Lbm, cpc, opc, production and quality control methods therefor, kit, graft material, and disease model

Abstract

To provide a limb bud mesenchymal cell population, osteoblast progenitor cell population, or chondrocyte progenitor cell population, capable of producing a bone tissue or cartilage tissue of interest with high efficiency and high purity, preparation methods therefor, and quality control methods therefor.SOLUTION: The present invention provides a limb bud mesenchymal cell population, which is derived from mammalian lateral plate mesoderm cells, and is PRRX1 protein-positive.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority based on Japanese Patent Application No. 2019-169278, filed on September 18, 2019, and Japanese Patent Application No. 2020-62441, filed on March 31, 2020, the entire disclosures of which are incorporated herein by reference.

[0002] The present invention relates to a limb bud mesenchymal cell population, a chondrogenic precursor cell population, an osteoprogenitor cell population, a method for preparing and controlling the quality thereof, a transplant material, and a disease model.

[0003] In this specification, "CPC" refers to a chondrocyte precursor cell or a chondrocyte precursor cell population, "LBM" refers to a limb bud mesenchymal cell or a limb bud mesenchymal cell population, and "OPC" refers to an osteoblast precursor cell or an osteoblast precursor cell population. In addition, "PRRX1 positive" refers to PRRX1. high "PRRX1 negative" may be written as "PRRX1 low It is sometimes written as follows. [Background technology]

[0004] Mesenchymal stem cells, which are one type of somatic stem cells, have the ability to differentiate into cells such as osteoblasts and chondrocytes, and are being considered for application in regenerative medicine, but they have problems with low differentiation induction efficiency and limited self-proliferation ability. In addition, conventional differentiation induction methods using pluripotent stem cells focus on directly inducing them into the desired tissue cells, but the low induction efficiency and low quality are considered problems (Non-Patent Documents 1 and 2).

[0005] Furthermore, if the quality of the osteoblasts or chondrocytes is poor, the quality of the resulting bone tissue or cartilage tissue will be reduced, making it impossible to transplant them into the living body. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Stem Cell Reports. 2015 Mar 10;4(3):404-18. [Non-Patent Document 2] Nat Biotechnol. 2015 Jun;33(6):638-45. Summary of the Invention [Problem to be solved by the invention]

[0007] One object of the present invention is to provide a limb bud mesenchymal cell population, an osteoprogenitor cell population, or a chondrocyte precursor cell population capable of producing the desired bone tissue or cartilage tissue with high efficiency and high purity, a method for preparing them, and a method for quality control thereof.

[0008] Another object of the present invention is to provide a kit for obtaining the limb bud mesenchymal cell population, osteoprogenitor cell population, chondrocyte precursor cell population, or chondrocytes or cartilage tissue that can be derived therefrom, from mammalian pluripotent stem cells.

[0009] Furthermore, it is another object of the present invention to provide a transplant material for treating bone- or cartilage-related diseases.

[0010] Furthermore, another object of the present invention is to provide a disease model relating to bone or cartilage. [Means for solving the problem]

