Method of assessing wound healing potency of mesenchymal stem population and related methods of selecting mesenchymal stem cells and identifying tissue as starting material for producing mesenchymal stem cell population

Abstract

To provide methods that can be used to identify donors of MSC-containing tissues or donors of MSC populations that are suitable for subsequent therapeutic applications, such as wound healing.SOLUTION: The present invention relates to a method of assessing the wound healing potency of a mesenchymal stem cell (MSC) population. In addition, the present invention concerns a method of selecting an MSC for producing a stem cell population under cGMP conditions, a method of selecting an MSC population for producing a stem cell population for subsequent pharmaceutical administration. Further the present invention relates to a method of selecting an MSC population for generating a master cell bank and a method for identifying a tissue suitable as starting material for producing an MSC for pharmaceutical use. The methods include a step of determining in a medium the level of at least two proteins selected from the group consisting of angiopoietin 1, transforming growth factor β, vascular endothelial growth factor and hepatocyte growth factor secreted in a medium by the MSC.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority based on U.S. Provisional Application No. 62 / 912,374, filed on October 8, 2019, the content of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a sequence listing in a computer - readable form, which is hereby incorporated by reference.

[0003] Field of the Invention The present invention relates to a method for assessing the wound - healing efficacy of a mesenchymal stem cell population. Further, the present invention relates to a method for selecting a mesenchymal stem cell population for generating a stem cell population under cGMP conditions and a method for selecting a mesenchymal stem cell population for generating a stem cell population for subsequent pharmaceutical administration. Further, the present invention relates to a method for selecting a mesenchymal stem cell population for generating a master cell bank and a method for identifying a tissue suitable as a starting material for generating a mesenchymal stem cell population for pharmaceutical use. The present invention also relates to the use of at least one protein for assessing the wound - healing efficacy of a mesenchymal stem cell population. The present invention also relates to the use of at least one protein for selecting a mesenchymal stem cell population for generating a stem cell population under cGMP conditions. Further, the present invention relates to the use of at least one protein for selecting a mesenchymal stem cell population for generating a stem cell population for subsequent pharmaceutical administration. The present invention relates to the use of at least one protein for selecting a mesenchymal stem cell population for generating a master cell bank and for selecting a mesenchymal stem cell population for identifying a tissue suitable as a starting material for generating a mesenchymal stem cell population for pharmaceutical use. Further, the present invention relates to a method for identifying a suitable medium for inducing or improving the wound - healing properties of a mesenchymal stem cell population.

Background Art

[0004] Background of the Invention Mesenchymal stem cells (MSCs) have the ability of self-renewal and multi-lineage differentiation. Therefore, these cells are attractive and promising tools for regenerative medicine. MSCs can be isolated from various tissues, such as bone marrow stroma, adipose tissue, dermis, placenta, umbilical cord blood, or various umbilical cord tissues, such as Wharton's jelly, the subendothelial layer of the umbilical vein, or the amniotic tissue of the umbilical cord (Mitchell, K.E. et al. (2003) Stem Cells 21, 50-60; U.S. Patent No. 5,919,702; U.S. Patent Application No. 2004 / 0136967; Romanov, Y.A. et al. (2003) Stem Cells 21, 105-110; Covas, D.T. et al. (2003) Braz. J. Med. Biol. Res. 36, 1179-1183; US2006 / 0078993). Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord were first reported in U.S. Patent Application No. 2006 / 0078993 (leading to U.S. Patents Nos. 9,085,755, 9,737,568, and 9,844,571), as well as the corresponding international patent application WO2006 / 019357. Furthermore, a population of such mesenchymal stem cells derived from the amniotic membrane of the umbilical cord was recently described in U.S. Application No. 20181 / 27721 or the corresponding international application WO 2018 / 067071.

[0005] The mesenchymal stem cell population described in US Application No. 20181 / 27721 or the corresponding international application WO 2018 / 067071 has the advantage that more than 99% of the stem cells in this population express three MSC markers, CD73, CD90, while lacking the expression of CD34, CD45, and HLA-DR. Thus, this highly homogeneous and well-defined cell population is an ideal candidate for clinical trials and cell-based therapies, for example, because it fully meets the generally accepted criteria for using human MSCs in cell therapy, such as those defined by Dominici et al, "Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement", Cytotherapy (2006) Vol. 8, No. 4, 315-317; Sensebe et al., "Production of mesenchymal stromal / stem cells according to good manufacturing practices: a review", Stem Cells Research & Therapy 2013, 4:66); Vonk et al., Stem Cells Research & Therapy (2015) 6:94; or Kundrotas Acta Medica Lituanica. 2012. Vol. 19 No. 2. P. 75-79. As described in international application WO 2018 / 067071, this mesenchymal stem cell population can be used in an undifferentiated state, for example, for wound healing, for example, for the treatment of burns or chronic diabetic wounds. Alternatively, this mesenchymal stem cell population can be differentiated into, for example, insulin-producing β-islet cells and then administered to patients suffering from insulin deficiency, for example, diabetes, for example, by transplantation (see also international application WO2007 / 046775 in this regard).

[0006] The process for generating this mesenchymal stem cell population, which is also described in international application WO 2018 / 067071, is suitable for implementation under Good Manufacturing Practice (GMP) conditions required for such allogeneic cell-based therapies. However, GMP-compliant production requires quality control of the manufactured drug product, regardless of whether the drug product is a small organic molecule, a biological molecule, or a cell population as in the case of the mesenchymal stem cell population of international application WO 2018 / 067071. Therefore, it would be desirable to make available quality control assays for GMP-compliant production of the mesenchymal stem cell population of international application WO 2018 / 067071.

[0007] In this regard, it is known that mesenchymal stem cells, like any other biological material, have inherent variability. For example, studies of the cell biology of mesenchymal stem cells have identified various factors that affect their lifespan and proliferative capacity. It has been described that issues such as the origin of the donor tissue, the donor's age, the environmental background, and the isolation method affect the overall quality of mesenchymal stem cells (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. (supra) 2016 elucidated the individual variability in mesenchymal stem cells by comparing three different isolation methods of MSCs from umbilical cord for cell banking in order to identify whether there are advantages regarding cell viability, lifespan in culture, proliferative potential, and differentiation ability. Since at least two of the three studied protocols processed the same samples under highly controlled experimental conditions, the authors reported that the results revealed that some of the observed variability, including readings of doubling time and lifespan, is clearly donor-specific. In further studies, 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 the gene expression of immunomodulatory molecules varies among samples of Wharton's jelly mesenchymal stem cells (WJ-MSC) and that there is no specific pattern.In co-culture, all WJ-MSCs were able to inhibit the proliferation of CD3+ T cells activated by mitogens, although to varying degrees, and each PBMC responded at different inhibition levels. The authors suggested that each WJ-MSC exhibited unique behavior and differed with respect to the pattern of cytokine mRNA expression and immunomodulatory capacity. The authors also hypothesized that variability between samples could play a role in the effectiveness of therapeutically utilized WJ-MSCs.

[0008] Considering these results, MSCs derived from the amnion of the umbilical cord are also likely to have intrinsic variability with respect to the production of specific molecules, which could then affect their suitability for therapeutic applications, such as wound healing or diabetes. Therefore, it would be advantageous to make available methods for identifying, for example, suitable MSC populations or MSC-containing donor tissues for use for wound healing purposes. Such methods would ideally be useful for generating master cell banks (required for manufacturing cell therapy drug products) or for manufacturing stem cell populations for subsequent pharmaceutical administration under cGMP conditions.

[0009] Accordingly, it is an object of the present invention to provide methods that can be used, for example, to identify donors of MSC-containing tissues or donors of MSC populations that are suitable for subsequent therapeutic applications, such as wound healing.

Summary of the Invention

[0010] This object is achieved by a method and use having the features of the independent claims.

[0011] In a first aspect, the present invention provides a method for assessing the wound healing efficacy of a mesenchymal stem cell population, comprising determining the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population in the medium.

[0012] 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, comprising determining the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0013] 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 pharmaceutical administration, comprising determining the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0014] In a fourth aspect, the present invention provides a method for selecting a mesenchymal stem cell population for generating a master cell bank, comprising determining the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0015] In a fifth aspect, the present invention provides a method for identifying a tissue suitable as a starting material for generating a mesenchymal stem cell population for pharmaceutical use, comprising determining the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by a sample of tissue or cells isolated from the tissue.

[0016] In a sixth aspect, the present invention provides the use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for assaying the wound healing efficacy of a mesenchymal stem cell population.

[0017] In a seventh aspect, the present invention provides the use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), 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.

[0018] In an eighth aspect, the present invention provides the use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), 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 pharmaceutical administration.

