Bone marrow mesenchymal stem cell-derived cell population and method for preparing the same

Abstract

CD90 + , CD105 + , CD45 - The present invention provides a cell population enriched for MSC-derived cells that exhibit high TIMP-1 secretion and low MMP13 gene expression. Such cell populations are useful as pharmaceuticals in contexts where it is desirable to utilize the trophic or immunosuppressive therapeutic effects of MSCs but avoid activities associated with MSCs' ability to undergo phenotypic differentiation. The cell populations of the present invention are useful in treating conditions selected from the group consisting of osteoarthritis, myocardial infarction, meniscal cartilage injury (e.g., meniscus tear), ligament injury (e.g., ligament rupture), skin injury, and soft tissue injury. They may also be used in the treatment of diseases selected from the group consisting of blood disorders, graft-versus-host disease, and inflammatory diseases.

Description

[Technical Field]

[0001] The present invention relates to cell populations that can be used as pharmaceuticals. The present invention also relates to methods for preparing cells for use as pharmaceuticals. The present invention further relates to methods for selecting mesenchymal stem cell (MSC)-derived cells for therapeutic use, and to therapeutic methods using MSC-derived cells. [Background technology]

[0002] MSCs have been proposed as therapeutic agents for use in a wide range of applications, including tissue repair and regeneration and the alleviation of conditions such as graft-versus-host disease. The means by which MSCs achieve these therapeutic actions in vivo are incompletely understood, but are generally thought to involve differentiation to generate new cells that contribute to tissue replacement, trophic effects that stimulate repair by neighboring cells, and suppression of local immune responses. Summary of the Invention [Problem to be solved by the invention]

[0003] One factor limiting the adoption of MSC-based therapies is the ability to generate therapeutically effective numbers of cells. Many therapies require large numbers of cells, which are most efficiently generated through long-term cell culture. However, it has been shown that the properties associated with the therapeutic use of MSCs can change significantly over time in culture. For example, the ability of MSCs to differentiate into multiple phenotypes (pluripotency), a key stem cell characteristic, is progressively lost in sustained cell culture, while other therapeutically relevant properties may be retained. Thus, such sustained culture, which is desirable for generating large numbers of cells, appears incompatible with preserving the properties that contribute to the therapeutic activity of MSCs in vivo or generating the homogeneous populations required for stable cell-based medicines. [Means for solving the problem]

[0004] According to a first aspect of the present invention, CD90+ , CD105 + , CD45 - and High TIMP-1 secretion Low MMP13 gene expression A cell population enriched for MSC-derived cells for pharmaceutical use is provided.

[0005] The cell population for medical use defined by the first aspect of the present invention is capable of stimulating trophic repair, as discussed in more detail elsewhere herein. Such cell population for medical use may be referred to as a "cell population of the present invention" and the constituent cells of such a population may be referred to as "cells of the present invention." The cell population of the present invention may be capable of stimulating trophic repair, as discussed in more detail elsewhere herein. + , CD105 + , CD45 - and may be free of (or substantially free of) cells with high TIMP-1 secretion and high MMP13 gene expression.

[0006] The medicament may be used in promoting tissue repair and / or in promoting immunosuppressive therapy.

[0007] According to a second aspect of the present invention, Culturing MSCs to generate MSC-derived cells; CD90 + , CD105 + , CD45 - and High TIMP-1 protein secretion Low MMP13 gene expression Enriching the population of MSC-derived cells; and Formulating cells for pharmaceutical use The present invention provides a method for preparing cells for use as a pharmaceutical, comprising:

[0008] A medicament produced by formulating cells prepared by the method of the second aspect of the present invention may be referred to as a "medicament of the present invention." + , CD105 + , CD45- and may be free of (or substantially free of) cells with high TIMP-1 secretion and high MMP13 gene expression.

[0009] According to a third aspect of the present invention, assessing the levels of MMP13 gene expression and TIMP-1 protein secretion by the cells; and Selecting cells for use in therapeutic applications that do not require phenotypic differentiation potential when the cells exhibit low levels of MMP13 gene expression and high levels of TIMP-1 protein secretion. The present invention provides a method for selecting MSC-derived cells for therapeutic use, comprising:

[0010] In a suitable embodiment, the method according to the third aspect of the invention may optionally further comprise the step of selecting the cells for use in a therapeutic application requiring phenotypic differentiation potential if the cells exhibit high levels of both MMP13 gene expression and TIMP-1 protein secretion.

[0011] It will be appreciated that the method of the third aspect of the invention may be practiced by selecting a cell population having desired characteristics. Examples of suitable techniques, including techniques for enrichment of desired cell types, are described elsewhere in this disclosure.

[0012] According to a fourth aspect of the present invention, CD90 + , CD105 + , CD45 - and High TIMP-1 protein secretion Low MMP13 gene expression Methods of treatment are provided that include providing a therapeutically effective amount of cells to a subject in need of treatment.

[0013] The subject may be one in need of tissue repair and / or in need of immunosuppressive treatment.

[0014] According to a fifth aspect of the present invention, CD90+ , CD105 + , CD45 - and High TIMP-1 secretion Low MMP13 gene expression A pharmaceutical composition is provided that includes a cell population enriched for MSC-derived cells and a pharmaceutically acceptable excipient.

[0015] The pharmaceutical composition of the present invention is + , CD105 + , CD45 - and may be free of (or substantially free of) cells with high TIMP-1 secretion and high MMP13 gene expression. [Brief explanation of the drawings]

[0016] [Figure 1] Growth and phenotypic characteristics of MSCs from four patients. (A) The number of population doublings achieved after each passage using MSCs from each patient was recorded until growth ceased (for PN242, data were collected until passage 30, during which cells continued to grow). (B) Population doubling times are shown for all four patients up to passage 17. (C) Typical cell morphology is shown for PN251 MSCs at passages 4 and 16 (growth arrest occurred at passage 17 in this patient) and for PN242 at passage 19 (growth arrest was not observed in this patient). Size bar = 200 μM. (D) The percentage of cells expressing MSC markers CD90 and CD105 and hematopoietic stem cell markers CD34 and CD45 was determined by fluorescence-activated cell sorting. As discussed further elsewhere herein, the cells of the present invention demonstrate a combination of CD90- and CD105-positive and CD45-negative characteristics. [Figure 2] Macroscopic appearance of tissue-engineered cartilage generated using MSCs from four patients at multiple passages. MSCs from passages 2 to 16 were seeded onto polyglycolic acid scaffolds and then induced to undergo chondrogenic differentiation. Each image shows replicate samples from each patient at each passage examined. [Figure 3]Biochemical properties of tissue-engineered cartilage generated using MSCs from four patients at multiple passages. As shown in Figure 2, MSCs from each patient across various passages were used for cartilage tissue engineering. (A-D) The correlation between the dry weight or glycosaminoglycan (GAG) content of the tissue-engineered cartilage constructs at the time of tissue engineering and MSC population doublings is shown for each patient. In all graphs, each point represents the average value of multiple tissue-engineered replicates generated using cells from a single patient at a single passage. (E) The correlation between the dry weight of the tissue-engineered cartilage constructs at the time of tissue engineering and the passage number of the MSCs is shown for all four patients. Each point represents the result of a single replicate sample. (F) The correlation between the GAG ​​content of the tissue-engineered cartilage constructs at the time of tissue engineering and the passage number of the MSCs is shown for all four patients. Each point represents the result of a single replicate sample. In all graphs, the line represents a linear model fitted to the points, while the Spearman's rank correlation coefficient and its significance are shown in the upper right corner. [Figure 4] Trophic repair of meniscal cartilage and immune regulation by multiple-passage MSCs from four patients. For trophic repair of meniscal cartilage, undifferentiated MSCs were seeded onto a collagen scaffold, inserted between two fragments of ovine meniscus, secured with clips, and cultured for 30 days. Histomorphometry was used to measure the degree of connectivity between the two fragments of meniscal tissue. (A-C) Typical examples of high, medium, and low connectivity of meniscal tissue. Connectivity was measured at the midpoint of the arrow. Size bar = 1 mm. (D-G) The correlation between % connectivity of meniscal tissue and population doubling of MSCs is shown for each patient. In all graphs, each point represents the average of multiple experimental replicates using cells from one patient at a single passage. (H) The correlation between % connectivity of meniscal tissue and MSC passage number is shown for all four patients. Each point represents the result of a single replicate sample. In all correlation graphs (A-H), the lines represent a linear model fitted to the points, while the Spearman rank correlation coefficient and its significance are shown in the upper right corner. (I) Immune regulation was determined as the % inhibition of T cell proliferation, measured as the disappearance of Cell-Trace Violet from labeled cells quantified by FACS. Each point is the result of pooled triplicate wells. [Figure 5]Transcriptomics and Proteomics. (A) Overview of the analytical steps performed. Transcriptomics and proteomics data were processed and integrated according to data standards. The integrated data were further analyzed using Ingenuity pathway analysis to identify key genes and proteins that change across passages, map these to biological functions, and predict possible upstream regulators (see Figures 6 and 7). (B) Principal component analysis (PCA) score plot of the first two principal components of all genes and proteins. There is patient segregation (see blue and orange ellipses) that may also be related to changes in osteogenic potential (see Figure S2), while passage-related changes are primarily captured in PC2. (C) Venn diagram of significant variables after a two-way ANOVA test reveals 338 genes and proteins that vary significantly and independently of patient group variation. (D) PCA of the 338 significant variables across passages. Scores of the first two principal components are shown (accounting for approximately 70% of the variance). As expected, there is significant segregation across passages. Heatmap of the regulation data of 338 significant proteins and genes, represented by rows and clustered by Ward's hierarchical clustering. Columns represent biological samples, with each row being a patient sample. They are ordered by passage: red is passage 1, green is passage 5, cyan is passage 10, and purple is passage 15. [Figure 6]Ingenuity pathway analysis of integrated transcriptomics and proteomics data. (A) Ingenuity pathway analysis identified FOXM1 and four other key regulatory genes that fluctuated with increasing passage number (see Figure S5). FOXM1 data from transcriptomics analysis show inhibition of gene expression with increasing passage number. Each bar represents the mean ± SEM of results using MSCs from four individual patients. (B) Ingenuity pathway analysis of the integrated transcriptomics and proteomics dataset predicts inhibition of the FOXM1 canonical pathway for five of the six identified downstream regulators. (C) Ingenuity pathway analysis identified multiple genes and proteins that mapped to the search terms "cell motility," "cell migration," or "wound healing." Most of these identified genes and proteins did not fluctuate significantly across passages as determined by ANOVA. The most highly expressed genes and proteins are listed for each search term. (D) Transcriptomic analysis of CXCL12 data shows sustained gene expression with increasing passage number. Each bar represents the mean ± SEM of MSCs from four individual patients. (E) Proteomic analysis of CXCL12 data shows sustained protein secretion with increasing passage number. Each bar represents the mean ± SEM of MSCs from four individual patients. [Figure 7]Gene and Protein Markers of In Vitro MSC Senescence. The MMP13 gene, which is lost during in vitro senescence of MSCs, was selected as a marker for early-passage cells. Meanwhile, TIMP1 protein secretion, which is independent of in vitro senescence, was selected as a marker for MSCs. (A) MMP13 data from transcriptomics analysis shows a decrease in gene expression with increasing passage number. Each bar represents the mean ± SEM of MSCs from four individual patients. (B) MMP13 data from qPCR analysis shows a decrease in gene expression with increasing passage number. Each bar represents the mean ± SEM of MSCs from four individual patients. (C) TIMP-1 data from proteomics analysis shows sustained protein secretion with increasing passage number. Each bar represents the mean ± SEM of MSCs from four individual patients. (D) TIMP-1 data from ELISA analysis shows sustained protein secretion with increasing passage number. Each bar represents the mean ± SEM of results using MSCs from four individual patients. (E) ELISA analysis of TIMP-1 secreted by MSC / collagen scaffold constructs shows sustained secretion of protein with increasing passage number for live constructs, but decreased secretion for constructs frozen and thawed under conditions that reduce their viability. This pattern of high TIMP-1 secretion and low MMP13 gene expression is characteristic of the cells of the present invention. In particular, this (combined with the CD90-positive, CD105-positive, CD45-negative expression profile discussed elsewhere herein) indicates cells suitable for use in therapeutic applications that do not require phenotypic differentiation potential. Such applications may instead utilize the cells' trophic or immunosuppressive therapeutic activity. For each cell source, results are shown for one live construct compared to one frozen construct. [Figure 8] (Related to Figure 3). Guidance template image for osteogenic scoring. MSCs were induced to undergo osteogenic differentiation and then stained with Alizarin Red. Each slide was scored by two independent observers as - (see CTL, control), +, ++, +++, or ++++. Size bar = 200 μM. [Figure 9](Related to Figure 3). Osteogenic Differentiation of MSCs from Four Patients at Multiple Passages. MSCs from each patient over various passages were induced to undergo osteogenic differentiation in vitro and scored as shown in Figure 8. (A-D) The correlation between MSC osteogenic score and cumulative population doublings is shown for each patient. In all graphs, each point represents the average of multiple replicates using cells from one patient at a single passage. (E) The correlation between MSC osteogenic score and passage number is shown for all four patients. Each point is the result of a single replicate sample. In all graphs, the line represents a linear model fitted to the points, while the Spearman's rank correlation coefficient and its significance are shown in the upper right corner. [Figure 10] (Related to Figure 3). Guidance template image for adipogenic scoring. MSCs were induced to undergo adipogenic differentiation and then stained with Oil Red O. Each slide was scored by two independent observers as - (see CTL, control), +, ++, +++, or ++++. Size bar = 200 μM. [Figure 11] (Related to Figure 3). Adipogenic Differentiation of MSCs from Four Patients at Multiple Passages. MSCs from each patient over various passages were induced to undergo adipogenic differentiation in vitro and scored as shown in Figure S3. (A-D) The correlation between MSC adipogenic score and cumulative population doublings is shown for each patient. In all graphs, each point represents the average of multiple replicates using cells from one patient at a single passage. (E) The correlation between MSC adipogenic score and passage number is shown for all four patients. Each point represents the result of a single replicate sample. In all graphs, the line represents a linear model fitted to the points, while the Spearman's rank correlation coefficient and its significance are shown in the upper right corner. [Figure 12](Related to Figures 5 and 6). Ingenuity pathway analysis of genes and proteins that change with increasing passage. Ingenuity pathway analysis identified FOXM1 (see Figure 6) and four other key regulatory genes that change with increasing passage number. (A) Association of MYOC data from transcriptomics analysis with increasing passage number and Ingenuity pathway analysis of the integrated transcriptomics and proteomics dataset. Each bar represents the mean ± SEM of results using MSCs from four individual patients. (B) Association of NUPR1 data from transcriptomics analysis with increasing passage number and Ingenuity pathway analysis of the integrated transcriptomics and proteomics dataset. Each bar represents the mean ± SEM of results using MSCs from four individual patients. (C) Association of VEGF data from transcriptomics analysis with increasing passage number and Ingenuity pathway analysis of the integrated transcriptomics and proteomics dataset. Each bar represents the mean ± SEM of results using MSCs from four individual patients. (D) Association of PTGER2 data with increasing passage number from transcriptomics analysis and Ingenuity pathway analysis of the integrated transcriptomics and proteomics dataset. Each bar represents the mean ± SEM of results using MSCs from four individual patients. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is based, at least in part, on the inventors' discovery, disclosed for the first time in this document, that in vivo cultured MSCs exhibit a selective loss of pluripotency, but not trophic or immunosuppressive activity, prior to senescence. This discovery, and the knowledge that many of the therapeutic uses of MSCs and MSC-derived cells arise as a result of the trophic or immunosuppressive activity of such cells, rather than through phenotypic differentiation of such cells, opens new possibilities for providing therapeutic uses for cultured cells that have previously been ignored for such applications. The ability to use these large-scale cultures therapeutically offers significant advantages in that it allows for the generation of much larger numbers of therapeutically useful cells than previously thought possible.

