp Ile Phe Gln Val Val Lys 1185 1190 1195 1200 Ala Leu Arg Lys Ala Arg Pro Gly Met Val Ser Thr Phe Glu Gln Tyr 1205 1210 1215 Gln Phe Leu Tyr Asp Val Ile Ala Ser Thr Tyr Pro Ala Gln Asn Gly 1220 1225 1230 Gln Val Lys Lys Asn Asn His Gln Glu Asp Lys Ile Glu Phe Asp Asn 1235 1240 1245 Glu Val Asp Lys Val Lys Gln Asp Ala Asn Cys Val Asn Pro Leu Gly 1250 1255 1260 Ala Pro Glu Lys Leu Pro Glu Ala Lys Glu Gln Ala Glu Gly Ser Glu 1265 1270 1275 1280 Pro Thr Ser Gly Thr Glu Gly Pro Glu His Ser Val Asn Gly Pro Ala 1285 1290 1295 Ser Pro Ala Leu Asn Gln Gly Ser 1300 <210> 10 <211> 254 <212> PRT <213> Human() <400> 10 Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu Ser Trp Ala Ile Lys Glu Glu His Val Ile 20 25 30 Ile Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met 35 40 45 Phe Asp Phe Asp Gly Asp Glu Ile Phe His Val Asp Met Ala Lys Lys 50 55 60 Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu 65 70 75 80 Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu Glu 85 90 95 Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr Asn Val Pro Pro 100 105 110 Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg Glu Pro Asn 115 120 125 Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val Val Asn Val 130 135 140 Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Thr Gly Val Ser Glu Thr 145 150 155 160 Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe His Tyr Leu 165 170 175 Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg Val Glu His 180 185 190 Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe Asp Ala Pro 195 200 205 Ser Pro Leu Pro Glu Thr Thr Glu Asn Val Val Cys Ala Leu Gly Leu 210 215 220 Thr Val Gly Leu Val Gly Ile Ile Ile Gly Thr Ile Phe Ile Ile Lys 225 230 235 240 Gly Val Arg Lys Ser Asn Ala Ala Glu Arg Arg Gly Pro Leu 245 250

Claims

1. A method of assessing the wound healing potency of a mesenchymal stem cell population, wherein the mesenchymal stem cell population is an umbilical cord mesenchymal stem cell population, wherein the umbilical cord mesenchymal stem cell population is an umbilical cord amniotic membrane mesenchymal stem cell population, wherein the method comprises determining the level of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) secreted into the culture medium by the mesenchymal stem cell population into the culture medium, and wherein a secreted level of all four proteins equal to or exceeding a threshold value is indicative of the wound healing potency of the mesenchymal stem cell population, wherein the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

2. A method of identifying a tissue suitable as starting material for producing a mesenchymal stem cell population for pharmaceutical use, wherein the mesenchymal stem cell population is an umbilical cord mesenchymal stem cell population, wherein the umbilical cord mesenchymal stem cell population is an umbilical cord amniotic membrane mesenchymal stem cell population, wherein the method comprises determining the level of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) secreted into the culture medium by a sample of the tissue or cells isolated from the tissue into the culture medium, wherein the tissue is umbilical cord or umbilical cord amniotic membrane, and wherein a secreted level of all four proteins equal to or exceeding a threshold value is indicative of the tissue or isolated cells being suitable as starting material for producing a mesenchymal stem cell population for pharmaceutical use, wherein the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

3. A method of selecting a population of mesenchymal stem cells for producing a population of stem cells under cGMP conditions, wherein the population of mesenchymal stem cells is a population of mesenchymal stem cells of the umbilical cord, wherein the population of mesenchymal stem cells of the umbilical cord is a population of mesenchymal stem cells of the umbilical cord amnion, wherein the method comprises determining the level of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) secreted into the culture medium by the population of mesenchymal stem cells into the culture medium, and wherein a secreted level of all four proteins equal to or exceeding a threshold value indicates that the population of mesenchymal stem cells is suitable as starting material for producing a population of mesenchymal stem cells under cGMP conditions, wherein, the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

4. A method of selecting a population of mesenchymal stem cells for producing a population of stem cells for subsequent pharmaceutical administration, wherein the population of mesenchymal stem cells is a population of mesenchymal stem cells of the umbilical cord, wherein the population of mesenchymal stem cells of the umbilical cord is a population of mesenchymal stem cells of the umbilical cord amnion, wherein the method comprises determining the level of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) secreted into the culture medium by the population of mesenchymal stem cells into the culture medium, and wherein a secreted level of all four proteins equal to or exceeding a threshold value indicates that the population of mesenchymal stem cells is suitable as starting material for producing a population of mesenchymal stem cells for subsequent pharmaceutical administration, wherein the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

5. A method of selecting a population of mesenchymal stem cells for generating a master cell bank, wherein the population of mesenchymal stem cells is a population of mesenchymal stem cells of the umbilical cord, wherein the population of mesenchymal stem cells of the umbilical cord is a population of mesenchymal stem cells of the umbilical cord amnion, wherein the method comprises determining the level of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF) and Hepatocyte Growth Factor (HGF) secreted by the population of mesenchymal stem cells into the culture medium, and wherein a secreted level of all four proteins equal to or exceeding a threshold value indicates that the population of mesenchymal stem cells is suitable for generating a master cell bank, wherein, the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

6. The method of any one of claims 1 to 5, wherein the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or 500 pg / ml.