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

[0020] In a tenth aspect, the present invention provides the use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for identifying a suitable tissue as a starting material for generating a mesenchymal stem cell population for pharmaceutical use.

[0021] In an eleventh aspect, the present invention provides a method of identifying a suitable medium for inducing or improving the wound healing properties of a mesenchymal stem cell population, the method comprising determining in a medium the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF), secreted into the medium by the mesenchymal stem cell population.

[0022] In a twelfth aspect, the present invention provides the use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) proteins for identifying a suitable medium for inducing or improving the wound healing properties of a mesenchymal stem cell population. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings.

[0024]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Mode for Carrying Out the Invention

[0025] Detailed Description of the Invention The present invention relates to several methods that are all suitable for validation for various stages of a GMP-compliant manufacturing process of a mesenchymal stem cell population for therapeutic use and / or as quality control. These methods all use a step of determining the level of at least one protein selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF secreted into the medium by the mesenchymal stem cell population in the medium.

[0026] Surprisingly, it has been found that the step of determining the secretion levels of Ang-1, TGFβ, VEGF, and HGF in the medium of the MSC population is an appropriate criterion for several aspects of the GMP-compliant manufacturing process of the MSC population. The determination of the secretion levels of Ang-1, TGFβ, VEGF, and HGF in the medium in which the MSC population is cultured or stored can be used to assess the wound healing efficacy of the MSC population, to identify a suitable (donor) tissue as a starting material for generating an MSC population with appropriate wound healing efficacy, to select an MSC population for cGMP-compliant production, or to select an MSC population for later pharmaceutical administration or for generating a master cell bank. Furthermore, the determination of the secretion levels of Ang-1, TGFβ, VEGF, and HGF can be used to identify a suitable medium for inducing or improving the wound healing properties of the mesenchymal stem cell population.

[0027] Note that the involvement of Ang-1, TGFβ1, 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 Cells 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 mesenchymal stem cells, and the results showed that "Ang1-MSC significantly promoted wound healing, increased epithelial and dermal regeneration, and enhanced angiogenesis compared to MSC, Ad-Ang1, or sham treatment". Notably, the authors Li et al. stated that MSC alone does not produce sufficient Ang-1, and for this reason, the authors inserted the Ang-1 gene into MSC to obtain genetically modified cells.

[0028] Regarding the involvement of transforming growth factor β, such as TGFβ1 (SEQ ID NO:2), TGFβ2, and TGFβ3, in wound healing, specifically in the healing of chronic / non-healing wounds, see, for example, Ramirez et al., "The Role of TGFb Signaling in Wound Epithelialization", Advances In Wound Care, Volume 3, Number 7, 2013, 482-491 or Pakyari et al., Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing, Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.

[0029] Regarding the involvement of VEGF (SEQ ID NO:3) in wound healing, specifically 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.

[0030] Regarding HGF (SEQ ID NO: 4) in wound healing, specifically in the healing of chronic / non-healing wounds, see, for example, Yoshida et al., "Neutralization of Hepatocyte Growth Factor Leads to Retarded Cutaneous Wound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation" J. Invest. Dermatol. 120:335-343, 2003, Li, Jin-Feng et al. "HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through β1-Integrin / ILK Pathway." BioMed Research International 2013(2013):470418, or Conway et al, "Hepatocyte growth factor regulation: An integral part of why wounds become chronic". Wound Rep Reg (2007) 15 683-692.

[0031] Wound healing efficacy can mean the potential, ability, competence, or effectiveness to facilitate or accelerate wound healing. In the present invention, when the secretion levels (also referred to as concentrations) of one, two, three, or all four of Ang-1, TGFβ, VEGF, and HGF are equal to or higher than the specific thresholds defined herein for each of these proteins, the MSC population is considered to have, for example, sufficient wound healing efficacy, or the tissue is considered suitable as a starting material for generating a pharmaceutically suitable MSC population (see also Example 2). Thus, the assessment of wound healing includes the determination of the secretion levels of Ang-1, TGFβ, VEGF, and HGF, and the subsequent determination of whether the specific thresholds have been reached or exceeded. The assessment of the wound healing efficacy of an MSC population can be performed at different stages of MSC culture. To identify tissues suitable as starting materials for generating an MSC population that has wound healing efficacy and may thus be suitable for later pharmaceutical use, the wound healing efficacy can be directly assessed in the tissue prior to MSC culture. For suitability for pharmaceutical application, the MSC population may have to be generated under current Good Manufacturing Practice (cGMP) conditions. Thus, determining the secretion levels of Ang-1, TGFβ, VEGF, and HGF prior to generating the MSC population may be suitable for selecting an MSC population for generating an MSC population under cGMP conditions. Further, an MSC population for later pharmaceutical administration can be selected using the methods described herein.

[0032] The MSC population selected according to the present invention can also be used to generate a master cell bank. In this regard, the selected MSC population can be further characterized and tested for integrity and for contaminants such as bacteria, fungi, mycoplasma, and viruses prior to cryopreservation. After preparation, the MSC master cell bank can enable the amplification of a specific MSC population for the formation of cultures for further research or manufacturing processes whenever needed. For example, an MSC master cell bank containing MSCs with wound healing properties can enable the amplification of a specific MSC population that secretes an ideal amount of Ang-1, TGFβ, VEGF, and HGF for wound healing.

[0033] The secretion levels of Ang-1, TGFβ, VEGF, and / or HGF are used as selection criteria in the methods described herein. Secretion levels equal to or exceeding the threshold values may indicate, for example, (i) the wound healing efficacy of the MSC population, or (ii) a tissue or isolated cell population suitable as a starting material for generating the MSC population. In the present invention, the threshold value for Ang-1 can 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 threshold value for Ang-1 is about 500 pg / ml. The threshold value for TGFβ can 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 threshold value for TGFβ is about 500 pg / ml. For VEGF, the threshold value can be about 80 pg / ml, about 100 pg / ml, about 120 pg / ml, about 140 pg / ml, about 160 pg / ml, about 180 pg / ml, or about 200 pg / ml, and the threshold value for VEGF is preferably about 100 pg / ml. For HGF, the threshold value can be about 80 pg / ml, about 100 pg / ml, about 120 pg / ml, about 140 pg / ml, about 160 pg / ml, about 180 pg / ml, or about 200 pg / ml. Preferably, the threshold value for HGF is about 100 pg / ml. In one example of the present invention, the following threshold levels (threshold values) are used: - For angiopoietin 1 (Ang-1), a threshold value of about 400 pg / ml, - For transforming growth factor β (TGFβ), a threshold value of about 400 pg / ml, - For vascular endothelial growth factor (VEGF), a threshold value of about 80 pg / ml, - For hepatocyte growth factor (HGF), a threshold value of about 80 pg / ml. In another example of the present invention, the following threshold levels (threshold values) are used: - For angiopoietin 1 (Ang-1), a threshold of about 500 pg / ml, - For transforming growth factor β (TGFβ), a threshold of about 500 pg / ml, - For vascular endothelial growth factor (VEGF), a threshold of about 100 pg / ml, - For hepatocyte growth factor (HGF), a threshold of about 100 pg / ml. In these two examples, for instance, in order to consider the MSC population to have appropriate wound healing properties or to consider the tissue to be an appropriate starting material for generating a pharmaceutically suitable MSC population, the secretion levels of all four proteins are equal to or exceed their respective thresholds of secretion level / concentration (see Example 2). Here, it should be noted that the concentrations determined in the present invention, and thus the threshold levels, are preferably absolute concentrations.