[0011] The present invention provides the following limb bud mesenchymal cell population, chondrocyte precursor cell population, osteoblast precursor cell population, preparation methods and quality control methods thereof, kits, transplant materials, and disease models. Item 1. A limb bud mesenchymal cell population derived from mammalian lateral plate mesoderm cells and positive for PRRX1 protein. Item 2. A limb bud mesenchymal cell population according to Item 1, which satisfies at least one condition selected from the group consisting of CD44 positivity, CD140B positivity, and CD49f negativity. Item 3. A step of inducing differentiation of pluripotent stem cells into lateral plate mesoderm cells; and Item 3. A method for preparing a limb bud mesenchymal cell population according to Item 1 or 2, comprising a step of culturing the lateral plate mesoderm cells induced to differentiate by the above step under conditions of Wnt signal activation and FGF signal inactivation. Item 4. A method for preparing an osteoprogenitor cell population, comprising the step of culturing the limb bud mesenchymal cell population according to Item 1 or 2 in an environment free of a Wnt signal activator. Item 5. A method for preparing a mammalian chondrocyte precursor cell population, comprising the step of culturing the limb bud mesenchymal cell population according to Item 1 or 2 under an environment that activates Wnt signaling. Item 6. A method for preparing a mammalian chondrocyte precursor cell population according to Item 5, comprising the step of culturing the limb bud mesenchymal cell population according to Item 1 or 2 under an environment that activates Wnt signaling and under conditions that activate FGF signaling. Item 7. A method for quality control of a mammalian limb bud mesenchymal cell population, comprising a step of determining whether or not the mammalian limb bud mesenchymal cells satisfy at least one condition selected from the group consisting of CD44 positivity, CD140B positivity, and CD49f negativity. Item 8. A method for quality control of a mammalian chondroprogenitor cell population, comprising the step of determining whether or not the mammalian chondroprogenitor cells satisfy at least one condition selected from the group consisting of CD90 positivity and CD140B positivity. Item 9. A mammalian chondrocyte precursor cell population that satisfies at least one condition selected from the group consisting of CD90 positivity and CD140B positivity. Item 10. A population of mammalian chondrocyte precursor cells according to Item 9, which has been cryopreserved. Item 11. A mammalian osteoprogenitor cell population having a RUNX2 positivity rate of 95% or more. Item 12. A population of mammalian osteoprogenitor cells according to Item 11, which has been cryopreserved. Item 13. A kit for inducing limb bud mesenchymal cells (LBM) from mammalian pluripotent stem cells, comprising the following (i) to (iii): (i) Culture medium for inducing pluripotent stem cells into primitive streak cells (ii) Medium for inducing primitive streak cells to lateral plate mesoderm cells (iii) Culture medium for inducing limb bud mesenchymal cells from lateral plate mesoderm cells Item 14. A kit for inducing chondrocytes from mammalian pluripotent stem cells, comprising the following (i) to (iv): (i) Culture medium for inducing pluripotent stem cells into primitive streak cells (ii) Medium for inducing primitive streak cells to lateral plate mesoderm cells (iii) Culture medium for inducing limb bud mesenchymal cells from lateral plate mesoderm cells (iv) Culture medium for inducing chondrocyte progenitor cells from limb bud mesenchymal cells Item 15. A kit for inducing chondrocytes from mammalian pluripotent stem cells, comprising the following (i) to (v): (i) Culture medium for inducing pluripotent stem cells into primitive streak cells (ii) Medium for inducing primitive streak cells to lateral plate mesoderm cells (iii) Culture medium for inducing limb bud mesenchymal cells from lateral plate mesoderm cells (iv) Culture medium for inducing chondrocyte progenitor cells from limb bud mesenchymal cells (v) Medium for inducing chondrocytes from chondroprogenitor cells Item 16. A kit for inducing RUNX2-positive osteoprogenitor cells from mammalian pluripotent stem cells, comprising the following (i) to (iv): (i) Culture medium for inducing pluripotent stem cells into primitive streak cells (ii) Medium for inducing primitive streak cells to lateral plate mesoderm cells (iii) Culture medium for inducing limb bud mesenchymal cells from lateral plate mesoderm cells (vi) Culture medium for inducing RUNX2-positive osteoprogenitor cells from limb bud mesenchymal cells Item 17. A transplant material comprising the mammalian chondroprogenitor cell population according to Item 9 or 10, or chondrocytes or cartilage tissue differentiated from the chondroprogenitor cell population. Item 18. A transplant material comprising the mammalian osteoprogenitor cell population according to Item 11 or 12, or osteoblasts or bone tissues differentiated from the osteoprogenitor cell population. Item 19. A cartilage-related disease model comprising a chondroprogenitor cell population induced from iPS cells derived from a patient with a cartilage-related disease and satisfying at least one condition selected from the group consisting of CD90 positivity and CD140B positivity, or chondrocytes induced from the chondroprogenitor cell population. Item 20. A bone-related disease model comprising osteoprogenitor cells having a RUNX2 positivity rate of 95% or more induced from iPS cells derived from a patient with a bone-related disease, or osteoblasts induced from the osteoprogenitor cell population. Effect of the Invention

[0012] According to the present invention, by controlling the quality of a limb bud mesenchymal cell population and a chondroprogenitor cell population, high-quality cartilage tissue and transplant materials for cartilage regeneration can be obtained.