[0018] Moreover, the inventors have identified a set of characteristic markers that allow cells that retain trophic or immunosuppressive activity but are incapable of phenotypic differentiation to be distinguished from cells that retain phenotypic differentiation potential (with or without trophic or immunosuppressive activity). This characteristic expression profile (CD90 + , CD105 + , CD45 - These cells, which have high TIMP-1 secretion and low MMP13 gene expression, have not been identified before, and this identification indeed provides significant advantages for the medical use of MSC-derived cells.

[0019] It is recognised that some of the cell populations for medical use defined according to the first aspect of the invention retain trophic or immunosuppressive activity but lack phenotypic differentiation potential, ensuring that these therapeutic activities can be achieved by such cells without generating new cell populations at the site of injury, which is advantageous as it reduces or avoids the potential for undesirable outcomes that may potentially be associated with in situ differentiation of MSCs or MSC-derived cells (an advantage shared by other aspects of the invention).

[0020] Moreover, it has been recognized that the therapeutic properties of cultured MSCs (or MSC-derived cells) change over time. Cells grown for extended periods (or for higher passages) may be able to alleviate pain in injured joints, while cells grown at lower passage numbers may exacerbate pain. A given group of cells may contain cell populations at many different stages in this process; thus, it is important to be able to distinguish between such populations and select only those required for the desired therapeutic use (especially the more "senescent" populations).

[0021] Although differences in the properties of cultured MSCs have been recognized, prior to the disclosure of the present invention, there was no reliable method for determining that the "switch" had occurred to a cell or cell population that had therapeutic trophic or immunosuppressive activity but lacked complex or undesirable properties (e.g., phenotypic differentiation or the potential for exacerbating joint pain). + , CD105 + , CD45 - By identifying the importance of the marker profile of high TIMP-1 secretion and low MMP13 gene expression, the inventors have enabled reliable and stable selection of such advantageous cells and populations.

[0022] Identification of this marker profile avoids the need for selection based on criteria associated with significant failure, such as passage number. By way of example only, these include inter-individual variability in the point at which this change occurs (as is appropriate for both allogeneic and autologous cell sources), variability in cell division rates in cell culture, and considerations that are not relevant in modern bioreactor-based culture conditions, such as the impact of authenticity on passage number.

[0023] CD90 + , CD105 + , CD45 - A cell population enriched for MSC-derived cells with high TIMP-1 secretion and low MMP13 gene expression represents a cell population with improved homogeneity, which is important in the ability to deliver predictable and reproducible cell-based medicines in practice.

[0024] It is understood from the results disclosed herein that the identification of cell populations based on low or high MMP13 gene expression is particularly important in that it allows for the differentiation of cell populations that have trophic or immunosuppressive therapeutic activity but lack phenotypic differentiation potential (e.g., chondrogenesis) from cell populations that retain phenotypic differentiation potential (e.g., chondrogenesis). Such cells are derived from the cells of the present invention (CD90 + , CD105 + , CD45 - In contrast to CD90, which is a type of inflammatory cytokine that secretes TIMP-1 and has low MMP13 gene expression, + , CD105 + , CD45 - TIMP-1 secretion and MMP13 gene expression may be elevated.

[0025] In view of the above, the present invention provides: Nutritional repair and immune regulation are maintained in continuous cultures MSC-derived cells retain the ability to exert these therapeutic mechanisms after extensive cell expansion, Provide a means by which cells with such desirable characteristics can be selected from cells with less advantageous properties (e.g., retention of phenotypic differentiation potential). It is recognized that this is the first time that this has been identified.

[0026] MSCs are used in a wide range of clinical applications for tissue regeneration (e.g., bone and cartilage; cardiovascular disease) and disease modification, including hematological disorders, graft-versus-host disease, and inflammatory disorders. Some such clinical approaches are based on differentiation, while others rely on trophic or immunoregulatory functions.

[0027] Several studies have described a loss of differentiation potential with increasing in vitro passaging of MSCs, and other studies suggest that in vivo aging also results in a loss of differentiation potential after ex vivo isolation of senescent cells. These findings, coupled with the parallel finding that in vitro aging of MSCs shortens telomere length, have been interpreted as indicating a rapid aging process that impairs cellular function and limits their clinical utility.

[0028] Having found for the first time that cells derived from extensively passaged MSCs retain their trophic and immunosuppressive activities even when pluripotency is lost, the inventors recognized that such cells may still be capable of providing useful therapeutic effects. Furthermore, they identified a characteristic pattern of markers that allows such therapeutically effective cells to be recognized, selected after cell culture, and incorporated into pharmaceuticals.

[0029] The invention will now be further described with reference to the following sections.

[0030] Cell population of the present invention For the purposes of the present invention, the following considerations may be interpreted as applicable to cell populations according to the first aspect of the invention, as well as to cell populations selected in the method of the second aspect of the invention, or for use in the therapeutic method of the fourth aspect of the invention.

[0031] The cells of the present invention can be autologous or allogeneic cells. The cells of the present invention may not have pluripotency. Those skilled in the art will recognize many methods for determining pluripotency or the absence of pluripotency, including but not limited to those discussed in the Examples.

[0032] Characterization of cells for cell markers The therapeutically useful MSC-derived cells referred to in various embodiments of the present invention exhibit characteristic expression patterns for a number of markers, including cell surface markers (CD90, CD105, and CD45) and functional markers (TIMP-1 and MMP13).

[0033] Cell surface markers CD90 CD90, also known as Thy-1, is an N-glycosylated glycophosphatidylinositol (GPI)-anchored cell surface protein that contains a single V-like immunoglobulin domain. As discussed further below, the therapeutically useful MSC-derived cells described in this disclosure characteristically contain CD90 + is.

[0034] CD105 CD105, also known as endoglin, is a type I membrane glycoprotein. It is located on the cell surface where it functions as part of the TGF-β receptor complex. As discussed further below, the therapeutically useful MSC-derived cells described in this disclosure characteristically contain CD105 + is.

[0035] CD45 CD45, also known as protein tyrosine phosphatase receptor type C (PTPRC), is a type I transmembrane protein involved in numerous cell signaling pathways. As discussed further below, the therapeutically useful MSC-derived cells described in this disclosure characteristically contain CD45 - is.

[0036] Characterization of cells that are positive or negative for cell surface markers The expression (or absence of expression) of cell surface markers, including CD90, CD105, and CD45, by a cell population can be examined by any suitable means known to those skilled in the art. By way of example only, cell surface markers can be labeled and detected with an appropriate reagent, such as an antibody specific to the cell marker of interest. Such antibody labeling techniques can be used in combination with cell selection techniques, as further described below.

[0037] Cells are positive for a particular cell surface marker (e.g., CD90 or CD105) if, after appropriate immunofluorescence labeling, they have a certain level of fluorescence that is greater than 90% of cells treated with a corresponding control antibody, e.g., an isotype-matched control antibody (denoted as "+ve" or "+ve"). + ") can be considered.

[0038] Cells are negative for a particular cell surface marker (e.g., CD45) if, after appropriate immunofluorescence labeling, they have a level of fluorescence that is less than 10% of cells treated with a corresponding control antibody, e.g., an isotype-matched control antibody (denoted as "-ve" or " - ") can be considered.

[0039] Functional Cell Markers TIMP-1 TIMP-1, also known as TIMP metallopeptidase inhibitor 1, is a member of the tissue inhibitor of metallopeptidase family. It is a glycoprotein that functions as an inhibitor of matrix metalloproteinases. As discussed further below, the therapeutically useful MSC-derived cells described in this disclosure characteristically have high levels of TIMP-1 protein secretion.

[0040] MMP13 MMP13 (matrix metallopeptidase 13, also known as collagenase 3) is a metalloproteinase enzyme involved in the degradation of extracellular membrane components. It is encoded by the MMP13 gene in humans. As discussed further below, the therapeutically useful MSC-derived cells described in this disclosure characteristically have low MMP13 gene expression.

[0041] The relevance of this marker in allowing the selection of cell populations that have trophic and immunosuppressive activity but lack phenotypic differentiation potential has not been reported previously. As reported herein, the inventors have demonstrated that this marker can be used to select other cell populations (CD90 + , CD105 + , CD45 - The cells of the present invention (CD90 + , CD105 + , CD45 - We found that this is key to distinguishing between thyroid cancer and thyroid cancer (high TIMP-1 secretion and low MMP13 gene expression).

[0042] Characterization of cells high or low for functional cell markers The cells of the present invention are characterized as having high TIMP-1 protein secretion.

[0043] The expression and secretion of TIMP-1 can be examined using any technique known to those skilled in the art that is suitable for assessing protein secretion.By way of example only, TIMP-1 protein expression can be examined using an enzyme-linked immunosorbent assay (ELISA) technique.An example of a suitable ELISA for TIMP-1 secretion, which can be used to determine whether the cells of interest are positive for TIMP-1 secretion in the manner required for the cells of the present invention, is further described in the Examples.

[0044] The cells of the present invention are characterized as having low MMP13 gene expression.

[0045] MMP13 gene expression can be evaluated by examining the level of transcripts that indicate MMP13 expression in cells. Preferably, MMP13 gene expression can be evaluated by MMP13-specific reverse transcription PCR. Particularly suitable methods based on this technique are further described in the Examples.

[0046] A cell population can be identified as having high TIMP-1 secretion based on the amount of this protein secreted by a given number of cells over a period of time. Preferably, high secretion of TIMP-1 protein can be indicated by secretion of 100 ng / ml (or more) of TIMP-1 by cells (initially seeded at a density of 2.25 million cells / well) over 24 hours. Further details of suitable conditions that can be used in such an assessment are provided in the Examples, in connection with the study reported in Figure 7E.