[0034] Any pharmaceutically suitable MSC population can be used in the present invention. Thus, the MSC population can be derived from any mammalian tissue or compartment / body part known to contain MSCs. In an exemplary example, the MSC population can be a MSC population of the umbilical cord, a placental MSC population, a MSC population of the umbilical cord-placental attachment, a MSC population of umbilical cord blood, a MSC of bone marrow, or a MSC population derived from adipose tissue. The MSC population of the umbilical cord can be derived from any compartment of the umbilical cord tissue containing MSCs, for example, amnion, perivascular MSC population, Wharton's jelly MSC population, amnion MSC population of the umbilical cord, or a mixed MSC population of the umbilical cord, that is, a population of MSCs containing stem cells of two or more of these compartments. The MSCs of these compartments, and their isolation, are known to those skilled in the art and are described, for example, in Subramanian et al "Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton's Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells", PLoS ONE 10(6):e0127992, 2015, and the references cited therein, Van Pham et al. "Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications", Cell Tissue Bank (2016) 17:289-302, 2016. The mixed MSC population of the umbilical cord can be obtained, for example, by removing the arteries and veins from the umbilical cord tissue, mincing the remaining tissue and Wharton's jelly, and culturing the umbilical cord tissue (by tissue explants) in the culture medium of the present invention.The mixed MSC population of the umbilical cord can also be obtained by culturing the whole umbilical cord tissue with intact umbilical blood vessels as tissue explants under the conditions described by Schugar et al. in "High harvest yield, high expansion, and phenotype stability of CD146 mesenchycal stromal cells from whole primitive human umbilical cord tissue. Journal of biomedicine & biotechnology. 2009:2009:789526" (culturing in serum-supplemented DMEM containing 10% fetal bovine serum, 10% equine serum, and 1% penicillin / streptomycin). In this regard, note that the MSC population of the umbilical cord placental attachment part can be isolated as described by Beeravolu et al. in "Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta." J Vis Exp. 2017;(122):55224. In the examples of the present invention, the MSC population is derived from the umbilical cord or the amnion of the umbilical cord (see Examples 1 to 4). The MSC population of the amnion of the umbilical cord is highly defined and homogeneous. Thus, in one aspect of the present invention, the mesenchymal stem cell population described in International Application WO 2018 / 067071 is used. Thus, in a typical example of the method, at least about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the MSCs express the following markers: CD73 (SEQ ID NO.5), CD90 (SEQ ID NO.6), and CD105 (SEQ ID NO.7).Furthermore, in these examples, 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 can lack the expression of the following markers: CD34 (SEQ ID NO.8), CD45 (SEQ ID NO.9), and HLA-DR (SEQ ID NO.10). In a specific example, about 97% or more, about 98% or more, or about 99% or more of the MSC population expresses CD73, CD90, and CD105 while lacking the expression of CD34, CD45, and HLA-DR. In a preferred example, at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the 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, about 99% or more of the MSCs can lack the expression of CD34, CD45, and HLA-DR. In a specific example, about 97% or more, about 98% or more, or about 99% or more of the MSC population expresses CD73, CD90, and CD105 while lacking the expression of CD34, CD45, and HLA-DR.

[0035] In the present invention, the levels of Ang-1, TGFβ, VEGF, and HGF are typically determined in the supernatant of the medium in which the MSC population is stored, transported, or cultured. The MSC population can be stored for long or short periods. Examples of long-term storage media include, but are not limited to, glycerol and trehalose that enable storage at about -80°C, or cryoprotective substances that enable storage at about -195°C, such as dimethyl sulfoxide (DMSO). Short-term storage can include transportation to the administration site (e.g., a clinic or hospital) and / or storage for the period until the MSC population is administered to the subject. The excipient HypoThermosol® is an exemplary example of a short-term storage medium. This storage medium is suitable for transportation and enables MSC storage at about 2 - 8°C. Another example of a medium suitable for transportation is Plasmalyte. Examples of media suitable for MSC culture can include, but are not limited to, commercially available media such as CTS StemPro MSC SFM, MesenPRO RS medium, StemPro MSC SFM XenoFree. In one example of the present invention, the MSC cell culture medium can be the culture medium PTT6 described in International Application WO 2018 / 067071. Thus, according to the disclosure of International Application WO 2018 / 067071, the MSC cell culture medium can include Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 medium (F12), a serum-free basal medium such as M171, and fetal bovine serum (FBS). Thus, in one example, the medium can include DMEM at a final concentration of about 55 - 65% (v / v), F12 at a final concentration of about 5 - 15% (v / v), M171 at a final concentration of about 15 - 30% (v / v), and FBS at a final concentration of about 1 - 8% (v / v). The value of “%(v / v)” as used herein refers to the volume of an individual component relative to the final volume of the medium. This means, for example, that if DMEM is present in the medium at a final concentration of about 55 - 65% (v / v), 1 liter of the medium contains about 550 - 650 ml of DMEM.In other examples, the medium can contain DMEM at a final concentration of about 57.5 - 62.5% (v / v), F12 at a final concentration of about 7.5 - 12.5% (v / v), M171 at a final concentration of about 17.5 - 25.0% (v / v), and FBS at a final concentration of about 1.75 - 3.5% (v / v). In a further example, the medium can 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 medium can contain adjuvants that are advantageous for MSC culture. In the present invention, the MSC culture medium can contain, for example, epidermal growth factor (EGF). When present, EGF can be present in the culture medium at a final concentration of about 1 ng / ml to about 20 ng / ml. In some of these examples, the culture medium can contain EGF at a final concentration of about 10 ng / ml. The culture medium of the present invention can also contain insulin. When present, insulin can be present at a final concentration of about 1 μg / ml to 10 μg / ml. In some of these examples, the culture medium can contain insulin at a final concentration of about 5 μg / ml. The culture medium can further contain at least one of the following adjuvants: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In such examples, the culture medium can contain all three of adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3). In these examples, the culture medium can contain adenine at a final concentration of about 0.05 - about 0.1 μg / ml, hydrocortisone at a final concentration of about 1 - about 10 μg / ml, and / or 3,3',5-triiodo-L-thyronine sodium salt (T3) at a final concentration of about 0.5 - about 5 ng / ml. In this regard, note that culturing an MSC population in the medium described herein can increase the expression and / or secretion of at least one protein selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF.

[0036] In the method of the present invention, typically, after an appropriate period, it may be necessary to subject the medium containing MSCs to centrifugation in order to determine the concentrations of Ang-1, TGFβ, VEGF, and HGF. In this regard, the appropriate period can be any incubation period (when the cell population is stored or transported, for example, in a storage medium or transport medium, such as Hypothermosol) or culture period (when the cell population is cultured in a culture medium) suitable for the MSC population to secrete a detectable amount of protein. In an exemplary example, the appropriate period can be an incubation period or culture period of about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 46 hours, about 48 hours, about 50 hours, or about 54 hours. After centrifugation, the supernatant of the centrifuged medium can be subjected to an immunoassay to determine the level / concentration of the secreted protein. Any immunoassay suitable for detecting one or more proteins secreted into the medium can be applied in the present invention. Exemplary examples of suitable immunoassays for detecting proteins in the medium are enzyme-linked immunosorbent assay (ELISA) or singleplex assay. (For example, commercially available from BioVendor (Brno, Czech Republic) under the trade name Q-Plex, or from R&D Systems Inc (Minneapolis, USA)) The singleplex assay can be performed by placing two spots consisting of capture antibodies in a defined arrangement at the bottom of each well of a 96-well ELISA plate (in addition to the assay spot, the second spot is a positive control spot to ensure appropriate assay procedures). An example of a suitable assay for detecting multiple proteins in the medium is a multiplex assay. In such a multiplex assay, multiple analytes can be immobilized on a solid surface that spatially separates the analytes, such as an ELISA plate. Alternatively, the multiplex assay may be performed using analytes immobilized on beads or particles. In such cases, different beads / particles are utilized for the assay of each analyte.In an exemplary example, a bead-based multiplex assay can be used to detect Ang-1, TGFβ, VEGF, and HGF secreted into the medium (see Examples 1 and 4). Such multiplex assay systems for simultaneously detecting and quantifying multiple target analytes in complex samples, such as cell culture media, are commercially available from R&D Systems Inc (Minneapolis, USA), for example, as Luminex® assays and Luminex® High Performance assays.

[0037] The present invention also relates to the use of at least one protein selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF for assessing the wound healing efficacy of an MSC population. Further, the present invention relates to the use of at least one protein selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF for selecting an MSC population for generating a stem cell population under cGMP conditions. Accordingly, the present invention relates to the use of at least one protein selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF for selecting an MSC population for generating a stem cell population for subsequent pharmaceutical administration. The present invention further relates to the use of at least one protein selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF for selecting an MSC population for generating a master cell bank.

[0038] Furthermore, the present invention relates to the use of at least one protein selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF for selecting an MSC population for identifying a tissue suitable as a starting material for generating an MSC population for pharmaceutical use. In the present invention, such a tissue can be any mammalian tissue or compartment / body part known to contain MSCs. Examples of such tissues include, but are not limited to, a few known MSC-origin tissues such as bone marrow, adipose tissue, placental tissue, tissue of the placental attachment of the umbilical cord, umbilical cord tissue such as Wharton's jelly, and the amnion of the umbilical cord. In one example, the tissue is the umbilical cord or the amnion of the umbilical cord, and the MSC population generated from them can be the MSC population of the amnion of the umbilical cord.

