Highly purified mesenchymal stem cells

Abstract

To provide a highly purified, homogeneous cell population of rapidly proliferating human mesenchymal stem cells, a method for sorting out highly purified and highly homogeneous RECs, and an indicator that guarantees the cell performance.SOLUTION: A cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), wherein at least one of the following characteristics (a) and (b) is satisfied. (a) The variation coefficient of forward scattered light in flow cytometry is 35% or less. (b) The average cell size is 20 μm or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a highly purified cell population of human mesenchymal stem cells that is homogeneous and rapidly proliferating. [Background technology]

[0002] Mesenchymal stem cells (MSCs) are one of the somatic stem cells that have been widely used in clinical applications, following hematopoietic stem cells, because there are few ethical issues associated with cell collection and they have the ability to differentiate into various forms such as bone, cartilage, and fat. Because MSCs can be isolated using relatively simple procedures, they are widely used as biomaterials, primarily by inducing differentiation into cartilage or bone in vitro and then transplanting them into localized areas. Furthermore, when moving forward with clinical application, it will be essential for commercialization to be able to produce the necessary amount of cells that maintain a certain level of function.

[0003] However, if there is variation in the properties of the starting material (such as human bone marrow fluid), that is, if there are differences in the properties derived from the donor, the properties of the cell preparation as a product will be significantly affected. For this reason, it is important to obtain mesenchymal stem cells with high purity and little variation. The present inventors have isolated LNGFR- and Thy-1-positive cells from bone marrow aspirates by flow cytometry, obtained rapidly expanding clones (RECs), and established a purification and isolation method that can eliminate differences in the proliferation potential of donor-derived MSCs (Japanese Patent No. 6463029, WO2016 / 017795, Mabuchi Y. et al., Stem Cell Reports 1(2): 152-165, 2013). In this method, RECs are isolated and selected using, for example, Ror2 expression as an indicator.

[0004] A purified mesenchymal stem cell composition and a method for purifying a mesenchymal stem cell composition are also known (Patent No. 6025329), and this method purifies mesenchymal stem cells with a diameter of 150 μm or less. However, REC clones still exhibit variations in differentiation and proliferation potential. Even RECs with excellent proliferation potential inevitably deteriorate with passage, and effective quality control methods are needed. Furthermore, even when Ror2 expression is used as an indicator, there is still room for improvement in the correlation with proliferation potential. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6463029 [Patent Document 2] International Publication No. 2016 / 017795 Brochure [Patent Document 3] Patent No. 6025329 [Non-patent literature]

[0006] [Non-Patent Document 1] Mabuchi Y Morikawa S, Harada S; Niibe K, Suzuki S, Renault-Mihara F, Houlihan DD, Akazawa C, Okano H, Matsuzaki Y. LNGFR+ Thy-1+ Vcam-1hi+ cells reveal functionally distinct subpopulations in mesenchymal stem cells. Stem Cell Reports 1(2): 152-165, 2013. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, in the present invention, it was necessary to establish a method for selecting highly pure and highly uniform RECs and an index that guarantees cell performance. [Means for solving the problem]

[0008] As a result of investigations conducted by the inventors to solve the above-mentioned problems, they obtained cells that were positive for both LNGFR and Thy-1 from human bone marrow fluid, produced numerous REC clones from single cells, and repeatedly measured their differentiation and proliferation abilities. As a result, they found that the size and uniformity of the cells were more strongly correlated with the differentiation and proliferation abilities of each clone.

[0009] The inventors also analyzed the size and variation of the cells constituting each clone using forward scatter (FSC) as an indicator in flow cytometry, and found that the smaller the size and the smaller the FSC CV value, the better the proliferation and differentiation ability.They also found that by selecting each clone based on this indicator, it is possible to produce cells with a certain range of functions.