[0047] A cell population can be identified as having low MMP13 gene expression based on comparison with a suitable baseline.By way of example only, a housekeeping gene, such as Tata binding protein, can provide a suitable baseline.If cells demonstrate that the MMP13 expression level compared to a housekeeping gene (for example, Tata binding protein) is half or less than half of the relative expression by freshly isolated MSCs, these cell populations can be characterized as having low MMP13 gene expression.Suitable freshly isolated MSCs for comparison purposes can be derived from sources that are appropriately compatible with the cell source of the present invention.Expression fold comparison (whether MMP13 or suitable housekeeping gene) can be carried out using the method further described in Examples.

[0048] Cell enrichment and enriched cell populations The cell population according to the first aspect of the invention is enriched for the presence of therapeutically useful MSC-derived cells compared to naturally occurring cell populations, and the cells prepared in the method of the second aspect of the invention are enriched for the presence of such cells, and selection of cells according to the third aspect of the invention may similarly yield enriched cell populations.

[0049] Therapeutically useful MSC-derived cells that are enriched in the population have characteristic expression of markers (i.e., CD90 + , CD105 + , CD45 - These can be identified based on the criteria of high TIMP-1 protein secretion and low MMP13 gene expression.

[0050] Those skilled in the art understand the term "enrichment" when used in the context of a cell population. Enrichment can be achieved by positive selection of desired cells (i.e., cells with characteristic expression of a marker) or by elimination of undesired cells.

[0051] By way of example only, enrichment techniques may utilize culture conditions that support the growth of cells with desired traits (e.g., characteristic expression of markers discussed herein) and / or conditions that reduce the viability of cells lacking the desired trait. Cells grown under such conditions may be monitored to ensure that the desired population has been enriched. By way of example only, cell culture conditions that can be used to enrich for a population of therapeutically useful MSC-derived cells according to the present invention may include the use of 10% fetal bovine serum and supplementation with 5 ng / ml fibroblast growth factor (FGF2).

[0052] Alternatively, enrichment or selection can be understood in the context of techniques such as fluorescence-activated cell sorting (FACS), which allows for the selection of cells based on their expression of markers (e.g., CD90, CD105, and CD45). Unwanted cells can then be discarded.

[0053] Thus, the method for preparing cells for use as a medicament according to the second aspect of the invention comprises the step of detecting the markers listed (CD90 + , CD105 + , CD45 - The method may include selecting cells based on one or more of the following criteria: high MMP-1 secretion, high TIMP-1 secretion, and low MMP13 gene expression, thereby enriching the MSC-derived cells in the specified cell population. A suitable selection step may include selection based on low MMP13 gene expression, a marker that the inventors have found to be particularly useful in identifying cells that lack phenotypic differentiation potential but retain trophic activity.

[0054] Preferably, the enriched MSC-derived cell population may contain at least 80% CD90 and CD105 positive cells and 10% or less CD45 positive cells.

[0055] For purposes of the present invention, enriched cell populations are those with a characteristic marker profile to be enumerated (i.e., CD90 + , CD105 + , CD45 - and high TIMP-1 secretion and low MMP13 gene expression). Suitably, the enriched cell population may comprise at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells having the recited characteristic marker profile. The enriched cell population may comprise substantially 100% of cells having the recited characteristic marker profile (i.e., CD90 + , CD105 + , CD45 - The cells may be composed of cells with high TIMP-1 secretion and low MMP13 gene expression.

[0056] The cell population according to the first aspect of the invention, or the cell population selected in the method of the second aspect of the invention, may have the characteristic marker profile listed (i.e., CD90 + , CD105 + , CD45 - The IL-16 expression level may be substantially composed of only cells with the following characteristics: high TIMP-1 secretion and low MMP13 gene expression.

[0057] The cell population according to the first aspect of the invention, or the cell population selected in the method of the second aspect of the invention, may be a cell population that expresses CD90 + , CD105 + , CD45 - and may be free of or substantially free of cells with high TIMP-1 secretion and high MMP13 gene expression.

[0058] Cell enrichment or cell selection may be carried out by any suitable method, including but not limited to those methods discussed herein.

[0059] Cell selection and analysis With knowledge of the characteristic marker profile of the cells of the present invention, those skilled in the art can easily identify a method for selecting cells that exhibit this profile.Similarly, those skilled in the art can easily identify a method for examining a cell population to determine whether they exhibit a characteristic expression pattern.These include, but are not limited to, techniques selected from the group consisting of flow cytometry, fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS).

[0060] cell culture The present inventors have found that the trophic and immunosuppressive activity of the cells of the present invention is maintained through continuous cell culture. This has the advantage of enabling the generation of a large number of therapeutically effective cells. As presented elsewhere herein, the cells of the present invention can maintain therapeutically effective levels of trophic or immunosuppressive activity for up to 30 passages.

[0061] Thus, the cell populations of the present invention may comprise cells that have been subjected to at least 5 subcultures, at least 10 subcultures, at least 15 subcultures, at least 20 subcultures, at least 25 subcultures, or at least 30 subcultures.

[0062] Similarly, the method according to the second aspect of the invention may comprise culturing the cells for at least 5 subcultures, at least 10 subcultures, at least 15 subcultures, at least 20 subcultures, at least 25 subcultures, or at least 30 subcultures.

[0063] Medical Uses and Treatment Methods of the Invention The cell population of the first aspect of the invention, the medicament produced from the cells prepared according to the second aspect of the invention, the cells selected by the method of the third aspect of the invention, and the treatment method of the fourth aspect of the invention are all suitable for therapeutic use. In particular, the therapeutic use may be to promote tissue repair or to promote immunosuppressive therapy. Further details of such embodiments are provided below.

[0064] tissue repair The medicament, medical use or treatment method of the present invention can be used to promote tissue repair. The need for promoting tissue repair can occur as a result of any condition that causes tissue injury. For example, the need for promoting tissue repair can occur as a result of a condition selected from the group consisting of osteoarthritis, myocardial infarction, meniscal cartilage injury (e.g., meniscus tear), ligament injury (e.g., ligament rupture), skin injury and soft tissue injury. Tissue repair can be promoted by promoting trophic active treatment. Tissue repair can alleviate pathologies associated with diseased or damaged joints. This can be evaluated by appropriate means, such as histological examination (of biopsy or experimentally treated joints) or suitable imaging diagnostic techniques.

[0065] Suitably, tissue repair may include treatment of damaged tissue to promote tissue repair by increasing the rate of tissue repair and / or improving the quality of the repaired tissue resulting from the treatment. Promotion of tissue repair may be readily assessed relative to a suitable control.

[0066] The cells, therapeutic methods or medical uses of the present invention may be capable of promoting tissue repair by at least 10% compared to a suitable control. By way of example only, the cells, therapeutic methods or medical uses of the present invention may be capable of promoting tissue repair by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to a suitable control. In fact, the cells, therapeutic methods or medical uses of the present invention may be capable of promoting tissue repair by 100% or more compared to a suitable control.

[0067] Suitably, tissue repair may include treatment of damaged tissue selected from the group consisting of cartilage, cardiovascular tissue, bone, and soft tissue, such as skin.

[0068] Suitably, the tissue repair may include treatment of a condition selected from the group consisting of treatment of a cartilage tear, e.g., treatment of a meniscal cartilage tear; treatment of osteoarthritis, myocardial infarction, meniscal cartilage injury (e.g., meniscus tear), ligament injury (e.g., ligament rupture), skin injury, and soft tissue injury.

[0069] It is understood that the trophic activity of MSC-derived cells can contribute to tissue repair.Therefore, the medical use or treatment method utilizing the cell population or medicine of the present invention can be for use in tissue repair by stimulating trophic activity.This use can be contrasted with the previously described applications in which MSC or MSC-derived cells achieve therapeutic benefit through phenotypic differentiation (i.e., differentiation to obtain replacement cells that contribute to tissue repair).

[0070] Nutritional Activity The cells of the present invention have the trophic activity that they are derived from MSCs.The medical uses and treatment methods of the present invention, particularly for use in tissue repair, can utilize the trophic activity of such cells.In fact, the cells of the present invention can be selected for their trophic activity (and the absence of phenotypic differentiation potential).

[0071] Trophic activity can be considered to be the ability of MSCs or MSC-derived cells to induce neighboring cells to secrete active molecules that contribute to tissue repair or regeneration after transplantation. Trophic repair can occur as a result of the production of large amounts of growth factors and other mediators by MSCs or MSC-derived cells. Trophic activity has been shown to contribute to tissue repair or regeneration in the treatment of stroke, myocardial infarction, and meniscal cartilage repair, among other examples.

[0072] The method of the second or third aspect of the present invention may further comprise the step of assessing the cells to determine their trophic activity. Preferably, cells for use as pharmaceuticals may be selected based on their trophic activity. The cell population according to the first aspect of the present invention may be enriched for MSC-derived cells with trophic activity.

[0073] The presence or absence of trophic activity can be assessed by any suitable method known to those skilled in the art.Suitable methods can be used to examine the ability of cells to alleviate the pathology of diseased or damaged joints.By way of example only, the trophic activity of cells according to the first aspect of the present invention, or cells prepared in the method according to the second aspect of the present invention, can be assessed by examining their ability to bind separate cartilage sites.Preferably, the ability to promote such binding can be demonstrated in vitro.Alternatively, or in addition, such ability can be demonstrated in vivo.

[0074] Details of assays that allow for assessment of trophic activity by binding of separate cartilage sites are provided in "Repair of torn avascular meniscal cartilage using undifferentiated autologous mesenchymal stem cells: from in vitro optimization to a first-in-human study" (Whitehouse, et al., Stem Cells Translation Medicine, 2017; 6:1237-1248), the disclosure of which is incorporated by reference insofar as it relates to a model for studying binding of cartilage treated with test agents, and details of suitable techniques are provided in the Examples of this disclosure.

[0075] Therapeutic applications that can benefit from the trophic activity of the cells or medicaments, or medical uses or methods of treatment of the invention, correspond to therapeutic applications that would benefit from the promotion of tissue repair.

[0076] immunosuppressive treatment The cells of the invention also have immunosuppressive activity that is retained from the MSCs from which they are derived. Medical uses and treatment methods of the invention may utilize the immunosuppressive activity of the cells and may be for use in immunosuppressive therapy.

[0077] There are numerous therapeutic applications that have been found to utilize the immunosuppressive properties of MSCs or MSC-derived cells. Suitably, immunosuppressive therapy may be used in the treatment of a disease selected from the group consisting of hematological disorders, graft-versus-host disease and alloreactive immune disorders, autoimmune diseases or inflammatory diseases.

[0078] Immunosuppressive therapy using the cells, methods of treatment or medical uses of the present invention may be used in the treatment of patients undergoing stem cell transplantation, e.g., hematopoietic stem cell transplantation. Patients undergoing such transplantation may suffer from a blood disorder.

[0079] The ability of the cells, therapeutic methods, and medical uses of the present invention to achieve therapeutically effective immunosuppressive activity also makes them useful for treating graft-versus-host disease. In such embodiments, treatment with the cells or medicaments of the present invention may reduce the recipient's immune response, thereby mitigating the response to an allogeneic transplant.

[0080] There are also many conditions in which the onset or progression of the disease is mediated by the body's immune response. These include alloreactive immune diseases, autoimmune diseases, or inflammatory diseases. Such diseases may benefit from treatment with the cells or medicaments of the invention, and from the medical uses or methods of treatment of the invention.

[0081] In a suitable embodiment, the cells, medicaments or methods of treatment according to the invention are for use in the treatment of diseases due to immunosuppression of the immune response.

[0082] Immunosuppressive activity The method of the second or third aspect of the present invention may further comprise the step of evaluating the cells to determine their immunosuppressive activity. Preferably, cells to be used as a pharmaceutical may be selected based on their immunosuppressive activity. The cell population according to the first aspect of the present invention may be enriched for MSC-derived cells having immunosuppressive activity.

[0083] The presence or absence of immunosuppressive activity can be assessed by any suitable method known to those skilled in the art. The immunosuppressive activity of the cells or pharmaceuticals of the present invention can be compared to the activity of a suitable control. When assessed in a suitable assay, the cells or pharmaceuticals of the present invention may be capable of achieving at least 10% suppression of immune activity compared to a control. For example, the cells or pharmaceuticals of the present invention may be capable of achieving at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% suppression of immune activity compared to a suitable control. In fact, the cells or pharmaceuticals of the present invention may be capable of achieving 100% suppression of immune activity compared to a suitable control.

[0084] By way of example only, the immunosuppressive activity of cells, such as those prepared in the method of the first aspect of the invention, may be assessed by examining their ability to inhibit T cell proliferation. In suitable embodiments, immunosuppressive activity is demonstrated by the ability to inhibit T cell proliferation by at least 80%, consistent with the results presented in Figure 4I.