[0039] For example, to determine whether tissue from a particular donor is suitable as a starting material for generating an MSC population for pharmaceutical use, the tissue can be cultured directly, for example, as tissue explants. For such tissue explants, samples of each tissue (e.g., Wharton's jelly, placental amnion, or amnion of the umbilical cord) can be placed in tissue culture dishes and cultured in a suitable culture / growth medium described herein (see also U.S. Patent Nos. 9,085,755 or 9,737,568 in this regard). Then, after an appropriate culture period, cell outgrowth from the tissue (the migration of MSCs from the tissue to the surface of the culture dish) occurs, and then the culture medium can be analyzed for the secretion of Ang-1, TGFβ, VEGF, and HGF. Alternatively, first, an MSC population can be isolated from the selected tissue using known isolation methods, and then the isolated MSC population can be cultured in a suitable medium and checked for the secretion of Ang-1, TGFβ, VEGF, and HGF. Irrespective of the tissue, the use of Ang-1, TGFβ, VEGF, and HGF for the methods described herein may include determining in the medium at least one, at least two, at least three, or all four of said proteins secreted into the medium by an MSC population, or by a tissue sample, or by cells isolated from a tissue sample.

[0040] The present invention further relates to a method for identifying a suitable medium for inducing or improving the wound healing properties of a mesenchymal stem cell population. This method also includes determining the level of at least one protein selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF secreted into the cell culture medium by the MSC population. Accordingly, the present invention also relates to the use of at least one protein for identifying a suitable medium for inducing or improving the wound healing properties of mesenchymal stem cells. This use may include determining the levels of at least two, at least three, or all four proteins selected from the group consisting of Ang-1, TGFβ, VEGF, and HGF secreted into the cell culture medium by the mesenchymal stem cell population in the cell culture medium.

[0041] The present invention is further illustrated by the following non-limiting experimental examples.

[0042] The sequences of the polypeptides disclosed herein are shown in Table 1.

[0043] [Table 1] TIFF2025087789000003.tif249170TIFF2025087789000004.tif237170 [Examples]

[0044] Experimental Examples Example 1: Identification of suitable umbilical cords containing MSCs, assessment and selection of MSC populations obtained from umbilical cords to generate suitable pharmaceutical compositions for wound healing The MSCs were derived from fresh umbilical cord tissue collected at the University of Colorado Hospital.

[0045] Stage 1: Tissue Bank In the initial step, generally, after obtaining consent from the Institutional Review Board (IRB) of the facility from the tissue donor, umbilical cords are collected. The freshly collected umbilical cords are cut into 1 - 2 mm 3 sections and cryopreserved with speed control in vials containing 4 - 5 segments, and stored in isolation in the -196 °C liquid nitrogen (LN 2 ) gas phase (see Figure 1). Maternal blood samples collected within 7 days of childbirth are screened for infectious diseases, and the tissue is tested for microbial contamination. Intermediate passing criteria for Stage 1 include that tests for all infectious diseases except CMV are negative, the sterility test is negative, and the mother's questionnaire is acceptable. Although vaginal flora naturally contaminates the umbilical cord during childbirth, collection during antibiotic use sterilizes a part of it. Up to 100 umbilical cord samples can be collected in such a tissue bank. Sterile tissue is used in Stage 2.

[0046] Stage 2: Developmental culture Developmental culture is the outgrowth from umbilical tissue used to generate a pure MSC strain. 10 tissue segments are individually placed in a 6 - well plate and grown for approximately 10 - 20 days to generate passage 0 (P0) cells. The P0 cells are seeded into a 175 cm 2 flask and cultured to obtain passage 1 (P1) cells. The P1 cells are either frozen in CryoStor 5 at 1 - 3×10 6 cells / vial or seeded for culture. The passage 1 cells are seeded into a 175 cm 5 flask at 2 - 3×10 2 cells / 175 cm. During growth, the cell morphology is recorded. Cells derived from passage 2 (P2) are assayed by flow cytometry for MSC markers, and the supernatant is assayed for cytokine production. More specifically, the P2 cells are subjected to a multiplex assay (R&D Systems / Biotechne catalog number LXSAHM) for the following analytes, Ang - 1, VEGF, HGF, and a single - plex assay was performed for TGFβ. The assays were performed as follows.

[0047] Multiplex assay: (i) Standard substances were prepared by combining 100 μl of each standard substance into one microcentrifuge tube already containing an appropriate volume of complete PTT6 medium to make a total volume of 1000 μl. Standard substance S1 contained all the multiplex standard substances combined in one vial. To perform a three-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. The procedure was continued up to S6. PTT6 complete medium was used as the blank. (ii) Beads were prepared by gently vortexing the vial to resuspend. It was important to take care not to invert the vial. When using the entire plate, 500 μl of beads was combined with 5.0 ml of dilution solution RD2 - 1. When using fewer wells, the volume was adjusted accordingly. The vial or wells were light-shielded. (iii) If necessary: Sample preparation. All samples were used undiluted unless the sample concentration exceeded the highest standard value (S1). If it exceeded, the assay was repeated with appropriately diluted samples. PTT6 was used for dilution. All samples were measured in triplicate. (iv) After gently vortexing the beads, 50 μl was added to each well using a multi-channel pipette and reservoir. (v) 50 μl of standard substance or sample per well was added. Then, the plate was covered with a plate sealer and incubated at room temperature (RT) for 2 hours in an orbital shaker at 800 rpm while shielding from light. (vi) During sample incubation, a biotin antibody cocktail was prepared by gently vortexing the vial to resuspend it, taking care not to invert the vial. Then, 500 μl of the biotin antibody cocktail was combined with 5.0 ml of dilution solution RD2-1. The solution was thoroughly mixed. (vii) Streptavidin phycoerythrin (PE) was prepared by gently vortexing the vial to resuspend it (taking care not to invert the vial). Then, 200 μl of streptavidin-PE concentrate was combined with 5.35 ml of wash buffer. The solution was thoroughly mixed and shielded from light. (Viii) The plate was washed as follows: The plate was attached to a magnet and left for at least 1 minute. While the plate was still attached to the magnet, the plate was quickly inverted and then moved relatively forcefully downward (1 - 2 times) to decant the plate into the sink and empty the wells. Complete removal of the liquid by inversion was essential, but the plate was not blotted. The magnet was removed, and 100 μl of wash buffer was filled into the wells using a multi-channel pipette. Then, the magnet was attached again, left for 1 minute, and decanting was performed as before. This washing was repeated 3 times in total. (ix) 50 μl of diluted biotin antibody was added to each well using a multi-channel pipette. The plate was covered and incubated at RT for 1 hour on a shaker set at 800 rpm. Then, the plate was washed 3 times as before. (x) 50 μl of diluted streptavidin-PE was added to each well. The plate was covered and incubated at RT for 30 minutes on a shaker as before. Then, the plate was washed 3 times as before. (xi) 100 μl of washing buffer was added to each well, and the plate was incubated for 2 minutes at RT on a shaker as described above. Subsequently, the well contents were immediately transferred to a Costar 6509 96-well 4-plate using a multi-channel pipette set to 120 μl. The plate was then placed in the fitting mold of a Luminex 3D scanner. (xii) The plate was read and analyzed using Luminex 3D and Xponent software.

[0048] TGFβ1 single plex: (i) Standards were prepared by using 1.5 ml polypropylene tubes for dilution. For this purpose, 500 μl of standard S1 was pipetted into the S1 tube. 200 μl of complete PTT6 medium was added to tubes S2 - S6. Standards were serially diluted 1:3 by transferring 100 μl successively from S1 to S7 and mixing thoroughly and ensuring complete mixing. (ii) Beads were prepared by gently vortexing the vial for resuspension. It was important to take care not to invert the vial. When using the entire plate, 50 μl of beads was combined with 5.0 ml of microparticle diluent RD2 - 1. When using fewer wells, the volume was adjusted accordingly. The vial or wells were shielded from light. (iii) To make TGFβ1 immunoreactive (not done for standards, only for samples), 30 μl of activation reagent was added to 150 μl of supernatant. The solution was mixed thoroughly and incubated for 10 minutes at RT. All samples were used undiluted unless the sample concentration exceeded the highest standard value (S1). If it exceeded, the assay was repeated with appropriately diluted samples. PTT6 was used for dilution. All samples were measured in triplicate. (iv) After gently vortexing the beads, 50 μl was added to each well using a multi-channel pipette and reservoir. (v) 50 μl of the standard substance or sample was added per well. Subsequently, the plate was covered with a plate sealer and incubated at RT for 2 hours in an orbital shaker at 800 rpm while shielding from light. (vi) During the sample incubation, a biotin antibody cocktail was prepared by gently vortexing the vial to resuspend it. It was important to be careful not to invert the vial. Subsequently, 50 μl of the biotin antibody concentrate was combined with 5.0 ml of the biotin antibody diluent. The solution was thoroughly mixed. (vii) (Taking care not to invert the vial) Streptavidin - phycoerythrin (PE) was prepared by gently vortexing the vial to resuspend it. Subsequently, 55 μl of the 100× streptavidin - PE concentrate was combined with 5.35 ml of the wash buffer. The solution was thoroughly mixed and shielded from light. (Viii) The plate was washed as follows: The plate was attached to the magnet and left for at least 1 minute. While the plate was attached to the magnet, to decant the plate into the sink, the plate was quickly inverted and then moved downward relatively forcefully (1 - 2 times) to empty the wells. Complete removal of the liquid by inversion was essential, but the plate was not blotted. The magnet was removed, and 100 μl of the wash buffer was filled into the wells using a multi - channel pipette. Then, the magnet was attached again, left for 1 minute, and decanting was performed as before. This washing was repeated 3 times in total. (ix) 50 μl of the diluted biotin antibody was added to each well using a multi - channel pipette. The plate was covered and incubated at RT for 1 hour in a shaker set at 800 rpm. Subsequently, the plate was washed 3 times as before. (x) 50 μl of the diluted streptavidin - PE was added to each well. The plate was covered and incubated at RT for 30 minutes in a shaker as before. Subsequently, the plate was washed 3 times as before. (xi) 100 μl of washing buffer was added to each well, and the plate was incubated on a shaker at RT for 2 minutes as described above. Subsequently, the well contents were immediately transferred to a Costar 6509 96-well 4-plate using a multi-channel pipette set to 120 μl. The plate was then placed in the fitting mold of a Luminex 3D scanner. (xii) The plate was read and analyzed using Luminex 3D and Xponent software.