[0010] That is, the present invention is as follows. (1) A cell population of fast-proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) The average cell size is 20 μm or less. (2) The cell population according to (1), having a coefficient of variation of 30% or less. (3) The cell population according to (1) or (2), wherein the average cell size is 14 μm to 18 μm. (4) A method for evaluating the quality of a cell population of a fast-proliferating mesenchymal stem cell clone that is positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), wherein a cell population that satisfies at least one of the following characteristics (a) and (b) is determined to be of high quality: (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) The average cell size is 20 μm or less. (5) The method according to (4), wherein the coefficient of variation is 30% or less. (6) The method according to (4) or (5), wherein the average cell size is 14 μm to 18 μm. (7) A method for selecting a clinically applicable cell population from a cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), wherein a cell population that satisfies at least one of the following characteristics (a) and (b) is selected as a therapeutic mesenchymal stem cell population: (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) The average cell size is 20 μm or less. (8) The method according to (7), wherein the coefficient of variation is 30% or less. (9) The method according to (7) or (8), wherein the average cell size is 14 μm to 18 μm. [Effects of the Invention]

[0011] The present invention has enabled the selection of mesenchymal stem cell populations with uniform cell size and excellent proliferation and differentiation capabilities. The selected cell populations are of clinically applicable quality and are expected to be used in the treatment of myocardial infarction, cerebral infarction, spinal cord injury, bone or cartilage formation-related diseases, graft-versus-host disease (GVHD), liver cirrhosis, epidermolysis bullosa, and lower limb ischemia. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing an outline of a method for selecting RECs in the present invention. [Figure 2] FIG. 1 shows the results of measuring the CV value of forward scattered light (FSC) by flow cytometry. [Figure 3] FIG. 1 shows the results of evaluating cell proliferation ability and adipogenesis ability. [Figure 4] FIG. 1 shows the results of comprehensive analysis of REC clones. [Figure 5] FIG. 10 is a diagram showing a summary of the analysis results of the present invention. [Figure 6] FIG. 1 shows the results of comprehensive analysis of REC clones. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. 1. Overview In previous studies, the present inventors succeeded in isolating rapidly expanding cell clones (RECs) from LNGFR (CD271)-positive mesenchymal stem cells (CD271+ cells) or LNGFR (CD271) and Thy-1 (CD90)-copositive mesenchymal stem cells (CD271+CD90+ cells). These RECs can reach confluence in two weeks when seeded individually in a 96-well plate and cultured. Compared to mesenchymal stem cells obtained by conventional methods, they have over 1,000 times the proliferation, differentiation, and migration capabilities. Because they retain their migration ability, they can be administered intravenously, and are expected to be useful for treating serious systemic diseases such as osteochondrogenesis imperfecta.

[0014] However, even among such REC clones, variations in differentiation and proliferation ability are sometimes observed. The present invention provides cell clones with little variation and a method for selecting such cell clones.

[0015] The cell population containing the cell clone of the present invention is a population of mesenchymal stem cell clones that are co-positive for LNGFR (CD271) and Thy-1 (CD90) and rapidly proliferate, and satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) The average cell size is 20 μm or less.

[0016] 2. Human mesenchymal stem cell enrichment method In the present invention, mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90) can be obtained according to the method described in WO2009 / 31678, for example. The outline of the method is as follows.

[0017] First, mesenchymal stem cells are highly enriched by selecting a cell fraction positive for LNGFR (CD271) (CD271+) or co-positive for CD271 and CD90 (CD271+CD90+) from a cell population containing human mesenchymal stem cells. If the cell population containing human mesenchymal stem cells also contains hematopoietic cells, a step of selecting CD45 and CD235a co-negative (CD45-CD235a-) cells may be added to select non-hematopoietic cells.

[0018] Cell populations containing mesenchymal stem cells can be prepared by flow cytometry or affinity chromatography. The material from which this cell population can be obtained is not particularly limited, but examples include bone marrow and peripheral blood (including peripheral blood after administration of G-CSF), etc. The bone marrow may be from the spine, sternum, ilium, etc.