[0085] Therapeutic applications that may or may not require phenotypic differentiation potential In a third aspect of the present invention, there is provided a method by which suitable MSC-derived cells can be selected for therapeutic applications, the selection being based on the nature of the therapeutic application and on the expression of certain markers by the MSC-derived cells.

[0086] The method of the third aspect of the invention allows for a distinction to be made between cells for use in therapeutic applications that require phenotypic differentiation potential and cells for use in therapeutic applications that do not require phenotypic differentiation potential.

[0087] Therapeutic applications requiring phenotypic differentiation potential are those in which the therapeutic activity of MSC-derived cells is achieved by the ability of these cells to differentiate and the new cells generated to provide a therapeutic effect. Examples of such applications include in vitro tissue engineering, inducing MSCs to differentiate and thereby generate articular cartilage or bone, and the use of MSCs for transplantation into cartilage lesions or bone defects for the in situ generation of replacement tissue.

[0088] In contrast, there are numerous therapeutic applications in which MSC-derived cells do not require phenotypic differentiation potential to achieve a therapeutic effect. It is understood that therapeutic applications utilizing trophic effects to induce tissue repair do not require phenotypic differentiation potential, as it is the trophic factors provided by MSCs or MSC-derived cells that mediate such therapeutic activity. Similarly, it is recognized that therapeutic applications utilizing the immunosuppressive effects of MSC-derived cells do not require phenotypic differentiation potential. Examples of therapeutic applications utilizing trophic or immunosuppressive effects to achieve a required therapeutic activity are described elsewhere herein. Furthermore, the cell population of the first aspect of the present invention, the cells prepared according to the second aspect of the present invention, and the therapeutic method of the fourth aspect of the present invention are well suited for use in such applications that do not require phenotypic differentiation potential or pluripotency.

[0089] Cell formulation The cell population of the present invention can be formulated as a pharmaceutical composition.Indeed, the present disclosure also provides a composition comprising the cells of the present invention, and includes pharmaceutical compositions and formulations, for example, unit-dosage compositions that comprise a given number of cells for administration at a given dose or a fraction thereof.Pharmaceutical compositions and formulations generally include one or more pharmaceutically acceptable carriers or excipients as appropriate.In some embodiments, the composition includes at least one additional therapeutic agent.

[0090] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0091] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0092] In some embodiments, the choice of carrier will depend, in part, on the particular cell and / or method of administration. Accordingly, a variety of suitable formulations exist. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001 to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers, e.g., phosphate, citric acid and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins , such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0093] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixture thereof is typically present in an amount of about 0.001 to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in further detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0094] The formulation may comprise an aqueous solution. The formulation or composition may also comprise two or more active ingredients useful for the particular indication, disease, or condition being treated by the cells, preferably ingredients with complementary activities, where each activity does not adversely affect the other. Such active ingredients are preferably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.

[0095] In some embodiments, the pharmaceutical composition comprises the cells in an amount effective to treat or prevent a disease or condition, e.g., a therapeutically effective or prophylactically effective amount. In some embodiments, therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the treated subject. The desired dosage can be delivered by a single bolus of cells, multiple boluses of cells, or continuous infusion of cells.

[0096] Cells and compositions can be administered using standard administration techniques, formulations, and / or devices. Administration of cells can be autologous or heterologous. For example, immunoresponsive cells or progenitor cells can be obtained from one subject and administered to the same subject or a different compatible subject. Peripheral blood-derived immunoresponsive cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) can be administered by catheter administration, systemic injection, local injection, including intravenous injection, or local injection, including parenteral administration. When a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immunoresponsive cells) is administered, it is generally formulated into a unit injection form (solution, suspension, emulsion).

[0097] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to a subject using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[0098] In some embodiments, the composition is prepared as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, and in some embodiments, may be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide longer contact time with specific tissues. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerin, polyethylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0099] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, e.g., in a mixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, and / or coloring agents, depending on the route of administration and the desired preparation. In some embodiments, standard textbooks can be consulted to prepare suitable preparations.

[0100] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial agents, preservatives, antioxidants, chelating agents and buffers.Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol and sorbic acid.Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0101] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0102] The cell formulations of the present invention are + , CD105 + , CD45 - and may be free of or substantially free of cells with high TIMP-1 secretion and high MMP13 gene expression.

[0103] Dosages and Therapeutically Effective Amounts of the Cells of the Invention A therapeutically effective amount of the cells of the present invention can be selected based on the nature of the disorder being treated and based on the severity of the disease in the individual requiring treatment. Considerations such as the age and weight of the recipient can also influence the selection of an appropriate dose.

[0104] A therapeutically effective amount of the cells of the invention (eg, in the form of a pharmaceutical of the invention) can be provided in a single treatment frequency, or in multiple treatment frequencies.

[0105] Suitable numbers of therapeutically effective MSC-derived cells for use according to the present invention can be calculated based on the number of cells administered per injury site.

[0106] By way of example only, in the treatment of osteoarthritis or other knee injuries, a dose of 10-20 million cells per knee may prove therapeutically effective. This guidance may be adjusted depending on the organ being treated, i.e., for larger or smaller injury sites. The present disclosure provides, for example, the following embodiments. [1] For use as a medicine, CD90 + , CD105 + , CD45 - and ·TIMP-1 secretion + (high TIMP-1 secretion) and MMP13 gene expression - (Low MMP13 gene expression) A cell population enriched for MSC-derived cells. [2] The cell population of item 1, wherein the cells are substantially free of pluripotency. [3] Item 3. The cell population according to item 1 or 2, wherein the pharmaceutical is intended for use in tissue repair. [4] Item 4. The cell population according to any one of items 1 to 3, wherein the medicament is intended for use in treating a tissue selected from the group consisting of cartilage, cardiovascular tissue, and bone. [5] Item 5. The cell population according to Item 4, wherein the medicament is intended for use in treating a tissue selected from the group consisting of cartilage and bone. [6] Item 5. The cell population according to item 3 or 4, wherein the medicament is intended for use in treating a condition selected from the group consisting of osteoarthritis, myocardial infarction, meniscal cartilage injury (e.g., meniscus tear), ligament injury (e.g., ligament rupture), skin injury, and soft tissue injury. [7] 7. The cell population according to any one of paragraphs 3 to 6, wherein the medicament is intended for use in tissue repair by stimulating trophic repair. [8] Item 8. The cell population according to any one of items 1 to 7, wherein the medicament is intended for use in treating a disease selected from the group consisting of blood diseases, graft-versus-host disease, and inflammatory diseases. [9] Item 10. The cell population according to Item 8, wherein the medicament is intended for use in the treatment of a disease caused by immunosuppression of the immune response.

[10] The cell population is CD90 + , CD105 + , CD45 - and TIMP-1 secretion + and MMP13 gene expression - 10. The cell population of any one of paragraphs 1 to 9, comprising at least 15% cells which are

[11] Culturing MSCs to generate MSC-derived cells; MSC-derived cells: CD90 + , CD105 + , CD45 - and ·TIMP-1 secretion + (high TIMP-1 secretion) and MMP13 gene expression - (Low MMP13 gene expression) Concentrating the population, and Formulating cells for pharmaceutical use 1. A method for preparing cells for use as a pharmaceutical, comprising:

[12] Item 12. A medicament prepared by the method according to item 11 for use in tissue repair.

[13] Item 13. A medicament prepared by the method according to item 11 or 12 for use in treating tissue selected from the group consisting of cartilage, cardiovascular tissue and bone.

[14] Item 14. A medicament prepared by the method according to any one of items 11 to 13 for use in treating a condition selected from the group consisting of osteoarthritis, myocardial infarction, meniscal cartilage injury (e.g., meniscus tear), ligament injury (e.g., ligament rupture), skin injury, and soft tissue injury.

[15] 15. A medicament prepared by the method of any one of items 12 to 14 for use in tissue repair by stimulating trophic repair.

[16] Item 16. A medicament prepared by the method according to any one of items 11 to 15 for use in treating a disease selected from the group consisting of blood diseases, graft-versus-host disease, and inflammatory diseases.

[17] Item 17. A medicament prepared by the method according to item 16 for use in treating diseases caused by immunosuppression of immune responses.

[18] assessing the levels of MMP13 gene expression and TIMP-1 protein secretion by the cells; and Selecting cells for use in therapeutic applications that do not require phenotypic differentiation potential when the cells exhibit low levels of MMP13 gene expression and high levels of TIMP-1 protein secretion. 2. A method for selecting MSC-derived cells for therapeutic use, comprising:

[19] 20. The method of paragraph 18, further comprising selecting the cells for use in a therapeutic application requiring phenotypic differentiation potential if the cells exhibit high levels of both MMP13 gene expression and TIMP-1 protein secretion.

[20] 20. The method of paragraph 19, wherein the therapeutic application requiring phenotypic differentiation potential is selected from the group consisting of in vitro tissue engineering and transplantation to generate new tissue by in vivo differentiation. [twenty one] 21. The method of any one of paragraphs 18 to 20, wherein the therapeutic application not requiring phenotypic differentiation potential is selected from the group consisting of promoting tissue repair through trophic activity and promoting immunosuppressive therapy. [twenty two] 22. The method of any one of paragraphs 18 to 21, wherein the cells are derived from MSCs by passaging of the MSCs. [twenty three] CD90 + , CD105 + , CD45 - and TIMP-1 protein secretion + (high TIMP-1 secretion) and MMP13 gene expression - (Low MMP13 gene expression) A method of treatment comprising providing a therapeutically effective amount of cells to a subject in need of treatment. [twenty four] Item 24. The method of treatment according to Item 23, which aims to promote tissue repair. [twenty five] Item 25. The therapeutic method according to Item 23 or 24, which is intended for immunosuppressive treatment.

[26] The donated cells are CD90 + , CD105 + , CD45 - 26. The method of any one of Items 23 to 25, wherein the subject is a subject of the present invention, and the subject is free from or substantially free from cells with high TIMP-1 secretion and high MMP13 gene expression.

[27] CD90 + , CD105 + , CD45 - and High TIMP-1 secretion and Low MMP13 gene expression A pharmaceutical composition comprising a cell population enriched for MSC-derived cells and a pharmaceutically acceptable excipient.

[28] CD90 + , CD105 + , CD45 - The cell population according to any one of Items 1 to 10, or the pharmaceutical composition according to Item 27, wherein the cell population is a cell population that exhibits high TIMP-1 secretion and high MMP13 gene expression and is substantially free of cells.

[29] CD90 + , CD105+ , CD45 - Item 29. The cell population or pharmaceutical composition according to Item 28, wherein the cell population or pharmaceutical composition does not contain cells that secrete high amounts of TIMP-1 and have high expression of the MMP13 gene.

[0107] The invention will now be further described with reference to the following examples. [Example]

[0108] Bone marrow-derived mesenchymal stromal cells (MSCs) are defined as stem cells in part by their multipotentiality, but more recently, they have been shown to possess trophic and immunoregulatory properties that may be important for promoting tissue repair. To assess the hierarchical importance of MSC pluripotency relative to its trophic / immunoregulatory functions, we performed a detailed analysis of how these properties change as cells age in vitro. Using in vitro differentiation and repair models and integrating transcriptomic and proteomic data with Ingenuity pathway analysis, we show that MSC pluripotency declines with increasing passage, whereas trophic repair and immunoregulatory capabilities are maintained even after extensive in vitro cellular aging. These findings support the view that trophic repair and immunoregulation are central properties of MSCs and, therefore, are hierarchically more important than transient pluripotency.

[0109] introduction The concept of multipotent mesenchymal stem cells (MSCs) was established by Caplan in the early 1990s (Caplan, 1991) following the seminal work of Friedenstein in the 1960s and 1970s (Friedenstein et al., 1970; Friedenstein et al., 1968; Friedenstein et al., 1966), which demonstrated the presence of osteogenic progenitor cells in bone marrow. Building on this early work, there have been numerous studies demonstrating the ability of MSCs to differentiate in vitro, with clear evidence of skeletal lineage pluripotency ( Kolf et al., 2007 ), osteogenesis ( Gronthos et al., 1994 ; Pound et al., 2006 ; Pound et al., 2007 ; Simonsen et al., 2002 ), chondrogenesis ( Dickinson et al., 2017 ; Johnstone et al., 1998 ; Kafienah et al., 2002 ; Kafienah et al., 2007b ; Kafienah et al., 2006 ; Martin et al., 1997 ; Solchaga et al., 2005 ; Yoo et al., 1998 ), and adipogenesis ( Dickinson et al., 2017 ; Mirmalek-Sani et al., 2006 ; Munir et al., 2017) and more limited evidence of totipotency, including endodermal and ectodermal pathways (Caplan, 1991; Kuroda et al., 2011). Some of these studies have demonstrated that clonal populations of bone marrow MSCs retain multipotency (Dickinson et al., 2017; Muraglia et al., 2000), suggesting that at least some individual MSCs in heterogeneous marrow-derived populations exhibit stem cell properties.