[0049] Stage 3: Master Cell Bank Next, three cell lines that meet the Stage 2 intermediate acceptance criteria were seeded at 20 - 40×10 6 viable cells into a Terumo Quantum bioreactor and grown for approximately 7 - 14 days. After Quantum, the cells were tested for sterility, endotoxin, mycoplasma, human pathogen viruses, and adventitious viruses. The cells were cryopreserved at 9 - 10×10 6 cells / vial (50 - 70 vials per batch). MSC was grown in a flask in Terumo Quantum in a culture medium PTT6 formulated as follows for 1000 ml: 500 ml of PTT6 basal medium, 236 ml of M171, 236 ml of DMEM F12, 25 ml of fetal bovine serum, 0.1 ml of 0.1 mg / ml epidermal growth factor [final concentration 10 ng / ml], 0.35 ml of insulin [final concentration 5 μg / ml], and incubated at 37°C in 5% CO 2 ₂.

[0050] Stage 4: Therapeutic Cultures 1×, 3×, and 5×10 6 individual MSCs that tested positive for Ang-1, TGFβ, VEGF, and HGF were bottled in Hypothermosol® and stored at 2 - 8°C prior to pharmaceutical administration.

[0051] Example 2: Protein Secretion Level Analysis for Identifying Suitable Donor Umbilical Cords For analysis, 10 umbilical cords were collected from different donors. These umbilical cords were used to generate 10 individual MSC populations derived from the amnion of the umbilical cords.

[0052] The goal of the experiment was to determine the secretion levels of Ang-1, VEGF, HGF, and TGFβ1 (here, TGFβ1) in order to rule out conclusions about the variability of the secretion profiles of individual MSC populations and to identify MSC populations with sufficient secretion of Ang-1, VEGF, HGF, and TGFβ1.

[0053] Accordingly, the individual MSC populations were cultured according to the present invention, and after stage 2, the secretion levels of Ang-1, VEGF, HGF, and TGFβ (here, TGFβ1) were determined for each MSC population as described in Example 1. The results of the secretion level analysis are shown in Figure 2, where protein-specific thresholds (500 pg / ml for Ang-1 and TGFβ, and 100 pg / ml for VEGF and HGF, respectively) are indicated by the horizontal black lines.

[0054] As can be seen from the results, the secretion levels of TGFβ1 were higher than twice the threshold in all of the 10 individual MSC populations. Thus, for all individual MSC populations, the threshold of 500 pg / ml for TGFβ1 was exceeded. The plotted secretion levels of HGF showed that 5 out of 10 samples, namely MSC population 8049365 with a secretion level of approximately 600 pg / ml, MSC population 8049369 with a secretion level of approximately 300 pg / ml, MSC population 8049372 with a secretion level of approximately 1450 pg / ml, MSC population 8049373 with a secretion level of approximately 380 pg / ml, and MSC population 8049384 with a secretion level of approximately 190 pg / ml, exceeded the threshold of 100 pg / ml. The plotted secretion levels of Ang-1 showed that all individual MSC populations exceeded the threshold of 500 pg / ml. The secretion levels of VEGF showed that they exceeded the threshold of 100 pg / ml for all samples except 8049358, 8049359, and 8049370. In this regard, the secretion level of 8049359 was only approximately 50 pg / ml, and the levels of 8049358 and 8049359 were close to zero.

[0055] The results indicated that the protein secretion levels varied among different individual MSC populations, and it was confirmed that MSCs had individual secretion profiles. To identify MSC populations with sufficient secretion of Ang-1, VEGF, HGF, and TGFβ1, the secretion levels of these proteins were analyzed.

[0056] In this regard, MSC shows sufficient secretion of Ang-1, TGFβ, VEGF, and HGF when the levels of these proteins exceed their respective thresholds. Therefore, only MSC populations 8049365, 8049369, 8049372, 8049373, and 8049384 (each obtained from different donor umbilical cords) are MSC populations that exceed the thresholds given for each of the four selected proteins, and thus they show sufficient secretion of Ang-1, TGFβ, VEGF, and HGF. For subsequent experiments (preparation of the master cell bank and production of cells for pharmaceutical purposes), MSC population 8049372 was selected herein.

[0057] Example 3: Analysis of Secretion Level Stability MSC population 8049372, which showed sufficient secretion of the proteins Ang-1, TGFβ, VEGF, and HGF in Example 2, was used to analyze the stability of the protein secretion level. Therefore, the MSC population was further cultured until the cells were passaged 4 times. Then, two samples of the MSC population after stage 4 (MSC in well 1 and MSC in well 2) were analyzed for the secretion levels of Ang-1, VEGF, HGF, and TGFβ (here, TGFβ1) as described in Example 1. Then, the protein secretion levels of MSC population 8049372 determined in this experiment (after stage 4) were compared with the secretion levels of MSC population 8049372 determined in Example 2 (after stage 2). In this way, the changes in the secretion levels at different time points can be clarified. Thereby, conclusions can be drawn regarding the stability of the protein secretion level and thus whether the protein secretion is sufficient over time. The results are shown in Figure 3.

[0058] After stage 2, MSC population 8049372 showed a secretion level of about 2200 pg / ml for TGFβ1. After the cells were passaged two more times, MSC population 8049372 after stage 4 showed, on average, about 2345 pg / ml TGFβ1. Thus, the secretion level of MSC population 8049372 increased by about 7% after the cells were passaged two more times. The secretion level of HGF decreased by about 33% after the cells were passaged two more times, from about 1450 pg / ml after stage 2 to an average of about 959 pg / ml after stage 4. MSC population 8049372 after stage 4 showed, on average, about 1827 pg / ml Ang-1, which is about a 9% decrease relative to 2000 pg / ml Ang-1 after stage 2. The secretion level of VEGF increased by about 43% after the cells were passaged two more times, from about 150 pg / ml after stage 2 to an average of about 215 pg / ml after stage 4.

[0059] The results indicate that the secretion levels of the proteins Ang-1, VEGF, HGF, and TGFβ (here, TGFβ1) vary even after the cells are passaged two more times. However, each of the respective thresholds selected herein still exceeds that for all of the proteins analyzed even after the cells are passaged two more times. Thus, the results of this experiment indicate that MSC population 8049372 maintained relative protein secretion stability and thus maintained wound healing efficacy. Thus, this result indicates that the wound healing efficacy of the MSC population can be stable for a certain period. Based on this result, MSC population 8049372 is a suitable candidate as a starting material for generating a master cell bank or for preparing a pharmaceutical composition for later administration to a subject.

[0060] Example 4: Identification of a Suitable Medium for Inducing or Improving the Wound Healing Properties of Mesenchymal Stem Cells For this experiment, various isolated MSC populations from the amnion of the umbilical cord were cultured in PTT4, PTT6, or DMEM / F12 as described in International Application WO 2018 / 067071 and then analyzed for the secretion of wound healing marker proteins for comparison.