[0013] The limb bud mesenchymal cell population obtained by the present invention and the osteoprogenitor cell population or chondrocyte precursor cell population inducible therefrom, or the chondrocytes or cartilage tissue induced from the chondrocyte precursor cell population, can be used as a transplant material for treating diseases related to bone or cartilage. The present inventors have confirmed that the transplant material of the present invention is a safe transplant material that does not cause benign or malignant tumors.

[0014] By using the osteoprogenitor cell population or chondroprogenitor cell population of the present invention, high-quality bone tissue or cartilage tissue can be obtained.

[0015] By using the kit of the present invention, a limb bud mesenchymal cell population, an osteoprogenitor cell population, a chondrocyte precursor cell population, or chondrocytes or cartilage tissue that can be derived therefrom can be easily obtained from pluripotent stem cells such as iPS cells.

[0016] When the limb bud mesenchymal cell population, chondrocyte precursor cell population, and osteoblast precursor cell population of the present invention are induced from iPS cells of a patient with a bone- or cartilage-related disease, they are useful for producing a bone disease model or a cartilage disease model, and further for producing a model of a bone-related disease or a cartilage-related disease.

[0017] The disease model of the present invention can be used to study the pathology of bone- or cartilage-related diseases and support the development of new therapeutic agents. [Brief description of the drawings]