[0019] During cell preparation, if the material is in the form of a cell mass containing mesenchymal stem cells, the material can be subjected to physical treatment such as pipetting or enzymatic treatment with trypsin, collagenase, etc. If the material contains red blood cells, it is preferable to hemolyze the red blood cells in advance. The cell population prepared as described above is used to select CD271+ cells or CD271+CD90+ cells.

[0020] Methods for selecting CD271+ cells or CD271+CD90+ cells include, for example, methods using antibodies. The antibodies are anti-CD271 and / or anti-CD90 antibodies, which can select CD271+ cells or CD271+CD90+ cells. When using flow cytometry for selection, live cells can be quickly selected by using anti-CD271 antibodies labeled with different fluorescent dyes such as FITC, PE, or APC, or by combining anti-CD271 and anti-CD90 antibodies. In addition to flow cytometry, CD271+CD90+ cells can also be selected using magnetic beads or affinity chromatography. Before using these methods, dead cells may be removed by reacting a cell population with a fluorescent dye (e.g., PI) that stains dead cells and removing the fluorescently stained cells.

[0021] 3.REC cell enrichment Next, the selected LNGFR-positive cells or LNGFR and Thy1 co-positive cells are cultured as single cells (clones), and by selecting the lot with the fastest proliferation rate, highly purified human mesenchymal stem cells (REC: Rapidly Expanding Clone) with excellent proliferation, differentiation, and migration capabilities are obtained. Figure 1 illustrates the REC isolation process using the monoclonal culture method.

[0022] Mononuclear cells were prepared from human bone marrow or adipose tissue / placental chorion. The bone marrow mononuclear cells were stained with anti-LNGFR alone or with anti-LNGFR and anti-Thy1. Next, LNGFR-positive cells or LNGFR-positive and Thy1-positive cells were clonally sorted into 96-well culture plates using flow cytometry (cell sorter). That is, one cell was seeded per well. After two weeks of single-cell culture, the culture plates were photographed under a microscope, and wells that had reached confluence or semi-confluence were selected. The cells contained in these wells were designated as RECs.

[0023] Here, "fast proliferation" and "high-speed proliferation" mean that when cells are seeded one cell per well of a 96-well culture plate and cultured, the culture plate has a proliferation rate such that the plate becomes confluent or semi-confluent two weeks or earlier after the start of culture (doubling time is 26±1 hours). Confluence is a state in which cultured cells cover 90% or more of the surface of the culture vessel (culture surface). Semi-confluence is a state in which cultured cells cover 70-90% of the surface of the culture vessel (culture surface). The size and type of culture equipment used can be adjusted appropriately depending on the cell proliferation rate. Moderately / slowly expanding cells, i.e., cells that do not become semi-confluent or confluent after two weeks of single cell culture, are discarded. RECs collected from each well selected as RECs are transferred to culture flasks individually and cultured until they become confluent (expansion culture). The expanded cells are then collected separately. RECs from one well constitute one lot, and these are used for the selection described below.

[0024] Here, the cells of the present invention are obtained by clonal sorting in which one cell is seeded per well, and therefore the genetic traits of the expanded cells are all the same. Therefore, in the present invention, the entire cell population may be referred to as a "clone," or each individual cell constituting the cell population may be referred to as a "clone."

[0025] In the present invention, RECs to be used for selection can be evaluated in advance using a REC marker (anti-Ror2). For example, after the expansion culture, adherent and proliferated cells are collected from all the lots, and a portion (1 to 3 × 10) of each lot is cultured. 5Approximately 100 cells are selected and single-stained with an anti-Ror2 monoclonal antibody. A technique for single-staining with an anti-Ror2 monoclonal antibody is known (WO2016 / 17795). In summary, the proportion of REC marker-positive cells in the collected cells is determined by flow cytometry analysis using the REC marker. The proportion can be determined by quantitative PCR to quantify Ror2 mRNA expression, or by manually determining the proportion using a microscope. Lots (cell populations) with a positive proportion above a certain value (e.g., 65%) are considered acceptable and can be used in the selection process described below.