[0061] Culture protocol for the culture of isolated MSCs · Seed 5 million MSCs in a 100 mm tissue culture dish and leave in DMEM / F12 / 10% FCS for 24 hours. · Discard the medium, add PTT4, PTT6 / DMEM / F12 to the culture and leave for 24 hours. · Discard the medium and wash the cells with PBS. · Add 10 ml of DMEM to the culture and leave for 24 hours. · Discard the medium and add 5 ml of DMEM to the culture. · After 24 hours of culture, collect the conditioned medium, centrifuge to remove cell debris, and aliquot the supernatant into tubes for storage at -80 °C and subsequent analysis of marker protein secretion by cytokine assay.

[0062] Analysis of secretion levels in the supernatants of PTT4 medium, PTT6 medium, and DMEM / F12 medium Secretion level analysis was performed on MSC supernatants. Measurements and analysis were performed using Luminex 200 and Xponent software.

[0063] Except for samples of placenta supernatants, each sample was tested in triplicate. The goal of this experiment was to generate cytokine profiles of MSCs cultured in either PTT4 or PTT6 and compare the profiles of MSCs derived from different source tissues (outer umbilical cord layer, Wharton's jelly, placenta MSCs). Cytokine measurements were performed as follows. The profiles will reveal which stem cell populations grown in which medium secrete more of the cytokines of interest, i.e., which medium is appropriate for inducing or promoting the wound healing properties of MSCs.

[0064] Multiplex analysis Information on multiplex: R&D Systems / Bio-techne catalog number LXSAHM. This kit has lot number L123680, expiration date 08 / 28 / 18, and is for the following analytes: · Ang-1, angiopoietin · VEGF, vascular endothelial growth factor · HGF, hepatocyte growth factor

[0065] Information on TGFβ1 singleplex: R&D Systems / Bio-techne: · Base kit, catalog number LTGM00, lot number P156217, received 02 / 27 / 18, expiration date 08 / 30 / 18 · TGFβ1 component, catalog number LTGM100, lot number P161760, received 02 / 27 / 18, expiration date 11 / 27 / 19

[0066] Information on multiplex: R&D Systems / Bio-techne catalog number LXSAHM. This kit has lot number L123999, expiration date 09 / 25 / 18, and is for the following analytes: · Ang-1, angiopoietin · VEGF, vascular endothelial growth factor · HGF, hepatocyte growth factor

[0067] Data input Raw data output is in PDF and Excel formats. The data in Excel format is used to process the data.

[0068] Procedure Protein detection in MSC supernatants was performed according to detailed protocol information. As part of this experiment, the protocol had one modification not to use Std.8 of the multiplex kit. The reason for omitting Std.8 is that the R&D Systems protocol itself uses only standards 1 - 6. Additionally, Std.8 was only validated for 2 out of the 6 analytes that make up the multiplex: HGF. In the case of HGF, that analyte falls in the central region of the standard curve. Since the standards are reconstituted using growth medium, standard curves were constructed with both PTT4 and PTT6. Test samples grown in either PTT4 or PTT6 were extrapolated from their respective standard curves. Results were extrapolated by the Luminex software from standard curves specific to the analyte generated by the same software: the analysis algorithm was set to Logistic 5P Weighted with weighted analysis using 1 / y2 for weighting.

[0069] Sample 1. DMEM / F12 medium and PTT6 medium and PTT4 medium (not exposed to MSC) 2. Supernatants of the MSCs being tested 3. Optional: supernatants from different donors; derived from CR001A, C, D, and G

[0070] Results for Ang-1 are shown in Figure 4A, indicating that MSCs from the amnion of the umbilical cord produce more Ang-1 when grown in PTT6 than when grown in DMEM / F12 or PTT4. Results for VEGF are shown in Figure 4B, indicating that MSCs from the amnion of the umbilical cord produce more VEGF when grown in PTT6 than when grown in DMEM / F12 or PTT4. Results for HGF are shown in Figure 4C, indicating that MSCs from the amnion of the umbilical cord produce more HGF when grown in PTT6 than when grown in DMEM / F12 or PTT4. Finally, results for TGFβ1 are shown in Figure 4D, indicating that MSCs from the amnion of the umbilical cord produce more TGFβ1 when grown in PTT6 than when grown in DMEM / F12 or PTT4.

[0071] From the above experiments, the following can be concluded. When MSCs are cultured in PTT6 medium, the secretion of Ang-1, TGFβ1, VEGF, and HGF by the MSC population is significantly increased compared to the secretion levels in PTT4 or commercially available culture media, such as DMEM / F12. PTT6 medium has the highest ability to induce or improve the wound healing properties of the MSC population by promoting the secretion of all of Ang-1, TGFβ1, VEGF, and HGF (the involvement of which in wound healing is known as described herein). Thus, determination of the secretion levels of Ang-1, TGFβ1, VEGF, and HGF can be used to identify a suitable medium for inducing or improving the wound healing properties of a mesenchymal stem cell population.

[0072] It will be readily 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.

[0073] The patents and publications mentioned in this specification all indicate the level of those skilled in the art to which the present invention pertains. All patents and publications are hereby incorporated by reference into this specification to the same extent as if each individual publication had been specifically and individually indicated to be incorporated by reference.

[0074] The invention illustrated in this specification can be suitably implemented in the absence of any factors or limitations not specifically disclosed herein. Accordingly, terms such as "comprising," "including," "containing," etc. should be construed expansively and non - limitatively. Further, the terms and expressions used in this specification are for the purpose of description rather than limitation, and in using such terms and expressions, there is no intention to exclude equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention as set forth in the claims. Thus, although the invention has been specifically disclosed by way of preferred embodiments and optional features, it should be understood that modifications and variations of the disclosed embodiments of the invention may occur to 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 herein in a broad and comprehensive manner. Narrower species and sub - genera included within the scope of the comprehensive disclosure also each form part of the invention. This includes a comprehensive description of the invention that includes any conditions or negative limitations that remove any subject matter from the genus, regardless of whether the material being removed is specifically described herein. Further, when a feature or aspect of the invention is described by a Markush group, those skilled in the art will recognize that the invention is also thereby described by individual members of the Markush group or subgroups of members. Further aspects of the invention will become apparent from the following claims.

[0075] The present invention is further characterized by the following clauses.

[0076] 1. A method for assessing the wound healing efficacy of a mesenchymal stem cell population, comprising the step of determining in a medium the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0077] 2. A method for identifying a tissue suitable as a starting material for producing a mesenchymal stem cell population for pharmaceutical use, comprising the step of determining in a medium the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by a sample of tissue or cells isolated from the tissue.

[0078] 3. A method for selecting a mesenchymal stem cell population for producing a stem cell population under cGMP conditions, comprising the step of determining in a medium the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0079] 4. A method for selecting a mesenchymal stem cell population for producing a stem cell population for subsequent pharmaceutical administration, comprising the step of determining in a medium the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0080] 5. A method for selecting a mesenchymal stem cell population for generating a master cell bank, comprising the step of determining in a medium the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0081] 6. A method according to any one of items 1 to 5, comprising the step of determining in a medium the levels of at least two, at least three, or all four proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0082] 7. A secretion level equal to or exceeding a threshold value (i) the wound healing efficacy of the mesenchymal stem cell population; or (ii) a tissue or isolated cells suitable as a starting material for producing the mesenchymal stem cell population is shown by a method according to any one of items 1 to 6.

[0083] 8. The method of item 7, wherein the threshold value for angiopoietin 1 (Ang-1) is about 400 pg / ml or about 500 pg / ml.

[0084] 9. The method of item 7 or 8, wherein the threshold value for transforming growth factor β (TGFβ) is about 400 pg / ml or about 500 pg / ml.

[0085] 10. The method according to any one of items 7 to 9, wherein the threshold value for vascular endothelial growth factor (VEGF) is about 80 pg / ml or about 100 pg / ml.

[0086] 11. The method according to any one of items 7 to 10, wherein the threshold value for hepatocyte growth factor (HGF) is about 80 pg / ml or about 100 pg / ml.

[0087] 12. The secretion levels of all four proteins are equal to or exceed their respective thresholds, where the thresholds are - for angiopoietin 1 (Ang-1), a threshold of about 400 pg / ml, - for transforming growth factor β (TGFβ), a threshold of about 400 pg / ml, - for vascular endothelial growth factor (VEGF), a threshold of about 80 pg / ml, - for hepatocyte growth factor (HGF), a threshold of about 80 pg / ml for any of the methods of items 8 - 11.

[0088] 13. The secretion levels of all four proteins are equal to or exceed their respective thresholds, where the thresholds are - for angiopoietin 1 (Ang-1), a threshold of about 500 pg / ml, - for transforming growth factor β (TGFβ), a threshold of about 500 pg / ml, - for vascular endothelial growth factor (VEGF), a threshold of about 100 pg / ml, - for hepatocyte growth factor (HGF), a threshold of about 100 pg / ml for any of the methods of items 8 - 11.