[0018] [Figure 1] 1 shows an outline of the differentiation induction protocol of the present invention. [Diagram 2] Schematic diagram of a targeting vector for producing a PRRX1 reporter. [Diagram 3] 1 shows a protocol for inducing differentiation into PRRX1-positive cells via the lateral plate mesoderm lineage. [Figure 4] The results of immunostaining for HAND1 (a marker for lateral plate mesoderm) are shown. [Diagram 5] The results of immunostaining for CDX2 (a marker for paraxial mesoderm) are shown. [Figure 6] 1 shows a protocol for inducing differentiation into PRRX1-positive cells via the lateral plate mesoderm lineage. [Figure 7] The activators and / or inhibitors of various signals used (16 combinations) are shown. [Figure 8] Fluorescence images of tdTomato are shown. [Figure 9] The measurement results of qPCR (quantitative RT-PCR) for PRRX1 are shown. [Figure 10] The results of qPCR (quantitative RT-PCR) for HAND1 (a marker for lateral plate mesoderm) are shown. [Figure 11] The results of qPCR (quantitative RT-PCR) for ISL-1 (a marker for lateral plate mesoderm) are shown. [Figure 12] The results of qPCR (quantitative RT-PCR) for FOXF1 (a marker for lateral plate mesoderm) are shown. [Figure 13] A preferred protocol for inducing PRRX1-positive cells from lateral plate mesoderm cells (day 2) is shown. [Figure 14] The results of flow cytometry are shown. [Figure 15] The results of immunostaining using a PRRX1 antibody are shown. [Figure 16] The expression profile of CD antigens, etc. (1) is shown. [Figure 17] The expression profile of CD antigens, etc. (2) is shown. [Figure 18] The expression profile of CD antigens, etc. (3) is shown. [Figure 19] 1 shows a differentiation induction protocol. [Figure 20] Phase contrast microscopic images at each time point are shown. [Figure 21] The expression level of PRRX1 mRNA at each time point is shown. [Figure 22] The mRNA expression levels of lateral plate mesoderm markers (HAND1, ISL1, FOXF1) at each time point are shown. [Diagram 23] The results of immunostaining using a PRRX1 antibody are shown. [Figure 24] Phase contrast microscopic images at each time point are shown. [Diagram 25] The mRNA expression levels of PRRX1 and mesodermal markers (HAND1, ISL1, FOXF1) at each time point are shown. [Figure 26] The results of immunostaining using PRRX1 antibody are shown. [Figure 27] 1 shows a protocol for inducing differentiation into PRRX1-positive cells via the paraxial mesoderm lineage. [Figure 28] The results of immunostaining for HAND1 (a marker for lateral plate mesoderm) are shown. [Figure 29] The results of immunostaining for CDX2 (a marker for paraxial mesoderm) are shown. [Diagram 30] The activators and / or inhibitors of various signals used (six combinations) are shown. [Diagram 31] Fluorescence images of tdTomato are shown. [Diagram 32] The results of flow cytometry are shown. [Diagram 33]The results of qPCR (quantitative RT-PCR) measurements of PRRX1, a paraxial mesoderm marker (CDX2), and somite markers (MEOX1, PARAXIS) are shown. [Diagram 34] The activators and / or inhibitors of various signals used (16 combinations) are shown. [Diagram 35] The results of qPCR (quantitative RT-PCR) for RUNX2 are shown. [Diagram 36] 1 shows a differentiation induction protocol. [Figure 37] The measurement results of qPCR (quantitative RT-PCR) are shown. [Figure 38] The results of qPCR (quantitative RT-PCR) for RUNX2 are shown. [Figure 39] The results of immunostaining for RUNX2 are shown. [Diagram 40] The results of flow cytometry are shown. [Diagram 41] The results of Alizarin Red staining are shown. [Diagram 42] 1 shows a differentiation induction protocol. [Diagram 43] Phase contrast images under each condition are shown. [Diagram 44] The results of the coating study are shown below. (A) Stem Medium 3 was used to seed cells onto culture dishes coated with each coating agent. (B) Coating with clinical grade Matrix-511, the same as used for differentiation induction, was selected. [Diagram 45] The images are taken under a phase-contrast microscope at each passage number during subculture. PN: passage number. [Figure 46] The results of flow cytometry analysis and growth curves are shown. PN: passage number. [Figure 47] Comparison of proliferation potential of hCPCs induced from each pluripotent stem cell. [Figure 48] Development of a quality control method for LBM cells. (a) Chromosome karyotype of the 414C2 iPSC line, (b) karyotype of human CPCs (PN3) established from the 414C2 iPSC line. [Figure 49]Phase contrast images of cells before and after storage in liquid nitrogen are shown. (A) Before storage in liquid nitrogen, (B) After storage in liquid nitrogen. [Figure 50] RUNX2 expression in cells (hCPC-derived and LBM-derived) induced by differentiation of hCPC and human LBM (day 4) with RUNX2-positive osteoprogenitor cell differentiation-inducing medium. It was revealed that no increase in RUNX2 expression was observed in hCPC-derived cells. [Figure 51] Expression of chondrocyte markers (SOX5, SOX6, SOX9, COL2A1, ACAN) in each hCPC. (a) SOX9 expression in various iPS-derived hCPCs, (b) Protocol for treating hCPCs with Step 1 medium and Step 2 medium, (c) Assay results for cells with (complete) and without (w / o cytokines), (d) Results of mRNA expression of chondrocyte markers (SOX5, SOX6, SOX9, COL2A1, ACAN). [Figure 52] Preparation and staining of hyaline cartilage-like tissue. (a) Protocol for obtaining hyaline cartilage from hCPCs, (b) hyaline cartilage tissue on day 42 of step 3 treatment, (c) HE staining, Alcian Blue staining, Safranin O staining, and immunostaining for chondrocyte markers (SOX9, COL2), hypertrophic chondrocyte marker (COLX), and fibrous cartilage marker (COL1). [Figure 53] Safranin O staining of cartilage tissue induced from human LBM (day 4) and hCPCs. It was revealed that hCPCs had a higher ability to form cartilage tissue than human LBM. [Figure 54] Subcutaneous transplantation of hyaline cartilage-like tissue into NOD-SCID mice. (a) Protocol for inducing cartilage tissue from hCPCs and transplanting it subcutaneously, (b) Sectioning of hyaline cartilage-like tissue extracted from the subcutaneous tissue, (c) SafO staining of hyaline cartilage-like tissue derived from each iPS cell (414C2, Reporter) or ES cell (SEES6). [Figure 55] Experimental transplantation of human-derived cartilage tissue mass into cartilage defect site in SCID rats. (a) Preparation of cartilage tissue mass and transplantation into cartilage defect site, (b) Confirmation of survival of transplanted tissue 2 weeks after transplantation. [Figure 56] Subcutaneous transplantation of hyaline cartilage-like tissue into NOD-SCID mice. (a) Protocol for transplantation of hCPCs under the mouse kidney capsule, (b) Immunostaining of hyaline cartilage tissue obtained 8 weeks after transplantation, (c) Photograph taken immediately after subrenal capsule transplantation. [Figure 57] Preparation of cartilage plates. (a) Protocol for obtaining cartilage plates from hCPCs. (b) Photographs of hCPC spheroids immediately after casting, after 15 days of culture, and after 30 days of culture. [Figure 58] Safranin O staining of cartilage plate tissue formed in the mold. (a) Safranin O staining of cartilage plate tissue formed in the mold. (b) Safranin O staining of cartilage plate tissue formed in the mold after subcutaneous transplantation into a NOD-SCID mouse 3 weeks after transplantation. [Figure 59] Overview of quality control technology for chondroprogenitor cells (CPCs). We have developed a method for quality control of CPCs (= ability to form chondrocytes / cartilage tissue). To stably supply chondrocytes / tissues from CPCs, it is important to expand and culture hCPCs under conditions that satisfy at least one condition selected from the group consisting of CD90 positivity and CD140B positivity. [Figure 60] This is an overview of the quality control technology for limb bud mesenchymal cells (LBM). We have developed a method for quality control of LBM (=ability to form PRRX1-positive CPCs). In order to supply CPCs with high cartilage tissue formation ability from LBM, it is important to induce CPCs from "LBM that satisfies at least one condition selected from the group consisting of CD44-positive, CD99-positive, CD140B-positive, CD9-negative, CD49f-negative, and CD57-negative," and it is particularly important to induce CPCs from "LBM that satisfies at least one condition selected from the group consisting of CD44-positive, CD140B-positive, and CD49f-negative." [Figure 61](a)~(b) PRRX1 expression pattern of PRRX1-positive human chondroprogenitor cells (hCPCs). (c) Staining results of the cartilage tissue produced from them. hCPCs with each PRRX1 expression state were collected from the culture dish and induced to differentiate. The tissue formed after induced chondrocyte differentiation was fixed and stained with HE, Alcian blue, and Safranin O. The results showed that hCPCs maintained in a state with high PRRX1 expression levels had high chondrocyte / tissue formation ability, whereas hCPCs maintained in a state with low PRRX1 expression levels had low chondrocyte / tissue formation ability. In other words, there is a positive correlation between the expression level of PRRX1 and the ultimate ability to form cartilage tissue. [Figure 62] Differences in Alcian blue staining during induction of planar cartilage differentiation due to differences in PRRX1 expression levels. hCPCs with high PRRX1 expression levels (PRRX1 positive) or low PRRX1 expression levels (PRRX1 negative) were seeded onto culture dishes, and differentiation induction was performed according to the procedure described. After induction of chondrocyte differentiation, the cells were fixed and stained with Alcian blue. As a result, only hCPCs with high PRRX1 expression levels formed Alcian blue-positive nodules. [Figure 63] RNAseq was performed on the following two combinations, and genes that were elevated in samples with high PRRX1 expression were extracted. 901 genes were extracted from combination (1), and 317 genes were extracted from combination (2). 189 common genes were identified between the two combinations (= high-quality cell marker molecules). Combination (1): hCPC (PRRX1 positive peak) vs hCPC (PRRX1 negative peak), Combination (2): PRRX1 positive fraction in hCPC (double peak) vs PRRX1 negative fraction. [Figure 64]RNAseq was performed on the following two combinations, and genes that were elevated in samples with low PRRX1 expression were extracted. 728 genes were extracted from combination (3), and 479 genes were extracted from combination (4). 220 common genes were identified between the two combinations (= malignant cell marker molecules). Combination (3): hCPC (PRRX1 positive peak) vs hCPC (PRRX1 negative peak), Combination (4): PRRX1 positive fraction in hCPC (double peak) vs PRRX1 negative fraction. [Figure 65] When the specific genes of PRRX1-positive hCPCs, good marker molecules (189 genes) and bad marker molecules (220 genes), were analyzed using gene analytics software, it was found that the good marker molecules (189 genes) are genes that define hCPCs. [Figure 66] Analysis of the good marker molecules (189 genes) and the bad marker molecules (220 genes) using Enricher software revealed that the good marker molecules (189 genes) had expression patterns similar to those of transcription factors that characterize hCPCs. [Figure 67] Candidate genes for selecting PRRX1-negative or -positive hCPCs. Flow cytometry results for each surface antigen correlated with the expression level of PRRX1 identified by RNAseq. hCPCs with low PRRX1 expression (PRRX1-negative) or high PRRX1 expression (PRRX1-positive) were stained with antibodies against each surface antigen, and the expression levels were compared by flow cytometry. As a result, it was found that CD90 and CD140B were highly expressed in PRRX1-positive cases. [Figure 68](a)-(b) PRRX1 expression patterns of PRRX1-positive human limb bud mesenchymal cells (LBM) and hCPCs produced from them. (c) Cartilage tissue produced from each hCPC. hCPCs with each PRRX1 expression state were collected from the culture dish, and differentiation induction was performed according to the procedure described. The tissue formed after cartilage differentiation induction was fixed and stained with HE, Alcian blue, and Safranin O. As a result, it was found that hCPCs maintained in a state with high PRRX1 expression had high chondrocyte / tissue formation ability, while hCPCs maintained in a state with low PRRX1 expression had low chondrocyte / tissue formation ability. In other words, it can be seen that there is a positive correlation between the expression level of PRRX1 and its final ability to form cartilage tissue. [Figure 69] Candidate genes for selecting PRRX1-negative or -positive LBM. Flow cytometry results for each surface antigen correlated with the expression level of PRRX1 identified by RNAseq. LBM with low PRRX1 expression (PRRX1-negative) or high PRRX1 expression (PRRX1-positive) were stained with antibodies against each surface antigen, and the expression levels were compared by flow cytometry. As a result, it was found that CD44, CD99, and CD140B were highly expressed in PRRX1-positive cases, while CD9, CD49f, and CD57 were highly expressed in PRRX1-negative cases. [Figure 70] Type II collagen deficiency disease model 1. (a) Protocol for generating hCPCs from iPSC lines (ACGII-1, HCG-1) derived from patients with type II collagen deficiency-related diseases, (b) Immunostaining of PRRX1 expression in each hCPC, (c) Growth curve, (d) Results of cartilage matrix staining with Alcian blue staining, (e) Measurement of the expression of various cartilage differentiation marker genes (qPCR). [Figure 71] Type II collagen dyscrasia disease model 2. Compared to cells derived from healthy subjects (414C2), CPCs derived from patients with type II collagen dyscrasia-related diseases (ACGII-1, HCG-1) showed a decrease in intracellular glycogen. In addition, the normal orientation of ER observed in normal cells was not observed in patient-derived cells. [Figure 72] Osteogenesis imperfecta disease model. [Figure 73] 1 shows an overview of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] 1. Limb bud mesenchymal cells, chondrocyte precursor cells, RUNX2-positive osteoprogenitor cells In the present invention, mammalian limb bud mesenchymal cells (LBM) are multipotent cells that can differentiate into both chondroprogenitor cells and RUNX2-positive osteoprogenitor cells. On the other hand, chondroprogenitor cells are different from LBM in that they do not change into osteoblasts. Better chondrocytes / cartilage tissues can be obtained by inducing them from LBM quality-controlled by the present invention and CPC quality-controlled by the present invention. The quality control method of the present invention can obtain high-quality LBM and CPCs, and osteoprogenitor cells induced from LBM, osteoblasts or bone tissue induced from the osteoprogenitor cells, and chondrocytes or cartilage tissue induced from CPCs are of high quality suitable for transplantation.