[0026] 4. Selection of stem cell populations of the present invention In the present invention, by examining the cell proliferation ability, fat differentiation ability, expression level of REC-specific markers, and uniformity of cell size for each lot of REC clones and analyzing the correlation between each, it has become possible to select RECs with high purity and higher cell performance. In the present invention, the coefficient of variation (CV value) of forward scattered light and the average size of cells are used as indices for selection. Forward scatter is light scattered at a small angle in the forward direction relative to the axis of the laser beam. Forward scatter consists of scattered, diffracted, and refracted light from the cell surface, and provides information about the size of the sample.

[0027] The coefficient of variation (CV) is the standard deviation divided by the mean value, and is used to relatively evaluate the variation in data with different units, or the relationship between data and variation in relation to the mean value. In the present invention, cells with a CV value of 35% or less are selected. A cell population with a CV value of 35% or less is a cell population composed of cells of uniform size. Preferably, the CV value is 30% or less, 25% or less, or 20% or less. Furthermore, the average size of the cells in the cell population selected according to the present invention is 20 μm or less, preferably 18 μm or less, and in the range of 14 μm to 18 μm.

[0028] The present invention also provides a method for evaluating the quality of a cell population of LNGFR-positive cells or a fast-proliferating mesenchymal stem cell clone that is co-positive for LNGFR (CD271) and Thy-1 (CD90). In the present invention, a cell population that satisfies at least one of the following characteristics (a) and (b), preferably both characteristics, is determined to be of high quality. (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) The average cell size is 20 μm or less.

[0029] The cell population evaluated and selected in this manner is not limited in the number of cell clones constituting the population, and may be, for example, 0.8×10 7 ~1.2X10 7 It has about 100 cells. Furthermore, the cell population can be used clinically as a therapeutic mesenchymal stem cell population for the treatment of, for example, the following diseases, although the diseases are not limited to these. Genetic disorders (epidermolysis bullosa, hypophosphatasia, etc.) Bone and joint diseases (knee cartilage defects, osteoarthritis, herniated discs, etc.) Heart disease (myocardial infarction, ischemic heart failure, etc.) Liver disease (cirrhosis, non-alcoholic steatohepatitis, etc.)

[0030] Example The present invention will be explained in more detail below with reference to examples, although the scope of the present invention is not limited to these examples. [Example]

[0031] 1. Measurement of forward scatter (FSC) CV value by flow cytometry REC clones (clone numbers: 1 to 45) prepared in advance by a known method (WO2016 / 17795) and the scheme shown in FIG. 1 were used to measure the CV value of forward scattered light by flow cytometry. In flow cytometry, FSC is proportional to the surface area or size of the cell. In this example, the CV value of FSC was used as an index to evaluate the variation in cell size. Figure 2 shows the results of measuring the CV value of forward scattered light by flow cytometry.

[0032] (a) Individual REC clones were stained with PI, and a gate was set on the PI-negative live cell population to exclude dead cells from the analysis. (b) The PI-negative live cell population was developed on an FSC / SSC cytogram, and a gate (P1) was set on the main cell population to exclude debris and noise from the analysis. (c) The cell population within the P1 gate was expanded on an FSC histogram, a marker (M1) was set, and the CV value was measured.

[0033] 2. Evaluation of cell proliferation and adipogenesis (Figure 3) (1) Method for evaluating cell proliferation 1x10 5 REC cells were seeded onto a 100 mm culture dish and cultured at 37°C under 5% CO for 5 days. The cell number and average cell size were then measured using a cell counter. The culture medium used was DMEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with FBS, basic FGF, Hepes, and penicillin-streptomycin. (2) Method for assessing adipogenic potential 5x10 4 REC cells were seeded onto a 24-well plate and cultured at 37°C in a 5% CO environment for 2 days. The medium was then replaced with adipogenesis-inducing medium and cultured for an additional 14 days. After 14 days of culture, cells were stained with Oil Red 0 and the lipid droplet area was calculated by image analysis. The adipogenesis-inducing medium used was the culture medium (1) supplemented with dexamethasone, indomethacin, and IBMX.