[0089] 14. The mesenchymal stem cell population is selected from the group consisting of the mesenchymal stem cell population of the umbilical cord, the mesenchymal stem cell population of the placenta, the mesenchymal stem cell population of the umbilical cord-placenta attachment site, the mesenchymal stem cell population of umbilical cord blood, the mesenchymal stem cell population of bone marrow, and the mesenchymal stem cell population derived from adipose tissue, for any of the methods of items 1 - 13.

[0090] 15. The mesenchymal stem cell population of the umbilical cord is selected from the group consisting of the mesenchymal stem cell population of the amnion (AM), the perivascular (PV) mesenchymal stem cell population, the Wharton's jelly (WJ) mesenchymal stem cell population, the mesenchymal stem cell population of the amnion of the umbilical cord, and the mixed mesenchymal stem cell population (MC) of the umbilical cord, for the method of item 14.

[0091] 16. The method of item 2, wherein the tissue is umbilical cord or amnion of umbilical cord, and the mesenchymal stem cell population is the stem cell population of the amnion of umbilical cord.

[0092] 17. The method of item 15 or 16, wherein the mesenchymal stem cell population of the amnion of umbilical cord is a mesenchymal stem cell population in which at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90, and CD105.

[0093] 18. The method of item 17, wherein at least about 90% or more of the cells of the mesenchymal stem cell population lack the expression of the following markers: CD34, CD45, and HLA-DR.

[0094] 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 cells of the mesenchymal stem cell population express each of CD73, CD90, and CD105 and lack the expression of each of CD34, CD45, and HLA-DR.

[0095] 20. The method of any of the preceding items, wherein the medium is a cell culture medium or a storage medium.

[0096] 21. The method of item 20, wherein the storage medium is Hypothermosol or Plasmalyte.

[0097] 22. The method of any of items 1 to 19, wherein the medium contains Dulbecco's 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), a 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).

[0098] 23. The method of item 22, wherein the medium comprises Dulbecco's 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).

[0099] 24. The method of item 23, wherein the medium comprises Dulbecco's 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).

[0100] 25. The method of item 23 or 24, wherein the serum-free basal medium is M171.

[0101] 26. The method according to any one of items 22 - 25, wherein the medium further comprises epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml.

[0102] 27. The method of item 26, wherein the medium comprises epidermal growth factor (EGF) at a final concentration of about 10 ng / ml.

[0103] 28. The method according to any one of items 22 - 27, wherein the medium comprises insulin at a final concentration of about 1 μg / ml to 10 μg / ml.

[0104] 29. The method of item 28, wherein the medium comprises insulin at a final concentration of about 5 μg / ml.

[0105] 30. The method according to any one of items 22 - 29, wherein the medium further comprises at least one of the following adjuvants: adenine, hydrocortisone, and sodium 3,3',5-triiodo-L-thyronine (T3).

[0106] 31. The method according to any one of items 22 to 30, wherein the medium contains all three of adenine, hydrocortisone, and sodium 3,3',5-triiodo-L-thyronine (T3).

[0107] 32. The method according to item 30 or 31, wherein the medium contains 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-thyronine (T3) at a final concentration of about 0.5 to about 5 ng / ml.

[0108] 33. The culture of the mesenchymal stem cell population in the medium 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 the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ; specifically, TGFβ1), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) by the mesenchymal stem cell population, as compared to a reference medium that does not contain all of DMEM (Dulbecco's Modified Eagle Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (fetal bovine serum). The method according to any one of items 1 to 32.

[0109] 34. The method according to any one of items 1 to 33, wherein the cell culture medium is subjected to centrifugation after an appropriate culture period.

[0110] 35. The method according to item 34, wherein the appropriate culture 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.

[0111] 36. The method according to item 34 or 35, wherein the supernatant of the centrifuged cell culture medium is subjected to a multiplex assay.

[0112] 37. The method according to item 36, wherein the multiplex assay is bead-based.

[0113] 38. Use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for assaying the wound healing efficacy of a mesenchymal stem cell population.

[0114] 39. Use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for selecting a mesenchymal stem cell population for producing a stem cell population under cGMP conditions.

[0115] 40. Use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for selecting a mesenchymal stem cell population for producing a stem cell population for subsequent pharmaceutical administration.

[0116] 41. Use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for selecting a mesenchymal stem cell population for generating a master cell bank.

[0117] 42. Use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for identifying a tissue suitable as a starting material for producing a mesenchymal stem cell population for pharmaceutical use.

[0118] 43. The use according to item 42, wherein the tissue is umbilical cord or amnion of the umbilical cord, and the mesenchymal stem cell population is a stem cell population of the amnion of the umbilical cord.

[0119] 44. Use according to any one of items 38 to 41, comprising the step of determining in a cell culture medium the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the cell culture medium by a mesenchymal stem cell population.

[0120] 45. Use according to item 42 or 43, comprising the step of determining in a cell culture medium the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang 1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the cell culture by a sample of tissue or cells isolated from the tissue.

[0121] 46. Use according to any one of items 38 to 44, comprising the step of determining in a cell culture medium the level of at least two, at least three, or all four proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the cell culture medium by a mesenchymal stem cell population.

[0122] 47. A method for identifying a suitable medium for inducing or improving the wound healing properties of a mesenchymal stem cell population, comprising the step of determining the level of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

[0123] 48. Use of at least one protein selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) for identifying a suitable medium for inducing or improving the wound healing properties of a mesenchymal stem cell population.

[0124] 49. Use according to item 48, comprising the step of determining in the medium the levels of at least two, at least three, or all four proteins secreted into the medium by a mesenchymal stem cell population.

[0125] Sequence information SEQUENCE LISTING <110> CELLRESEARCH CORPORATION PTE. LTD. <120> A METHOD OF ASSESSING WOUND HEALING POTENCY OF A MESENCHYMAL STEM POPULATION AND RELATED METHODS OF SELECTING MESENCHYMAL STEM CELLS AND IDENTIFYING TISSUE AS STARTING MATERIAL FOR PRODUCING A MESENCHYMAL STEM CELL POPULATION <150> US 62 / 912,374 <151> 2019-10-08 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 498 <212> PRT <213> human <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 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 Ser Thr Leu Gly Ser Thr Ser Leu His Pro Val Ser Gln His Gly Asn 65 70 75 80 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> human <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 1010 1015 1020 Tyr Trp Lys Pro Glu Val Met Ile Ala Ala Gln Gly Pro Leu Lys 1025 1030 1035 Glu Thr Ile Gly Asp Phe Trp Gln Met Ile Phe Gln Arg Lys Val 1040 1045 1050 Lys Val Ile Val Met Leu Thr Glu Leu Lys His Gly Asp Gln Glu 1055 1060 1065 Ile Cys Ala Gln Tyr Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp 1070 1075 1080 Ile Glu Val Asp Leu Lys Asp Thr Asp Lys Ser Ser Thr Tyr Thr 1085 1090 1095 Leu Arg Val Phe Glu Leu Arg His Ser Lys Arg Lys Asp Ser Arg 1100 1105 1110 Thr Val Tyr Gln Tyr Gln Tyr Thr Asn Trp Ser Val Glu Gln Leu 1115 1120 1125 Pro Ala Glu Pro Lys Glu Leu Ile Ser Met Ile Gln Val Val Lys 1130 1135 1140 Gln Lys Leu Pro Gln Lys Asn Ser Ser Glu Gly Asn Lys His His 1145 1150 1155 Lys Ser Thr Pro Leu Leu Ile His Cys Arg Asp Gly Ser Gln Gln 1160 1165 1170 Thr Gly Ile Phe Cys Ala Leu Leu Asn Leu Leu Glu Ser Ala Glu 1175 1180 1185 Thr Glu Glu Val Val Asp Ile Phe Gln Val Val Lys Ala Leu Arg 1190 1195 1200 Lys Ala Arg Pro Gly Met Val Ser Thr Phe Glu Gln Tyr Gln Phe 1205 1210 1215 Leu Tyr Asp Val Ile Ala Ser Thr Tyr Pro Ala Gln Asn Gly Gln 1220 1225 1230 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 1250 1255 1260 Gly Ala Pro Glu Lys Leu Pro Glu Ala Lys Glu Gln Ala Glu Gly 1265 1270 1275 Ser Glu Pro Thr Ser Gly Thr Glu Gly Pro Glu His Ser Val Asn 1280 1285 1290 Gly Pro Ala Ser Pro Ala Leu Asn Gln Gly Ser 1295 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. 1. A method for assessing the wound healing efficacy of a mesenchymal stem cell population, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and an umbilical cord-placental attachment mesenchymal stem cell population, the method comprising determining in a culture medium the levels of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

2. 1. A method for identifying a suitable tissue as a starting material for generating a mesenchymal stem cell population for pharmaceutical use, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and an umbilical cord-placental attachment mesenchymal stem cell population, the method comprising determining in a culture medium the levels of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the tissue or a sample of cells isolated from the tissue.