[0020] The limb bud mesenchymal cells (LBM), chondroprogenitor cells and RUNX2-positive osteoprogenitor cells (hereinafter sometimes abbreviated as "LBM or differentiated cells thereof") of the present invention are derived from pluripotent stem cells. LBM and chondroprogenitor cells are PRRX1 protein positive, and RUNX2-positive osteoprogenitor cells are PRRX1 protein negative. In addition, the LBM of the present invention has proliferation properties, and for example, differentiation induction from LBM to a chondroprogenitor cell population can be performed in an environment that activates Wnt signals, and differentiation induction and proliferation can be performed simultaneously. The number of CPCs can be greatly increased by subculturing them in the presence of an FGF signal activator. The present inventors have confirmed that chromosomal abnormalities do not occur by subculturing CPCs. In addition, the present inventors have confirmed that CPCs whose number of cells has been greatly increased by subculturing do not develop either benign or malignant tumors when transplanted into a living body.

[0021] As used herein, "pluripotent stem cells" refer to cells that have both the ability to self-replicate (self-renewal ability) and the ability to differentiate into cells of multiple lineages (multipotency). The fact that a cell is a pluripotent stem cell can also be confirmed by the expression of stem cell markers such as Oct3 / 4 and Klf-4, instead of confirming that the cell has self-renewal ability and multipotency. "Progenitor cells" refer to cells that are in the process of differentiating from stem cells into functional cells, which are the final differentiation destination. RUNX2-positive osteoprogenitor cells are cells that can be induced to differentiate into osteoblasts, and by inducing differentiation from LBM into an osteoprogenitor cell population, an osteoprogenitor cell population with a RUNX2 positivity of 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, particularly 99.5% or more, and most preferably 100% RUNX2 positive can be obtained. By using an osteoprogenitor cell population with a high RUNX2 positivity, higher quality bone tissue can be produced. Furthermore, by preparing chondrocytes from the chondroprogenitor cells of the present invention, cartilage tissue of higher quality can be produced compared to the case of obtaining chondrocytes directly from pluripotent stem cells such as iPS cells or ES cells. In particular, by using chondroprogenitor cells whose quality has been controlled according to the quality control method of the present invention, cartilage tissue of high quality can be regenerated stably.