[0034] 3.Comprehensive analysis A comprehensive analysis was performed on 45 REC clones obtained in one sorting run (Fig. 4). When the FSC CV value, cell proliferation rate (proliferation potential), average cell size, and fat differentiation potential of each REC clone were examined, it was found that REC clones with low FSC CV values ​​(#3, #5, #15, #16, #17) had high proliferation and differentiation potential, but small average cell size. On the other hand, REC clones with high FSC CV values ​​(#7, #32, #39, #40, #44) were found to have low proliferation and differentiation potential and large average cell size. The average proliferation rate of clones with a CV of 30% to 35% was 6.4, that of clones with a CV of 25% to 30% was 9.2, and that of clones with a CV of less than 25% was 15.1. The average proliferation rate of clones with an average cell size of 18 μm to 20 μm was 7.0, that of clones with an average cell size of 16 μm to 18 μm was 12.7, and that of clones with an average cell size of less than 16 μm was 21.3. These results demonstrate that it is possible to select high-quality REC clones with both high proliferation and differentiation potential using the FSC CV value and average cell size as indicators.

[0035] 4. Summary of typical analysis examples (Fig. 5) Clone A, which has a low FSC CV value and a small average cell size, is a high-quality REC clone with both high adipogenic potential and proliferation potential. Clone B, which has a high FSC CV value and a large average cell size, is an unspecified REC clone with low adipogenic potential and proliferation potential. [Example]

[0036] Two bone marrow aspirate lots (# 18TL158166 and # 8F5040) were sorted by two methods (CD271 and CD90 co-positive and CD271 alone), and the resulting colony, REC, MEC, and SEC ratios were compared (Figure 6). In Figure 6, the left panel shows the results of cell sorting. The cells in the boxed area were seeded onto a 96-well plate, and the percentage of colonies obtained is shown in the "Colony" column in the table on the right. Among the colonies obtained, the percentages of rapidly growing colonies (REC), medium-growing colonies (MEC), and slowly growing colonies (SEC) are also shown in the table. Compared to sorting using both LNGFR and CD90 positivity as an indicator, sorting using LNGFR alone resulted in a higher percentage of colonies, but a lower percentage of RECs. However, we found that RECs could be isolated by sorting only LNGFR-positive cells.

Claims

1. A method for producing a clinically applicable cell population of human mesenchymal stem cells, comprising: (a) obtaining a population of mesenchymal stem cells that are LNGFR (CD271) positive or LNGFR (CD271) and Thy-1 (CD90) co-positive; (b) culturing the clones of the mesenchymal stem cells into single cells; (c) recovering and expanding rapidly growing clones; (d) measuring the coefficient of variation of forward scattered light by flow cytometry for the cell population after the expansion culture; and (e) selecting a cell population having a coefficient of variation of forward scattered light in flow cytometry of 35% or less; A manufacturing method comprising:

2. A manufacturing method as described in claim 1, wherein a cell population having a coefficient of variation of 30% or less is selected.

3. A manufacturing method described in claim 1 or 2, wherein in (c), if there are multiple clones that grow rapidly, the clones are recovered and expanded independently of each other.

4. The manufacturing method described in claim 3, wherein in (d), the cell populations after the expansion culture are subjected to the flow cytometry independently of each other.

5. A culture of a cell population of human mesenchymal stem cells, comprising: The cell population (a) obtaining a population of LNGFR (CD271)-positive or LNGFR (CD271) and Thy-1 (CD90)-co-positive mesenchymal stem cells; (b) culturing the clones of the mesenchymal stem cells into single cells; and (c) recovering fast-growing clones; is obtained by A culture having a coefficient of variation of forward scattered light in flow cytometry of 35% or less.

6. A culture described in claim 5, having a coefficient of variation of 30% or less.

7. A culture described in claim 5 or 6, wherein the average cell size is 20 μm or less.

8. A culture described in any one of claims 5 to 7, wherein the average cell size is 14 μm to 18 μm.

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