7. The method of claim 6, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or 500 pg / ml.

8. The method of claim 6, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or 100 pg / ml.

9. The method of claim 7, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or 100 pg / ml.

10. The method of claim 6, wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or 100 pg / ml.

11. The method of any one of claims 7 to 9, wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or 100 pg / ml.

12. The method of claim 6, wherein the secreted level of all four proteins equals or exceeds their respective threshold values, and wherein the threshold values are - the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml, - the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml, - the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml, and - the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml.

13. The method of claim 6, wherein the secreted level of all four proteins equals or exceeds their respective threshold values, and wherein the threshold values are - the threshold value for Angiopoietin 1 (Ang-1) is 500 pg / ml, - the threshold value for Transforming Growth Factor beta (TGF-beta) is 500 pg / ml, - the threshold value for Vascular Endothelial Growth Factor (VEGF) is 100 pg / ml, and - the threshold value for Hepatocyte Growth Factor (HGF) is 100 pg / ml. - the threshold for Hepatocyte Growth Factor (HGF) is 100 pg / ml.

14. The method of any one of claims 1 to 5, wherein 90% or more of the cells of the population of mesenchymal stem cells express each of the following markers: CD73, CD90, and CD105.

15. The method of claim 14, wherein 90% or more of the cells of the population of mesenchymal stem cells lack expression of the following markers: CD34, CD45, and HLA-DR.

16. The method of claim 14, wherein 91% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

17. The method of claim 14, wherein 92% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

18. The method of claim 14, wherein 93% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

19. The method of claim 14, wherein 94% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

20. The method of claim 14, wherein 95% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

21. The method of claim 14, wherein 96% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

22. The method of claim 14, wherein 97% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

23. The method of claim 14, wherein 98% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

24. The method of claim 14, wherein 99% or more of the cells of the population of mesenchymal stem cells express each of CD73, CD90, and CD105 and lack expression of each of CD34, CD45, and HLA-DR.

25. The method of any one of claims 1 to 5, wherein the medium is a cell culture medium or a storage medium.

26. The method of claim 25, wherein the storage medium is Hypothermosol or Plasmalyte.

27. The method of any one of claims 1 to 5, wherein the medium comprises Dulbecco’s Modified Eagle Medium (DMEM) at a final concentration of 55-65% (v / v), Ham’s F12 medium (F12) at a final concentration of 5-15% (v / v), serum-free basal medium at a final concentration of 15-30% (v / v), and fetal bovine serum (FBS) at a final concentration of 1-8% (v / v).

28. The method of claim 27, wherein the medium comprises Dulbecco’s Modified Eagle Medium (DMEM) at a final concentration of 57.5-62.5% (v / v), Ham’s F12 medium (F12) at a final concentration of 7.5-12.5% (v / v), serum-free basal medium at a final concentration of 17.5-25.0% (v / v), and fetal bovine serum (FBS) at a final concentration of 1.75-3.5% (v / v).

29. The method of claim 28, wherein the medium comprises Dulbecco’s Modified Eagle Medium (DMEM) at a final concentration of 61.8% (v / v), Ham’s F12 medium (F12) at a final concentration of 11.8% (v / v), serum-free basal medium at a final concentration of 23.6% (v / v), and fetal bovine serum (FBS) at a final concentration of 2.5% (v / v).

30. The method of claim 27, wherein the serum-free basal medium is M171.

31. The method of claim 27, wherein the medium further comprises epidermal growth factor (EGF) at a final concentration of 1 ng / ml to 20 ng / ml.

32. The method of claim 31, wherein the medium comprises epidermal growth factor (EGF) at a final concentration of 10 ng / ml.

33. The method of claim 27, wherein the medium comprises insulin at a final concentration of 1 pg / mL to 10 pg / mL.

34. The method of claim 27, wherein the medium comprises insulin at a final concentration of 5 pg / ml.

35. The method of claim 31, wherein the medium comprises insulin at a final concentration of 1 pg / mL to 10 pg / mL.

36. The method of claim 31, wherein the medium comprises insulin at a final concentration of 5 pg / ml.

37. The method of claim 27, wherein the medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt.

38. The method of claim 31, wherein the medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt.

39. The method of claim 33, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt.

40. The method of claim 35, wherein the culture medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt.

41. The method of claim 27, wherein culturing the population of mesenchymal stem cells in the culture medium results in an increase in expression and / or secretion of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) by the population of mesenchymal stem cells.

42. The method of any one of claims 1 to 5, wherein the culture medium is centrifuged after a suitable culture period.

43. The method of claim 42, wherein the suitable culture period comprises 12 hours, 24 hours, 36 hours, 46 hours, 48 hours, or 50 hours.

44. The method of claim 42, wherein the supernatant of the centrifuged cell culture medium is subjected to a multiplex assay.