[0022] The organisms from which the LBM or differentiated cells thereof of the present invention are derived include mammals, such as humans, mice, rats, cows, horses, pigs, rabbits, dogs, cats, goats, monkeys, and chimpanzees, with humans being preferred.

[0023] PRRX1 (paired related homeobox 1) protein is a transcription factor that contains a homeodomain and is known to be specifically expressed in the limb bud derived from the lateral plate mesoderm during development.

[0024] The nucleotide sequence of the cDNA of the PRRX1 gene of humans (Homo sapiens) and the amino acid sequence of the PRRX1 protein are registered in GenBank provided by the National Center for Biotechnology Information (NCBI) under the following accession numbers (if multiple revisions are registered, it is understood that the latest revision is referred to): Human PRRX1 gene: NM_006902 (NM_006902.5), NM_022716 (NM_022716.4) Human PRRX1 protein: NP_008833 (NP_008833.1), NP_073207 (NP_073207.1).

[0025] The fact that a cell is positive for the PRRX1 protein can be detected by a known method, for example, detection by a reporter gene whose expression is controlled by the transcription promoter sequence of the PRRX1 gene, detection by immunostaining using an antibody specific to the PRRX1 protein, etc.

[0026] The LBM or differentiated cells thereof of the present invention are derived from pluripotent stem cells. "Derived from pluripotent stem cells" means that the cells are induced to differentiate using pluripotent stem cells as raw material cells. For example, the LBM or differentiated cells thereof derived from pluripotent stem cells are LBM or differentiated cells thereof in which at least a part of the differentiation induction process has been performed outside of the body.

[0027] As pluripotent stem cells, preferred are pluripotent stem cells (cells that have the ability to differentiate into any somatic cell or germ lineage cell), such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

[0028] In a preferred embodiment, the LBM cells of the present invention are characterized by positive or negative expression of at least one antigen marker selected from the group consisting of CD9 negative, CD24 positive, CD29 positive, CD44 positive, CD46 positive, CD49f negative, CD55 positive, CD57 negative, CD58 positive, CD90 positive, CD99 positive, CD140A positive, CD140B positive, SSEA1 negative, SSEA3 negative, SSEA4 positive, TRA-1-60 negative, TRA-1-81 negative, CD73 negative, CD105 negative and CD166 negative.