[0110] More recently, increasing evidence has suggested that MSCs can support tissue repair through mechanisms not directly related to their multipotency (Prockop, 2007). Caplan described MSCs as having "trophic" capabilities, inducing neighboring cells to secrete active molecules after transplantation, for example, in the treatment of stroke, myocardial infarction, or meniscal cartilage repair (Caplan and Dennis, 2006). Trophic repair is most likely mediated by the production of large amounts of growth factors and other mediators by MSCs (Caplan and Correa, 2011; Caplan and Dennis, 2006; Kuroda et al., 2011; Prockop, 2009; Tolar et al., 2010). We have previously developed a therapeutic strategy for meniscal cartilage repair based on the trophic properties of MSCs (Pabbruwe et al., 2010b), which has shown some evidence of efficacy in preclinical and clinical trials (Whitehouse et al., 2017). A second mechanism of tissue repair independent of pluripotency is the ability of MSCs to suppress immune responses through various mechanisms, including downregulation of T cell proliferation (Dickinson et al., 2017; Keating, 2012; Spaggiari et al., 2007; Tolar et al., 2010; Uccelli et al., 2006; Uccelli et al., 2007). This important property of MSCs has been used clinically to support the engraftment of donated hematopoietic cells and prevent graft-versus-host disease (Lazarus et al., 2005; Tolar et al., 2010).

[0111] The increasing complexity in our understanding of MSC mechanisms of action, coupled with recent concerns about the lack of rigorous evidence regarding their physiological roles, has raised some doubts about whether they should be considered stem cells at all (Bianco et al., 2008; Caplan, 2017; Kuroda et al., 2011; Sipp et al., 2018). Despite this uncertainty, however, there remains a wide range of clinical applications for MSCs for tissue regeneration (e.g., bone and cartilage; cardiovascular disease) and disease modification, including hematological disorders, graft-versus-host disease, and inflammatory disorders (Squillaro et al., 2016). While some of these clinical approaches are based on differentiation, others rely on trophic or immunoregulatory functions (Caplan and Correa, 2011), and we still lack a clear understanding of how these various mechanisms of action interrelate.

[0112] Several studies have documented the loss of differentiation potential of MSCs with increasing in vitro passage (Bonab et al., 2006; Muraglia et al., 2000; Yang, 2018; Yang et al., 2018), and other studies suggest that in vivo aging also results in a loss of differentiation potential after ex vivo isolation of senescent cells (Choudhery et al., 2014; Ganguly et al., 2017; Stenderup et al., 2003). These findings, coupled with the parallel finding that in vitro aging of MSCs shortens telomere length (Baxter et al., 2004), have been interpreted as indicating a rapid aging process that impairs cellular function and limits their clinical utility. Conversely, comparable aging-related data on nutritional repair or immune regulation have not been reported. Therefore, it is currently unclear whether the decline in differentiation potential due to cellular senescence reflects a general decline in the functional capacity of MSCs as they approach senescence, or whether there is a selective loss of pluripotency.

[0113] We propose here that in vitro aging of MSCs provides a framework for understanding the connections between these various aspects of biology by identifying transient functional properties and core properties that are retained throughout the cell's lifespan. Thus, the goal of this study is to determine the relative hierarchical importance of differentiation potential and trophic repair / immunoregulation by determining the rate at which such functions decline with increasing numbers of in vitro aging MSCs.

[0114] result Patient characteristics and growth potential of MSCs Bone marrow was collected from patients undergoing arthroplasty for the treatment of traumatic knee injuries. All patients provided informed consent, and the study was conducted in full accordance with local ethical guidelines (Southmead Research Ethics Committee Ref. 078 / 01). Table S1 shows patient characteristics. All four patients were male, with a mean age of 49 years (range 38-70 years) at the time of surgery. MSCs were isolated from each bone marrow by adherence and grown under standard culture conditions until they could no longer proliferate. MSCs derived from PN241 and PN242 continued to proliferate for more passages than MSCs derived from PN251 and PN264, and PN242 cells showed no signs of growth arrest, even at passage 30 (Figure 1A and Table S1). Figure 1B shows the MSC population doubling time (PDT) for each patient at each passage up to passage 17. The PDT of MSCs from all four patients was similar at early passages and prolonged at later passages, with MSCs from one patient (PN264) showing particularly slow growth at higher passages. Early-passage MSCs had a typical stellate appearance, which was lost as the cells further senesced (Figure 1C, PN251). However, PN242 cells, when they continued to grow well, retained an elongated stellate appearance even at very high passages (Figure 1C, PN242).

[0115] Cell surface marker expression was determined by FACS using MSCs from all four patients at each of passages P1, P5, P10, and P15. Expression of the MSC markers CD105 and CD90 was maintained at >90% in late passages of MSCs from all four patients. The hematopoietic stem cell marker CD45 remained at <10% in all passages, whereas in two of the patients, expression of the hematopoietic stem cell marker CD34 increased slightly with increasing passage number (Figure 1D).

[0116] The trilineage differentiation potential of MSCs decreases with increasing passage MSCs from selected passages from each patient were examined for chondrogenic potential in a 3D cartilage tissue conversion assay, measuring the amount of cartilage converted, measured as dry weight of tissue, and the quality of the cartilage, measured as glycosaminoglycan content expressed as a percentage of dry weight. There was clear evidence of loss of chondrogenic potential due to in vitro aging of the cells. Typical macroscopic appearances of tissue-converted cartilage across various passages of MSCs from each patient can be seen in Figure 2, clearly demonstrating a decrease in the average size of cartilage constructs generated using late-passage MSCs. This macroscopic finding was supported by quantitative analysis. There was a significant negative correlation between the average dry weight of cartilage formed and the cumulative population doublings of MSCs recorded at the time of tissue conversion for three of the four patients, as well as a significant negative correlation between the average cartilage glycosaminoglycan content and the cumulative population doublings of MSCs recorded at the time of tissue conversion for all four patients (Figures 3A-D). Furthermore, both the dry weight and glycosaminoglycan content of all individual tissue-engineered cartilage constructs were significantly negatively correlated with the passage number of the MSCs used for tissue engineering (Figures 3E and 3F).

[0117] MSCs were also examined in monolayer culture for osteogenic potential by semiquantitative analysis of alizarin red staining. Representative images of staining patterns and the scoring system used are shown in Figure S1. There was some evidence of a gradual loss of MSC osteogenic potential, but the results were inconsistent between patients. In patient PN241, there was no apparent change in the mean osteogenic score with cumulative population doublings (Figure S2A). In patient PN242, there was a significant decrease in osteogenic potential with cumulative population doublings (Figure S2B). Although the change was not statistically significant, the osteogenic score in PN251 decreased to 0 by 10 population doublings and remained at 0 at higher doublings (Figure S2C). Patient PN264 showed no observable osteogenic activity, even at a low number of population doublings (Figure S2D). Overall, individual osteogenic scores of repeated assays did not significantly correlate with the passage number of MSCs used (fig. S2E), but the different patterns observed in each patient indicate a progressive, if variable, decline in osteogenicity with in vitro aging of the cells.

[0118] The adipogenic potential of MSCs was examined in monolayer cultures by semiquantitative analysis of Oil Red O staining. Representative images of the staining pattern and the scoring system used are shown in Figure S3. In patient PN251, there was a slight but significant decline in adipogenic potential with cumulative population doublings (Figure S4C), whereas in the other three patients, there was no significant change (Figures S4A, S4B, and S4C). Overall, the individual adipogenic scores of repeated assays did not significantly correlate with the passage number of the MSCs used (Figure S4E), and therefore, there was only very limited evidence of a decline in adipogenesis with in vitro aging of the cells.

[0119] Collectively, these data demonstrate a clear loss of differentiation potential in MSCs, with early passage cells tending to exhibit full tri-lineage potential, and late passage cells tending to retain adipogenic or adipogenic plus osteogenic, but not chondrogenic, potential. These data suggest that pluripotency is not a fundamental property of CD105+ve, CD90+ve, CD34-ve, CD45-ve MSCs, but rather a feature of freshly isolated MSCs that is lost with cell expansion in vitro.

[0120] MSC-mediated nutritional repair and immune regulation are maintained with increasing passages We used MSC in vitro binding of two fragments of meniscal cartilage as a model of trophic repair based on our previous work in this system in vitro, in an in vivo sheep model, and in patients with meniscal tears (Whitehouse et al., 2017). MSCs from all four patients, each at passage 17, were tested for their ability in our meniscal repair capacity assay. The variation in % meniscal binding in these studies was within the same range as previously described, as illustrated in representative histological images (Figure 4A–C). In contrast to our tripotential differentiation findings (see above), there was no significant change in the mean meniscal repair capacity with cumulative population doublings for any of the four patients (Figure 4D–G). Furthermore, the % binding measured for individual meniscal constructs showed no correlation with the passage number of MSCs used (Figure 4H).

[0121] In addition to stimulating tissue repair responses, MSCs can also suppress immunity by inhibiting T cell proliferation and other mechanisms. Therefore, we measured the percent inhibition of T cell proliferation by MSCs from selected passages in all four patients. As with meniscus repair, there was no significant change in the immunoregulatory capacity of MSCs with increasing passages (Figure 4I).

[0122] Taken together, our findings of meniscal trophic repair and inhibition of T-cell proliferation indicate that the protective and reparative effects of MSCs are fundamental features of CD105+ve, CD90+ve, CD34-ve, CD45-ve MSCs and are retained even after extensive expansion in vitro.

[0123] Transcriptomic and proteomic data reveal significant differences across passages that may be associated with loss of pluripotency We prepared mRNA from undifferentiated MSCs from all four patients at each passage. Based on growth and differentiation characteristics, we selected mRNA from passages P1, P5, P10, and P15 for gene array comparison. At each of these passages, we also collected conditioned medium for proteomic comparison. Genomic and proteomic data were combined and analyzed for patterns of changes in associated genes and proteins. The methodological approach to transcriptomics and proteomic analysis is illustrated in Figure 5A. The data structure was identified by principal component analysis (PCA). The score plot of the first two principal components is shown in Figure 5B. The changes observed across passages are explained by PC2, while PC1 captures considerable inter-patient variability, which can be stratified into two groups (shown by blue and orange ellipses). These two groups of patients also show differences in osteogenic potential (see above). To identify genes and proteins that change uniquely across passages, independent of this patient variability, we performed a two-way ANOVA for each variable, as described in the Methods section. Figure 5C shows the significant variables from this test. Only 338 genes and proteins are uniquely significant across passages, independent of patient-to-patient variation or any associated replicates. A PCA was calculated using these 338 genes and proteins. The first two principal components are shown in Figure 5D, along with a heatmap of the 338 selected variables. As expected, these genes and proteins showed very clear separation across all patient samples with passage, with a subset showing a decrease in their expression / abundance across passages (top half of the heatmap), while others show an increase in their expression / abundance (bottom half of the heatmap).

[0124] For each of these 338 significant proteins and genes, we calculated the fold change per passage and analyzed these data using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc., https: / / www.qiagenbioinformatics.com / products / ingenuity-pathway-analysis). Core analysis of IPA overlaid with the software's global molecular network identified numerous canonical pathways, functions, and upstream regulators that were significantly over-represented in this list and therefore associated with the loss of cellular pluripotency. The largest and most significant change was in the FOXM1 gene pathway, a master regulator of the cell cycle. There was clear evidence from the transcriptomics data for downregulation of the FOXM1 gene itself (Figure 6A), and IPA predicted that six of seven of its downstream effectors would also be downregulated (Figure 6B). Other upstream regulators predicted to be inactivated were the prostaglandin receptor PTGER2 and members of the VEGF family, while upstream regulators also predicted to be actively activated over passages included the growth regulator NUPR and the cytoskeleton regulator MYOC. Our quantitative transcriptomics analysis and associated IPA predictions of changes in the downstream effectors of these regulators are shown in Figure S5, although neither the changes nor the predicted downstream effects were as clear as for FOXM1.

[0125] Regulators of cell migration and wound healing may be related to the trophic properties of MSCs As shown in Figure 4, the trophic repair capacity of MSCs remains unchanged with increasing passage. To further explore this phenomenon, we hypothesized that (a) proteins and genes involved in regulating this function should not exhibit significant changes in abundance / expression across passages, and (b) functions related to cell motility, cell migration, and wound healing may be mechanistically involved in trophic repair. To test this hypothesis, we used the IPA database to identify proteins and genes involved in the three terms listed above. We then mapped such a list to our data, extracted overlapping variables, and assessed their significance across passages. The majority of proteins and genes mapping to these search criteria were found to be unchanged across passages and expressed at constant levels throughout all four passages of our MSC cultures (Figure 6B), supporting the hypothesis that genes and proteins predicted to be required for trophic repair continue to be expressed in senescent cells when pluripotency is lost but trophic repair capacity remains high. We considered CXCL12 (also known as stromal cell-derived factor 1) to be particularly important because it was associated with all three of our search criteria ( Fig. 6C ) and was consistently highly expressed at both the gene and protein levels ( Fig. 6D and E ).