45. The method of claim 44, wherein the multiplex assay is bead-based.

46. Use of all four proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for assessing wound healing potency of a population of mesenchymal stem cells of an umbilical cord, wherein the population of mesenchymal stem cells of an umbilical cord is a population of mesenchymal stem cells of an umbilical cord amnion, wherein the use comprises assaying the level of all four proteins secreted into a culture medium by the population of mesenchymal stem cells into the culture medium, and wherein a secreted level of all four proteins at or above a threshold value is indicative of wound healing potency of the population of mesenchymal stem cells, wherein the threshold value for angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for transforming growth factor beta (TGF-β) is 400 pg / ml or more, wherein the threshold value for vascular endothelial growth factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for hepatocyte growth factor (HGF) is 80 pg / ml or more.

47. Use of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) for selecting a mesenchymal stem cell population for producing a mesenchymal stem cell population under cGMP conditions, wherein the mesenchymal stem cell population is a mesenchymal stem cell population of the umbilical cord, wherein the mesenchymal stem cell population of the umbilical cord is a mesenchymal stem cell population of the umbilical cord amnion, wherein the use comprises determining the level of all four proteins secreted by the mesenchymal stem cell population into the culture medium, and wherein a secreted level of all four proteins equal to or exceeding a threshold value indicates that the mesenchymal stem cell population is suitable as starting material for producing a mesenchymal stem cell population under cGMP conditions, wherein, the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

48. Use of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) for selecting a mesenchymal stem cell population for producing a mesenchymal stem cell population for subsequent pharmaceutical administration, wherein the mesenchymal stem cell population is a mesenchymal stem cell population of the umbilical cord, wherein the mesenchymal stem cell population of the umbilical cord is a mesenchymal stem cell population of the umbilical cord amnion, wherein the use comprises determining the level of all four proteins secreted by the mesenchymal stem cell population into the culture medium, and wherein a secreted level of all four proteins equal to or exceeding a threshold value indicates that the mesenchymal stem cell population is suitable as starting material for producing a mesenchymal stem cell population for subsequent pharmaceutical administration, wherein the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

49. Use of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) for selecting a mesenchymal stem cell population for generating a master cell bank, wherein the mesenchymal stem cell population is an umbilical cord mesenchymal stem cell population, wherein the umbilical cord mesenchymal stem cell population is an umbilical cord amniotic membrane mesenchymal stem cell population, wherein the use comprises determining the level of all four proteins secreted by the mesenchymal stem cell population into a culture medium, and wherein a secreted level of all four proteins equal to or exceeding a threshold value indicates that the mesenchymal stem cell population is suitable for generating a master cell bank, wherein the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

50. Use of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) for identifying a tissue suitable as starting material for producing a mesenchymal stem cell population for pharmaceutical use, wherein the mesenchymal stem cell population is an umbilical cord mesenchymal stem cell population, wherein the umbilical cord mesenchymal stem cell population is an umbilical cord amniotic membrane mesenchymal stem cell population, wherein the tissue is umbilical cord or umbilical cord amniotic membrane, wherein the use comprises determining the level of all four proteins secreted by the mesenchymal stem cell population into a culture medium, and wherein a secreted level of all four proteins equal to or exceeding a threshold value indicates that the tissue is suitable as starting material for producing a mesenchymal stem cell population for pharmaceutical use, wherein the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

51. A method of identifying a medium suitable for inducing or improving wound healing properties of a mesenchymal stem cell population, wherein the method comprises determining the level of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) secreted by the mesenchymal stem cell population into the medium, wherein the mesenchymal stem cell population is an umbilical cord mesenchymal stem cell population, wherein the umbilical cord mesenchymal stem cell population is an umbilical cord amniotic membrane mesenchymal stem cell population, wherein the method comprises determining the level of all four proteins secreted by the mesenchymal stem cell population into the medium in the medium, and wherein a secretion level of all four proteins at or above a threshold value indicates that the medium is suitable for inducing or improving wound healing properties of a mesenchymal stem cell population, wherein the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

52. Use of all four proteins selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) for identifying a medium suitable for inducing or improving wound healing properties of a mesenchymal stem cell population, wherein the mesenchymal stem cell population is an umbilical cord mesenchymal stem cell population, wherein the umbilical cord mesenchymal stem cell population is an umbilical cord amniotic membrane mesenchymal stem cell population, wherein the use comprises determining the level of all four proteins secreted by the mesenchymal stem cell population into the medium in the medium, and wherein a secretion level of all four proteins at or above a threshold value indicates that the medium is suitable for inducing or improving wound healing properties of a mesenchymal stem cell population, wherein, the threshold value for Angiopoietin 1 (Ang-1) is 400 pg / ml or more, wherein the threshold value for Transforming Growth Factor beta (TGF-beta) is 400 pg / ml or more, wherein the threshold value for Vascular Endothelial Growth Factor (VEGF) is 80 pg / ml or more, and wherein the threshold value for Hepatocyte Growth Factor (HGF) is 80 pg / ml or more.

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