3. 1. A method for selecting a mesenchymal stem cell population for generating a stem cell population under cGMP conditions, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and an umbilical cord-placental attachment mesenchymal stem cell population, the method comprising determining in a culture medium the levels of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

4. 1. A method for selecting a mesenchymal stem cell population for generating a stem cell population for subsequent pharmaceutical administration, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and an umbilical cord-placental attachment mesenchymal stem cell population, the method comprising determining in a culture medium the levels of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

5. 1. A method for selecting a mesenchymal stem cell population for generating a master cell bank, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and an umbilical cord-placental attachment mesenchymal stem cell population, the method comprising determining in a culture medium the levels of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

6. The method of any one of claims 1 to 5, comprising determining in the culture medium the levels of at least three or all four proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the culture medium by the mesenchymal stem cell population.

7. A secretion level equal to or exceeding the threshold value is (i) the wound healing efficacy of a mesenchymal stem cell population; or (ii) A suitable tissue or isolated cells as starting material for generating a mesenchymal stem cell population. The method according to any one of claims 1 to 6, wherein

8. 8. The method of claim 7, wherein for Angiopoietin 1 (Ang-1), the threshold is about 400 pg / ml or about 500 pg / ml.

9. 9. The method of claim 7 or 8, wherein for transforming growth factor beta (TGFβ), the threshold is about 400 pg / ml or about 500 pg / ml.

10. The method of any one of claims 7 to 9, wherein for vascular endothelial growth factor (VEGF), the threshold value is about 80 mg / ml or about 100 pg / ml.

11. The method of any one of claims 7 to 10, wherein for hepatocyte growth factor (HGF), the threshold value is about 80 pg / ml or about 100 pg / ml.

12. The secretion levels of all four proteins were equal to or exceeded their respective thresholds, and the thresholds were - for angiopoietin 1 (Ang-1), a threshold of approximately 400 pg / ml; - for transforming growth factor beta (TGFβ), a threshold of approximately 400 pg / ml; - for vascular endothelial growth factor (VEGF), a threshold of approximately 80 pg / ml; - Hepatocyte growth factor (HGF) threshold of approximately 80 pg / ml The method according to any one of claims 8 to 11, wherein

13. The secretion levels of all four proteins were equal to or exceeded their respective thresholds, and the thresholds were - for angiopoietin 1 (Ang-1), a threshold of approximately 500 pg / ml; - for transforming growth factor beta (TGFβ), a threshold of approximately 500 pg / ml; - for vascular endothelial growth factor (VEGF), a threshold of approximately 100 pg / ml; - Hepatocyte growth factor (HGF) threshold of approximately 100 pg / ml The method according to any one of claims 8 to 11, wherein

14. The method of any one of the preceding claims, wherein the umbilical cord mesenchymal stem cell population is selected from the group consisting of an amniotic membrane (AM) mesenchymal stem cell population, a perivascular (PV) mesenchymal stem cell population, a Wharton's gelatin (WJ) mesenchymal stem cell population, an umbilical cord amniotic membrane mesenchymal stem cell population, and an umbilical cord mixed mesenchymal stem cell population (MC).

15. 3. The method of claim 2, wherein the tissue is an umbilical cord or an umbilical cord amniotic membrane and the mesenchymal stem cell population is an umbilical cord amniotic membrane stem cell population.

16. The method of claim 14 or 15, wherein the mesenchymal stem cell population of the amniotic membrane of the umbilical cord is a mesenchymal stem cell population in which at least about 90% or more of the cells of the stem cell population express each of the following markers: CD73, CD90, and CD105.

17. The method of claim 16, wherein at least about 90% or more of the cells of said mesenchymal stem cell population lack expression of the following markers: CD34, CD45, and HLA-DR.

18. The method of claim 16 or 17, wherein at least about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more of the cells of the mesenchymal stem cell population express each of CD73, CD90, and CD105, and lack expression of each of CD34, CD45, and HLA-DR.

19. 2. The method according to any one of the preceding claims, wherein the medium is a cell culture medium or a storage medium.

20. 20. The method of claim 19, wherein the storage medium is Hypothermosol or Plasmalyte.

21. 19. The method of any one of claims 1 to 18, wherein the culture medium comprises Dulbecco's 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).

22. 22. The method of claim 21, wherein the culture medium comprises Dulbecco's 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).

23. 23. The method of claim 22, wherein the culture medium comprises Dulbecco's 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).

24. 24. The method of claim 22 or 23, wherein the serum-free basal medium is M171.

25. The method of any one of claims 21 to 24, wherein the medium further comprises epidermal growth factor (EGF) at a final concentration of about 1 ng / ml to about 20 ng / ml.

26. 26. The method of claim 25, wherein the medium comprises epidermal growth factor (EGF) at a final concentration of about 10 ng / ml.

27. 27. The method of any one of claims 21 to 26, wherein the medium comprises insulin at a final concentration of about 1 μg / ml to 10 μg / ml.

28. 28. The method of claim 27, wherein the medium comprises insulin at a final concentration of about 5 μg / ml.

29. The method of any one of claims 21 to 28, wherein the medium further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-triiodo-L-thyronine sodium salt (T3).

30. 30. The method of any one of claims 1 to 29, wherein culturing the mesenchymal stem cell population in a medium as defined in any one of claims 21 to 29 results in increased expression and / or secretion by the mesenchymal stem cell population of at least one protein selected from the group consisting of Angiopoietin 1 (Ang-1), Transforming Growth Factor β (TGFβ; in particular TGFβ1), Vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF), compared to a reference medium not comprising all of DMEM (Dulbecco's Modified Eagle's Medium), F12 (Ham's F12 Medium), M171 (Medium 171), and FBS (Fetal Bovine Serum).

31. The method of any one of claims 1 to 30, wherein the cell culture medium is subjected to centrifugation after an appropriate culture period.

32. 32. The method of claim 31, wherein the suitable culture period comprises about 12 hours, about 24 hours, about 36 hours, about 46 hours, about 48 hours, or about 50 hours, preferably about 48 hours.

33. 33. The method of claim 31 or 32, wherein the centrifuged cell culture medium supernatant is subjected to a multiplex assay.

34. 34. The method of claim 33, wherein the multiplex assay is bead-based.

35. 1. Use of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) to assess the wound healing efficacy of a mesenchymal stem cell population, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and a umbilical cord-placental attachment mesenchymal stem cell population.

36. 1. Use of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) to select a mesenchymal stem cell population for generating a stem cell population under cGMP conditions, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and a mesenchymal stem cell population from the umbilical cord-placental attachment site.

37. 1. Use of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) to select a mesenchymal stem cell population for generating a stem cell population for subsequent pharmaceutical administration, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and a umbilical cord-placental attachment mesenchymal stem cell population.

38. 1. Use of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) to select a mesenchymal stem cell population for generating a master cell bank, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and an umbilical cord-placental attachment mesenchymal stem cell population.

39. 1. Use of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) to identify a suitable tissue as a starting material for generating a mesenchymal stem cell population for pharmaceutical use, wherein the mesenchymal stem cell population is selected from the group consisting of an umbilical cord mesenchymal stem cell population, a placental mesenchymal stem cell population, and a mesenchymal stem cell population from the umbilical cord-placental attachment site.

40. 40. The use of claim 39, wherein the tissue is an umbilical cord or an amniotic membrane of an umbilical cord and the mesenchymal stem cell population is an amniotic membrane of an umbilical cord stem cell population.

41. 39. The use of any one of claims 35 to 38, comprising determining in a cell culture medium the levels of at least three or all four proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the cell culture medium by the mesenchymal stem cell population.

42. 41. The use of claim 39 or 40, comprising determining in a cell culture medium the levels of at least three or all four proteins selected from the group consisting of angiopoietin 1 (Ang 1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the cell culture by a sample of tissue or cells isolated from the tissue.

43. A method for identifying a medium suitable for inducing or improving the wound healing properties of a mesenchymal stem cell population, comprising the step of determining the levels of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) secreted into the medium by the mesenchymal stem cell population.

44. The use of at least two proteins selected from the group consisting of angiopoietin 1 (Ang-1), transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) to identify a suitable medium for inducing or improving the wound healing properties of a mesenchymal stem cell population.

45. 45. The use of claim 44, comprising determining in the culture medium the levels of at least three or all four proteins secreted into the culture medium by the mesenchymal stem cell population.

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