[0126] Marker genes and proteins The IPA analysis outlined above demonstrated downregulation of the FOXM1 canonical pathway with increasing passage / loss of pluripotency and sustained expression of CXCL12 and other cell migration and wound healing genes and proteins with increasing passage. However, we believe this also warrants the identification of genes and proteins that may not be part of the canonical pathway or gene / protein family but can be used as specific markers of cellular senescence in vitro. Such markers would be useful for comparing cell studies from one laboratory to another or for determining the functionality of MSC populations used for therapeutic purposes. Therefore, we analyzed gene array and protein data to identify candidate markers.

[0127] Among the significantly variable genes identified by transcriptomics analysis, there was a significant decrease in the gene for matrix metalloproteinase 13 (MMP13; collagenase 3; Figure 7A and Table S2) with increasing passage and a significant increase in the gene for insulin-like growth factor binding protein 5 (IGF-binding protein 5; Table S3) with increasing passage. There were no significant changes in other MMP genes (Table S2) or IGFBP genes (Table S3), or in any genes in the transforming growth factor family (Table S4). Because the transcriptomics data indicated that the rate of decline in MMP13 gene expression with passage closely reflected the decline in chondrogenic potential (compare Figure 7A with Figure 3), we proceeded to validate these results using quantitative PCR to more precisely determine the changes in MMP13 gene expression with increasing passage. The results confirmed a sustained decrease in MMP13 gene expression with increasing passages (Figure 7B), demonstrating that loss of MMP13 gene expression can be used to help determine the degree of senescence of MSCs upon in vitro expansion.

[0128] In the proteomic dataset, we identified proteins expressed at the highest abundance across all passages (Table S5). The most abundant protein was tissue inhibitor of metalloproteinase 1 (TIMP1; Table S5 and Figure 7C). We further validated these results using an ELISA kit assay to more precisely determine changes in TIMP-1 protein secretion with increasing passages (Figure 7D), demonstrating that TIMP-1 expression is a defining characteristic of MSCs, independent of their degree of senescence during in vitro proliferation. Furthermore, when MSCs were seeded on collagen scaffolds and cultured for 24 hours, the cells continued to secrete high levels of TIMP-1 from the cell / scaffold construct into the culture medium; however, when the constructs were frozen and thawed under conditions that reduced viability, secreted TIMP levels were substantially reduced (Figure 7E).

[0129] We concluded that MSCs that express high levels of the MMP13 gene but secrete abundant TIMP-1 could potentially be used in therapeutic processes involving either phenotypic differentiation or trophic / immunoregulatory repair. However, MSCs that express low levels of the MMP-13 gene but secrete abundant TIMP-1 could only be used in processes involving trophic / immunoregulatory repair, but they cannot be reliably used in processes involving differentiation.

[0130] Consideration We demonstrated that the loss of MSC pluripotency with extensive in vitro passaging cannot be the result of a general loss of cellular function associated with MSC aging, because two other important properties of MSCs (trophic repair and immune regulation) were not significantly reduced even after a very large number of population doublings, and a reduced differentiation potential was observed over many passages before proliferation cessation. Furthermore, while gene and protein families in various important signaling pathways show clear changes that correlate with increasing passage number and loss of differentiation, genes and proteins known to be involved in wound repair and cell motility / migration do not fluctuate significantly with increasing passage number. Downregulation of the forkhead box M1 (FOXM1) canonical pathway showed a clear correlation with passage number, indicating a potential role for FOXM1 in the maintenance and subsequent loss of pluripotency. This finding is consistent with previous studies of its biological function. It is a proto-oncogene that is a key regulator of cancer stem cell survival (Nakano, 2014; Xie et al., 2010). It is also highly expressed in pluripotent and totipotent stem cells and has been shown to be important for maintaining stem cell potential (Besharat et al., 2018; Youn et al., 2017) and inducing totipotency through reprogramming (Jeong et al., 2017). CXCL12 (also known as SDF-1) was found to correlate with all three of our search criteria related to trophic repair, and its gene and protein levels were maintained even at very late passages, indicating a potential role for CXCL12 in MSC-mediated trophic repair. This finding is consistent with previous studies demonstrating its important role in spinal cord repair (Stewart et al., 2017) and myocardial repair (Dong et al., 2012), as well as in MSC-mediated induction of enhanced neuronal survival in vitro (Chalasani et al., 2003) and trophic mediation between endothelial and tumor cells (Rao et al., 2012).In addition to these mechanistic links, we identified two functional markers associated with in vitro senescence of MSCs: the MMP13 (collagenase 3) gene, which is downregulated with increasing passage, and the TIMP-1 protein, which is the most abundant protein in the secretome at all passages. The biological relevance of these two markers, identified independently of each other (one from transcriptomics data and the other from proteomics), is particularly intriguing because TIMP-1 is an inhibitor of MMP13 as well as other metalloproteinases.

[0131] Previous studies have clearly demonstrated that MSC pluripotency declines with aging both in vitro and in vivo (Bonab et al., 2006; Choudhery et al., 2014; Ganguly et al., 2017; Muraglia et al., 2000; Stenderup et al., 2003; Yang, 2018; Yang et al., 2018). Here, our findings that chondrogenesis and osteogenesis tend to decline at higher passage numbers, while adipogenesis is preserved, are consistent with the study of Yang et al. (Yang et al., 2018). Muraglia et al. (Muraglia et al., 2000) also demonstrated a loss of pluripotency with passage, but in their experiments, osteogenesis was preserved and adipogenesis was lost early. However, they dealt with MSC clonal cell lines, whereas the study reported here and the study by Yang et al. examined the whole MSC population.

[0132] Haynesworth et al. first described the unique cytokine expression pattern of MSCs (Haynesworth et al., 1996), demonstrating reduced cytokine production after stimulating differentiation using dexamethasone. Ten years later, Caplan and Dennis coined the term "trophic action," which they defined as "chemotactic, mitogenic, and differentiation-regulating effects emanating from cells as bioactive factors that act primarily on neighboring cells and never result in differentiation of the producing cells" (Caplan and Dennis, 2006). They cited the support for hematopoietic cell growth and differentiation provided by MSCs in the bone marrow microenvironment as a classic example of this effect. They also summarized evidence of MSCs providing trophic support in various cell therapy settings, including the treatment of stroke, myocardial infarction, and meniscal cartilage regeneration. Numerous other studies have documented the importance of MSC-induced trophic repair (Caplan and Correa, 2011; Caplan and Dennis, 2006; Kuroda et al., 2011; Prockop, 2009; Tolar et al., 2010). We exploited the trophic effects of MSCs to devise a novel method for treating intact meniscal cartilage tears using stem cell / collagen scaffold implants to promote the integration of damaged tissue. Our original in vitro studies demonstrated that the method relies on cell migration from the implant to the surrounding tissue and interaction with endogenous meniscal cells (Pabbruwe et al., 2009; Pabbruwe et al., 2010b). Importantly, we found that MSCs stimulated to undergo chondrogenic differentiation with transforming growth factor-β were less potent than undifferentiated MSCs in promoting meniscal repair (Pabbruwe et al., 2010b). We have now described the use of undifferentiated MSCs seeded onto collagen scaffolds to repair meniscal injuries in a sheep preclinical model and in the first human trials ( Whitehouse et al., 2017 ).In the current study, we used our in vitro semiquantitative meniscal chondrocyte attachment assay (Pabbruwe et al., 2010b) as a model of trophic repair and obtained the striking finding that MSCs cultured for up to 30 passages retained the same trophic repair capacity as very early passage cells. These functional data were supported by our analysis of genes and proteins involved in cell motility and migration and wound healing, which showed that, unlike those associated with differentiation, there were no significant changes in their expression with increasing passage.

[0133] The immunoregulatory effects of MSCs have been extensively described and involve various mechanisms, but it is clear that inhibition of T cell proliferation is a key component of their suppressive activity (Dickinson et al., 2017; Keating, 2012; Spaggiari et al., 2007; Tolar et al., 2010; Uccelli et al., 2006; Uccelli et al., 2007). Using a combination of anti-CD3 and anti-CD28 antibodies, human T cells can be potently stimulated to proliferate (Verhagen and Wraith, 2014). Their proliferation rate can be monitored by covalently labeling intracellular molecules with fluorescent dyes, which are then diluted 50% with each cell division and tracked by FACS (Quah et al., 2007). When MSCs are added to labeled cultures, stimulated T cells suppress lymphocyte proliferation, resulting in prolonged dye accumulation (Dickinson et al., 2017). In the current study, we used this method to measure the immunoregulatory activity of MSCs at early and late passages and found no loss of potency with increasing passage. In human MSCs, the T cell suppressive mechanism has been described as involving indoleamine 2,3-dioxygenase-mediated tryptophan degradation (Meisel et al., 2004). Collectively, these results demonstrate that immunoregulation, like trophic repair, is a fundamental property of MSCs that is not lost with increasing passage.

[0134] Previous studies have highlighted the loss of MSC pluripotency with increasing passage, a deficit assumed to be associated with the progression of the cells to a terminal senescent state (Bonab et al., 2006; Muraglia et al., 2000; Yang, 2018; Yang et al., 2018). However, the results described herein demonstrate that there is no apparent loss of trophic repair or immunoregulatory capacity even after extensive in vitro passaging, indicating that MSCs remain fully functional in this regard even just a few days before ceasing proliferation. We therefore conclude that a hierarchy of measurable MSC functions exists, with trophic repair and immunoregulation being central properties, whereas pluripotency is a transient property in vitro.

[0135] The term "mesenchymal stem cells" was coined by Caplan (Caplan, 1991), who described MSCs as "drugstores of injury" (Caplan and Correa, 2011), and more recently proposed changing their name to "medicinal signaling cells" (Caplan, 2017). Other studies have questioned the definition of MSCs as stem cells due to the lack of rigorous and conclusive biological evidence (Bianco et al., 2008;Javazon et al., 2004;Keating, 2012;Kuroda et al., 2011;Prockop, 2009), and all such studies have called for further experimental data before reaching a nomenclature conclusion. Others have been even more forthright in their conclusion that MSCs are not stem cells and have called for an immediate change in nomenclature to avoid the over-marketing of MSCs as a "miracle cure" (Caulfield et al., 2016; Sipp et al., 2017; Sipp et al., 2018). Prockop emphasized that the true nature of a stem cell should not be determined by its state at a single time point (Prockop, 2009). His insightful review provides useful context for the data reported herein. In this study, we examined the in vitro differentiation and trophic behavior of MSCs over many passages and thus concluded that while the properties of pluripotency are relatively transient, the trophic actions of such cells are apparently persistent from start to finish, until the time of growth arrest.

[0136] In conclusion, our findings highlight the hierarchical importance of trophic repair to pluripotency and provide further evidence supporting the definition of MSCs as repair cells rather than mesenchymal stem cells.

[0137] STAR method Reagent contact and resource sharing Further requests for reagents can be managed and accommodated by the lead contact, Anthony Hollander (A.Hollander@Liverpool.ac.uk).

[0138] Experimental model and subject details Isolation and expansion of human marrow-derived MSCs for in vitro testing The model used as the basis for all experiments reported herein was the long-term culture of human bone marrow-derived MSCs. Bone marrow plugs were harvested from the femoral heads of patients undergoing total hip replacement. All patients provided informed consent, and studies were performed in accordance with local ethical guidelines (North Bristol NHS Trust Research Ethics Committee). Patient details can be found in Table S1. Cells were suspended in stem cell growth medium consisting of low-glucose Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (v / v) fetal bovine serum (FBS, Thermo Scientific Hyclone, Loughborough, UK), 1% (v / v) Glutamax (Sigma), and 1% (v / v) penicillin / streptomycin (Sigma). The serum batch was selected to promote MSC growth and differentiation (Kafienah et al., 2007a). The medium was also supplemented with 10 ng / ml FGF-2 (Peprotech). This growth factor has previously been shown to enhance MSC proliferation rates in vitro (Bianchi et al., 2003; Solchaga et al., 2005), maintain MSCs as undifferentiated cells during proliferation (Kafienah et al., 2006; Martin et al., 1999), and enhance chondrogenic differentiation when FGF-2-expanded MSCs are subsequently exposed to differentiation conditions (Bianchi et al., 2003; Solchaga et al., 2005). Cell suspensions were separated from any bone in the samples by repeated washing with medium. Cells were centrifuged at 500 g for 5 minutes, and the supernatant / fat was removed. The resulting cell pellet was resuspended in medium and then plated over 1 cm. 2 1.5-2.0 x 10 per 5Nucleated cells were seeded at a seeding density of 1000. These flasks were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After 4 days, the first medium change was performed, followed by changes every other day until the adherent cells reached 90% confluence and were ready for passage.

[0139] Learn more about how Cell passaging and calculation of population doubling and doubling time At the end of each passage, MSCs were harvested, pooled, and counted using 0.25% trypsin-EDTA (Invitrogen) and then separated into different centrifuge tubes for reseeding and further growth, immediate use in measuring the percent meniscal cartilage attachment, storage in liquid nitrogen for subsequent use in differentiation protocols, and genomic and proteomic analyses. Cells for each patient were continuously passaged without freezing until growth arrest, defined as no detectable increase in cell number between passages (see Table S1). At each passage, the total number of MSCs harvested was determined. The first cell harvest after seeding with live bone marrow was interpreted as passage 0. The number of cells replated at the beginning of passage 1 was used as the baseline for calculating the first population doubling at the end of passage 1. Downstream analyses of MSCs were performed from passage 1 onward.

[0140] Population doubling (PD) numbers were calculated using the following formula: PD = [log(number of MSCs recovered)-log(number of MSCs seeded)] / log(2)] The PD of each passage was calculated and added to the PD of the previous passage to generate cumulative PD data for each passage.

[0141] Population doubling time (PDT) was calculated for each passage using the following formula: PDT = t × log(2) / log(recovered cells / seeded cells) (t = time from cell seeding to cell harvest)

[0142] Detection of cell surface phenotypic markers MSCs (100,000 cells from each patient at passages 1, 5, 10, and 15) were suspended in a 1:500 dilution of the live / dead cell dye Zombie (Biolegend) and incubated for 20 minutes in the dark. Nonspecific antigens were then blocked by incubating the cells in 1% (wt / vol) BSA (Sigma-Aldrich), 5% (vol / vol) FCS (Sigma-Aldrich), and 10% (vol / vol) human serum (Sigma-Aldrich) at room temperature for 1 hour. The cells were washed by centrifugation with three volumes of PBS, and the cell pellet was suspended in 100 μl of primary antibody solution containing 20–100 μg / ml of antibody in blocking solution. All primary antibodies were fluorescently labeled mouse anti-human IgG. Anti-CD105 fluorescein isothiocyanate (FITC), anti-CD90 phycoerythrin (PE), and anti-CD45-PE were obtained from R&D Systems, anti-CD34-FITC from BD Bioscience, and IgG1-FITC and IgG1-PE isotype controls from R&D Systems. After a 40-minute incubation at 4°C, cells were washed, suspended in 1 ml of PBS, and analyzed on a Canto flow cytometer (BD FACSCanto II) after exclusion of nonviable cells. Data were analyzed using FlowJo (Treestar). Positive expression was defined as a fluorescence level greater than 95% of the corresponding isotype-matched control antibody.

[0143] cartilage formation Cartilage tissue modification The chondrogenic potential of MSCs from each passage was assessed by performing 3D cartilage tissue engineering as previously described (Kafienah et al., 2007a). Briefly, 300,000 cells were dropwise placed onto 5 mm diameter × 2 mm thick polyglycolic acid (PGA) scaffold discs (Biomedical Structures, Warwick, RI, USA) pre-coated with 100 μg / ml fibronectin (Sigma). The constructs were then cultured in chondrogenic differentiation medium consisting of DMEM supplemented with 4500 mg / L glucose (Sigma-Aldrich), 100 nM dexamethasone, 80 μM ascorbic acid 2-phosphate, 1 mM sodium pyruvate, 1% (v / v) penicillin / streptomycin (all from Sigma-Aldrich), 1% insulin-transferrin-selenium-G (ITS), and 2 mM Glutamax-I (both from Invitrogen) supplemented with 10 ng / ml transforming growth factor-β3 (TGF-β3; R&D Systems). After 7 days, the medium was further supplemented with 10 μg / ml bovine pancreatic insulin (Sigma-Aldrich) until the end of culture. The constructs were incubated at 37°C on a rotating platform for a total of 35 days, with the medium changed every 3 days.

[0144] biochemical analysis Cartilage constructs were lyophilized and weighed at the end of the 35-day tissue modification period. The extracellular matrix was fully solubilized by overnight digestion with 2 mg / ml bovine pancreatic trypsin (Sigma-Aldrich), which was then boiled for 15 minutes to inhibit the enzyme's action (Dickinson et al., 2005). To obtain the dry weight of the extracellular matrix in the construct, the remaining undegraded scaffold material was lyophilized, weighed, and subtracted from the original dry weight. The amount of proteoglycan in the digest was measured as sulfated glycosaminoglycans (GAGs) using a dimethylmethylene blue (Sigma-Aldrich) colorimetric assay (Handley and Buttle, 1995).

[0145] Osteogenesis and adipogenesis Whole MSC populations or MSC clones were grown in monolayers to 50–70% confluence before osteogenic differentiation or 100% confluence before adipogenic differentiation. In both cases, cells were then cultured in α-MEM (Invitrogen) basal medium containing 10% FBS, 1% (v / v) penicillin / streptomycin, and 2 mM Glutamax-I. Cells stimulated to differentiate were cultured for 21 days in basal medium containing either osteogenic or adipogenic supplements (both from R&D Systems). After osteogenic differentiation, cells were fixed in 70% ethanol and stained with 40 mM Alizarin Red S (Sigma-Aldrich) at pH 4.1 for 5 minutes. After adipogenic differentiation, cells were fixed in 4% paraformaldehyde and stained with 0.3% Oil Red O (Sigma-Aldrich) for 30 minutes. The degree of differentiation was scored under blinded conditions and classified as − (no staining), +, ++, or +++ according to the increase in the site and number of mineralized deposits after osteogenic differentiation (see Fig. S1 ) and the increase in the number of lipid droplets after adipogenic differentiation (see Fig. S3 ).

[0146] Meniscus repair Preparation of ovine meniscal cartilage Sheep legs were purchased from Edge & Son Butchers, Wirral, Merseyside, and meniscal cartilage was removed under sterile conditions. Meniscal cartilage cylinders (5.0 mm diameter and 3.0 mm thickness) were collected from avascular (white zone) ovine menisci using a skin biopsy punch. These were rinsed and incubated for 20 minutes in phosphate-buffered saline (PBS; Invitrogen Ltd, Paisley, UK) containing 10% (v / v) penicillin / streptomycin (Sigma-Aldrich) and 1% (v / v) 250 μg / ml amphotericin B (Sigma-Aldrich). The viability of the fibrocartilage discs was maintained by culturing them in a basal medium consisting of low-glucose DMEM supplemented with 10 mM Hepes buffer (Sigma), 1% (v / v) penicillin / streptomycin, non-essential amino acids (NEAA; Sigma), 1% (v / v) Glutamax, and 10% amphotericin B at 37°C in a 5% CO environment. Explants were used in attachment experiments within 3 days of culturing.

[0147] cell seeding Collagen scaffolds (Ultrafoam collagen sponge; Bard, UK, www.barduk.com) were cut into 6 mm diameter discs and inoculated with 1 × 10 MSCs. 6 cells / cm 2 The scaffolds were seeded at a density of 1000 μg / cm². The suspension was applied dropwise onto the scaffolds placed in ultra-low attachment wells of a 24-well plate (Corning®, Acton, USA). After 4 hours, 1.5 ml of growth medium containing 10 ng / ml FGF-2 was added and changed daily. The seeded scaffolds were incubated at 37°C in an orbital shaker at 50 rpm for 48 hours.

[0148] Construction and culture of constructs Sandwich constructs of two ovine meniscal cartilage discs sandwiching a seeded scaffold were constructed using skin clips as previously described (Whitehouse et al., 2017). They were cultured in vitro in ultra-low attachment 6-well plates in growth medium with 10 ng / mL FGF-2 for 7 days, followed by 33 days in binding medium consisting of high-glucose DMEM supplemented with 10% (v / v) FBS, 1% (v / v) Glutamax, 1% (v / v) penicillin / streptomycin, insulin, and ascorbic acid-6-phosphate (50 μg / ml; Sigma). The medium was replenished twice weekly. Constructs were incubated at 37°C on a rotating platform throughout the culture period. At the end of the culture period, constructs were prepared for histological analysis by fixation in 10% (v / v) neutral-buffered formalin.

[0149] Histomorphometric analysis Histomorphometry was performed using methods we developed and characterized in previous studies (Pabbruwe et al., 2009; Pabbruwe et al., 2010a; Whitehouse et al., 2017). Fixed constructs were dehydrated and embedded in paraffin. Samples were cut into 4 μm sections and stained with hematoxylin and eosin (H&E) for morphological details. All tissue sections were scanned using a Leica Aperio slide scanner, and histomorphometric analysis was performed under blinded conditions using ImageScope software (Leica). Two perpendicular sections, one at the edge and one in the center of each construct, were used. For each section, the total length of the implant / meniscus interface and the length of any bonded sites at the interface were measured. The repair index was then determined as follows: % Bonding = Bonded interface length / total interface length x 100

[0150] immunosuppression Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteer donor blood samples. All patients provided informed consent, and the study was performed in accordance with local ethical guidelines (University of Liverpool Research Ethics subcommittee for Physical Interventions). PBMCs were isolated by centrifugation of blood over 1.077 g / mL Ficoll-Paque (GE Healthcare LifeSciences, Little Chalfont, UK) and cultured in RPMI-1640 containing L-glutamine and supplemented with 10% human AB serum and 1% (v / v) penicillin / streptomycin. PBMCs were stained with CellTrace™ Violet (ThermoFisher Scientific) to monitor T cell proliferation. The labeled PBMCs were then stimulated with 3.75 μg / ml anti-human CD3 (HIT3a) and 2 μg / ml anti-human CD28 (CD28.2) (both from Fisher Scientific Affymetrix eBioscience, Cheshire, UK) and cocultured for 72 hours with MSCs from four individual donors at passages 1, 5, 10, and 15. The T cell proliferation profile of each population was analyzed by flow cytometry after exclusion of nonviable cells stained with 7-aminoactinomycin D (7-AAD; BD Biosciences).

[0151] quantitative PCR Real-time quantitative PCR (RT-qPCR) was performed for MMP13 mRNA and the housekeeping gene Tata-binding protein (TBP) using the CellsDirect™ One-Step qRT-PCR Kit (ThermoFisher), which performed reverse transcription and PCR amplification in the same reaction tube. Primers specific for MMP13 (Hs00942584_m1) and TBP (Hs00427621_m1) were purchased from ThermoFisher TaqMan®. The reaction was initiated by synthesizing cDNA at 50°C for 15 minutes, followed by 95°C for 2 minutes to denature the RNA-cDNA hybrid and inactivate the reverse transcriptase. The thermal cycling program consisted of 50°C for 15 minutes, 95°C for 2 minutes, and 40 two-step cycles of 95°C for 10 seconds and 60°C for 30 seconds. MMP13 expression relative to TBP was determined for each MSC sample at each of the four time points, and results were normalized to time (passage 2), which was interpreted as a fold expression of 1.0.

[0152] TIMP-1 ELISA TIMP-1 protein in the secretome of MSCs was measured using the Quantikine® ELISA Kit for human TIMP-1 (R&D Systems). MSCs were seeded in 6-well plates at 2.25 million cells / well (3 replicates) and cultured in 2 ml of DMEM for 24 hours. The medium was then replaced with 1 ml of phenol-free culture medium and cultured for an additional 24 hours. Secretomes were then harvested and quantified using the ELISA kit at appropriate dilutions.

[0153] Genomic and proteomic data acquisition Transcriptomics Transcriptomics was performed by the Genome Research Center on mRNA extracted from all four patients at passages P1, P5, P10, and P15. MSCs were harvested at the end of each passage and 1 × 10 6Cells were isolated and resuspended in RNAprotect Cell Reagent (Qiagen). Cells were stored at -80°C until all donors and time-point samples were collected. RNA was then extracted at selected time points using the RNeasyPlus Mini Kit (Qiagen) according to the manufacturer's instructions. The RNA concentration in the extracts was determined using a NanoDrop2000 spectrophotometer (Thermo). The extracted RNA was stored at -80°C before analysis.

[0154] Ribosomal RNA depletion was performed using the Ribo-Zero™ H / M / R Kit (Illumina), followed by RNA-Seq library preparation using the NEB NextUltraDirectional RNA Library Prep Kit (Illumina). Paired-end sequencing of the RNA-Seq libraries was performed on the Illumina HiSeq4000 platform using V4 chemistry.

[0155] Proteomics Proteomics was performed on secretomes prepared from MSCs from all four patients at passages P1, P5, P10, and P15. MSCs were harvested at the end of each passage and 1 × 10 6 Cells were isolated and resuspended in 4 mL of serum-free, phenol red-free DMEM (Sigma) supplemented with 4500 mg / L glucose, 1% (v / v) Glutamax (Sigma), 1% (v / v) P / S (Sigma), and 2 mM Glutamax-I for 24 h at 37°C. Conditioned medium collected at the end of incubation was collected from each flask and stored at -80°C before analysis.

[0156] Proteomic analysis of secretome samples was performed at the University of Liverpool's Proteome Research Centre. Protein solutions were concentrated by sequentially adding 1 mL aliquots of each sample to 10 μL of Strataclean beads (Stratagene®, Hycor Biomedical Ltd., Edinburgh, UK). After each aliquot, the sample was vortexed for 1 minute and centrifuged at 2,000 × g for 2 minutes, and the protein-depleted supernatant was removed. After the final aliquot was added, the beads were washed twice with 1 mL of 25 mM ambic and then digested. For on-bead digestion, the beads were resuspended in 80 μL of 25 mM ambic and 5 μL of 1% (w / v) Rapigest (Waters Limited, Hertfordshire, UK) in 25 mM ambic was added. The samples were heated at 80°C for 10 min, then reduced by adding 5 μL of dithiothreitol (DTT, 9.2 mg / mL in 25 mM ambic) and heated at 60°C for 10 min. After cooling, 5 μL of iodoacetamide (33 mg / mL in 25 mM ambic) was added, and the samples were incubated at RT for 30 min in the dark. 1 μg of porcine trypsin (sequencing grade, Promega) was added, and the samples were incubated overnight at 37°C on a rotating mixer. The digest was acidified by adding 1 μL of trifluoroacetic acid (TFA) and incubated at 37°C for 45 min. The samples were then centrifuged at 17,200 × g for 30 min, and the supernatant was transferred to a 0.5 mL low-binding tube. These were centrifuged for an additional 30 min, and 10 μL was transferred to a total recovery vial for LC-MS analysis.

[0157] Data-dependent LC-MSMS analysis was performed on a QExactive HF quadrupole-Orbitrap mass spectrometer coupled to a Dionex Ultimate 3000 RSLC nano liquid chromatograph (Hemel Hempstead, UK). Sample digests (1–2 μL) were loaded onto a trapping column (AcclaimPepMap100C18, 75 μm × 2 cm, 3 μm packing, 100 Å) in 12 μL increments using a loading buffer of 0.1% (v / v) TFA, 2% (v / v) acetonitrile in water. -1 The trapping column was then loaded for 7 min at a flow rate of 0.05 mL / min. The trapping column was then aligned with an analytical column (EASY-Spray PepMap RSLC C18, 75 μm × 50 cm, 2 μm packing material, 100 Å) and eluted with 96.2% A (0.1% [v / v] formic acid): 3.8% B (0.1% [v / v] formic acid in water: acetonitrile [80:20] [v / v]) vs. 50% A: 50% B for 300 nL min. -1Peptides were eluted using a linear gradient over 90 min at a flow rate of 100 s, followed by a 5-min wash with 1% A:99% B, and the column was re-equilibrated to the starting conditions. The column was maintained at 40 °C, and the eluate was directly introduced into a coupled nanoelectrospray ion source operated in positive ion mode. The mass spectrometer was operated in DDA mode, and survey scans from m / z 350 to 2000 were acquired at a mass resolution of 60,000 (FWHM) at m / z 200. The maximum injection time was 100 ms, and the automatic gain control was set to 3e6. Sixteen of the most intense precursor ions, with charge states ranging from 2+ to 5+, were selected for MS / MS analysis with an isolation window of 2 m / z units. The maximum injection time was 45 ms, and the automatic gain control was set to 1e5. Peptide fragmentation was performed by high-energy collisional dissociation using a normalized collision energy of 30%. Dynamic exclusion of m / z values ​​was used with an exclusion time of 20 s to prevent repeated fragmentation of the same peptide. Raw mass spectrometry data files were imported into Progenesis QI for Proteomics v.2.0 software (Waters Ltd, Newcastle upon Tyne, UK) for alignment and peak detection. An aggregate file containing all peaks from all runs in the experiment was created to ensure there were no missing values. Data were filtered to remove charges of +1 and ≥ +8. msms fragmentation data were searched against the UniProt human-reviewed database using Mascot v.2.4.1 software (Matrix Science, London, UK). The precursor ion mass tolerance was set to 10 ppm, and the product ion tolerance was set to 0.01 Da. Methionine oxidation was selected as the dynamic modification, and carbamidomethylcysteine ​​as the fixed modification. One missed cleavage was allowed. The Mascot search yielded 2,594 proteins with a 2.17% FDR (percent similarity over homology). The FDR was set to 1%, and the 2,366 proteins were exported as an .xml file (FDR type: distinct psm) and imported into Progenesis, where peptides were assigned to proteins.Protein quantification was based on the average individual abundance for each protein per donor at each passage and comparison of differentially expressed proteins across the four passages.

[0158] Quantitative and statistical analysis Bioinformatics Data processing, integration, and analysis were performed by the Computational Biology Facility at the University of Liverpool. RNA-seq data were obtained as described above. Raw Fastq files were trimmed for the presence of Illumina adapter sequences using Cutadapt version 1.2.1, option -O3. Reads were further trimmed using Sickle version 1.200 with a minimum window quality score of 20. After trimming, reads shorter than 10 base pairs were removed. Sequence quality metrics were assessed using FastQC version 0.11.4. No samples were removed. Sequence data were aligned to the NCBI human genome database GRCh38 using Bowtie2 version 1.1.2 with recommended parameters (Langdon, 2015). Gene-level count data were generated from Bowtie2 alignments using htseq-count version 0.9.0. Rlog-transformed count data were generated using RlibraryDESeq2. These were filtered to remove genes with an average count less than 1. Statistical analyses were performed with R version 3.4.4, and graphical presentations were performed using the R package ggplot2.

[0159] Normalized proteomic and transcriptomic data were integrated, and preliminary exploratory analysis revealed relevant heterogeneity between patients. To distinguish between changes associated with patient heterogeneity and those associated with passage, variables differentially expressed across passages were calculated using a two-way ANOVA to account for patient variability as a confounding factor. This was followed by correction for multiple testing using the Benjamin-Hochberg method. Uniquely significant variables across passages (38 of 338 were proteins and 300 were genes) were selected for further analysis using the Ingenuity Pathway Database (IPA, Qiagen, Analysis 2015).

[0160] The binary logarithmic fold changes of 338 significant variables were calculated for one passage and used in IPA (Qiagen). Following the manual, the analysis predicted possible upstream regulators, potentially affecting pathways, and a number of significantly enriched functions and conditions. (Database availability date: December 9, 2018) We also used IPA to download knowledge conditions involved in selected key processes: (A) mesenchymal stem cell migration, (B) mesenchymal stem cell motility, and (C) wound healing. All such variables were mapped to our experimental data. We then assessed the number of variables within these conditions that changed over passages to contextualize the biological findings.

[0161] Principal component analysis was performed using the prcomp function in the statistical software R by mean centering and adjusting data by singular value decomposition.

[0162] statistics Differentiation variables (TE, GAG, and osteogenic and adipogenic scores) were compared across passages by calculating nonparametric Spearman correlations using the cor.test function in the stats package in R. Osteogenic and adipogenic potential were measured using semiquantitative data based on image analysis. These were converted to integers ranging from 0 to 1 for calculations.

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Table 1

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Table 2

[0165]

Table 3

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Table 4

[0167]

Table 5

Claims

A pharmaceutical composition comprising a cell population enriched with an MSC-derived cell population, wherein the MSC-derived cell population is ・CD90 ＋ 、CD105 ＋ 、CD45 － and; ・ TIMP-1 secretion ＋ (with high TIMP-1 secretion), where high TIMP-1 secretion means that TIMP-1 is secreted at an average of 100 ng / ml or more in 24 hours from cells seeded at a density of 2.25 million cells / well initially; ・ MMP13 gene expression － (low MMP13 gene expression), where low MMP13 gene expression means that the average level of the relative expression of MMP13 with respect to a housekeeping gene is less than or equal to half of the relative expression by the newly isolated MSC; and • having trophic activity, the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, which is intended for use in tissue repair.

3. The pharmaceutical composition according to claim 1 or 2, which is intended for use in the treatment of a tissue selected from the group consisting of cartilage, cardiovascular tissue and bone.

4. The pharmaceutical composition according to claim 3, which is intended for use in the treatment of a tissue selected from the group consisting of cartilage and bone.

5. The pharmaceutical composition according to claim 2 or 3, which is intended for use in the treatment of a condition selected from the group consisting of osteoarthritis, myocardial infarction, meniscus cartilage injury (e.g., meniscus tear), ligament injury (e.g., ligament tear), skin injury and soft tissue injury.

6. The pharmaceutical composition according to any one of claims 2 to 5, which is intended for use in tissue repair by stimulating trophic repair.

7. The pharmaceutical composition according to any one of claims 1 to 6, which is intended for use in the treatment of a disease selected from the group consisting of blood diseases, graft-versus-host disease and inflammatory diseases.

8. The pharmaceutical composition according to claim 7, which is intended for use in the treatment of a disease by immunosuppressing the immune response.

9. The pharmaceutical composition according to any one of claims 1 to 8, further comprising a pharmaceutically acceptable excipient.

10. The cell population is CD90 ＋ , CD105 ＋ , CD45 － , and is TIMP-1 secretion ＋ , and MMP13 gene expression － The pharmaceutical composition according to any one of claims 1 to 9, comprising at least 15% of cells that are

11. The cell population is CD90 ＋ , CD105 ＋ , CD45 － and does not contain cells with high TIMP-1 secretion and high MMP13 gene expression, where high TIMP-1 secretion refers to the secretion of at least 100 ng / ml of TIMP-1 on average in 24 hours from cells seeded at a density of 2.25 million cells / well initially, the pharmaceutical composition according to any one of claims 1 to 9.

12. At least 70% of the cells contained in the cell population enriched with the MSC-derived cell population are • CD90 +, CD105 +, CD45 -; • TIMP-1 secreting +; and • MMP13 gene expression -. The pharmaceutical composition according to any one of claims 1 to 10.

13. At least 80% of the cells contained in the cell population enriched with the MSC-derived cell population are CD90 positive and CD105 positive, and 10% or less are CD45 positive. The pharmaceutical composition according to any one of claims 1 to 9.

14. • Culturing MSCs to generate MSC-derived cells, • The MSC-derived cells are: - CD90 ＋ 、CD105 ＋ 、CD45 － ; and ・ TIMP-1 secretion ＋ (with high TIMP-1 secretion), where high TIMP-1 secretion means that TIMP-1 is secreted at an average of 100 ng / ml or more in 24 hours from cells seeded at a density of 2.25 million cells / well initially; and ・ MMP13 gene expression － (where MMP13 gene expression is low), and here, low MMP13 gene expression means that the average level of the relative expression of MMP13 with respect to a housekeeping gene is less than or equal to half of the relative expression by the newly isolated MSC. Concentrating into a population, and • Formulating the cells for use as a pharmaceutical composition A method for preparing the pharmaceutical composition according to any one of claims 1 to 3 or 5 to 8, comprising.

15. - Evaluating the levels of MMP13 gene expression, CD90, CD105, and CD45 expression, and TIMP-1 protein secretion by a cell population, and - If the cell population shows low-level MMP13 gene expression and high-level TIMP-1 protein secretion and is CD90 +, CD105 +, CD45 −, selecting this as a cell population for use in therapeutic applications that do not require phenotypic differentiation ability, where low-level MMP13 gene expression means that the average level of the relative expression of MMP13 to a housekeeping gene is less than or equal to half of the relative expression by freshly isolated MSCs, and high-level TIMP-1 secretion means that on average, 100 ng / ml or more of TIMP-1 is secreted in 24 hours from cells seeded at a density of 2.25 million cells / well initially, A method for selecting an MSC-derived cell population for therapeutic use, comprising the above.

16. The method according to claim 15, further comprising selecting this as a cell population for use in therapeutic applications that require phenotypic differentiation ability if the cell population shows both high-level MMP13 gene expression and TIMP-1 protein secretion.

17. The method according to claim 16, wherein the therapeutic application that requires phenotypic differentiation ability is selected from the group consisting of in vitro tissue modification and transplantation for generating new tissue by differentiation in vivo.

18. The method according to any one of claims 15 to 17, wherein the therapeutic application that does not require phenotypic differentiation ability is selected from the group consisting of promoting tissue repair by trophic activity and promoting immunosuppressive therapy.

19. The method according to any one of claims 15 to 17, wherein the cell population is derived from MSCs by passage of MSCs.

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