[0029] LBM is PRRX1 positive and has proliferation ability under an environment that activates FGF signal. In one embodiment of the present invention, the surface antigen of LBM preferably satisfies at least one, at least two, at least three, at least four, at least five or six conditions selected from the group consisting of CD44 positive, CD99 positive, CD140B positive, CD9 negative, CD49f negative and CD57 negative (FIG. 60), and in particular, it is preferable to satisfy at least one, at least two or three conditions selected from the group consisting of CD44 positive, CD140B positive and CD49f negative. In a preferred embodiment of the present invention, as long as it satisfies at least one, at least two or three conditions selected from the group consisting of CD44 positive, CD140B positive and CD49f negative, it may further satisfy one, two or three conditions selected from the group consisting of CD99 positive, CD9 negative and CD57 negative. When LBM satisfies at least one, at least two, at least three, at least four, at least five, or six conditions selected from the group consisting of CD44 positivity, CD99 positivity, CD140B positivity, CD9 negation, CD49f negation, and CD57 negation, it is preferable as a source for inducing differentiation into chondroprogenitor cells or osteoprogenitor cells. Therefore, by confirming whether or not LBM satisfies at least one, at least two, at least three, at least four, at least five, or six requirements selected from the group consisting of CD44 positivity, CD99 positivity, CD140B positivity, CD9 negation, CD49f negation, and CD57 negation, the quality of LBM as a source of chondroprogenitor cells or osteoprogenitor cells can be controlled.

[0030] In a preferred embodiment of the present invention, a mammalian chondroprogenitor cell population satisfying at least one or two conditions selected from the group consisting of CD90 positivity and CD140B positivity can produce or regenerate high-quality cartilage tissue. Therefore, the quality of a chondroprogenitor cell population can be controlled by examining whether or not it satisfies at least one or two conditions selected from the group consisting of CD90 positivity and CD140B positivity.

[0031] In the quality control method of the present invention, the presence or absence of expression of at least one surface antigen selected from the group consisting of CD44, CD99, CD140B, CD9, CD49f, and CD57 is detected in LBM, and the presence or absence of expression of at least one antigen selected from the group consisting of CD90 and CD140B is detected in CPC. In CPC, CD90 and CD140B are preferably positive. In LBM, of these six antigens, CD44, CD99, and CD140B are preferably positive, and CD9, CD49f, and CD57 are preferably negative. Here, the "positive" and "negative" expression of CD antigens is determined by placing the numerical value (negative histogram) obtained from a control (a sample not containing an antibody).

[0032] PRRX1 positivity / negative can be determined, for example, based on the values ​​of flow cytometry using PRRX1-tdTomato reporter cells. Specifically, when the measured value of tdTomato is 4x10 4 If the peak value is above 2x10, PRRX1 is positive. 4 If a peak appears at the following value, it can be determined that PRRX1 is negative. tdTomato is an example of a fluorescent label, and PRRX1 positivity / negative can be determined using the same criteria even when other labels are used.

[0033] Osteoprogenitor or chondroprogenitor cells can be produced in large quantities, cryopreserved and used for bone or cartilage production or regeneration when required.

[0034] The suitability of cryopreserved chondroprogenitor cells as a transplant material can be confirmed by the fact that the chondroprogenitor cells satisfy at least one, at least two, or three conditions selected from the group consisting of CD90 positivity and CD140B positivity. Therefore, the quality of the chondroprogenitor cells can be controlled by confirming whether or not they satisfy the requirements of CD90 positivity and CD140B positivity. When chondroprogenitor cells are induced to differentiate from iPS cells of a patient with a cartilage-related disease, the patient-derived chondroprogenitor cells serve as a disease model for the cartilage-related disease. This disease model can be used for screening of therapeutic agents for the cartilage-related disease, and is preferably cryopreserved.

[0035] Similarly, when osteoprogenitor cells are induced to differentiate from iPS cells of a patient with a bone-related disease, the patient-derived osteoprogenitor cells serve as a disease model for the bone-related disease. This disease model can be used for screening therapeutic agents for the bone-related disease, and is similarly preferably cryopreserved.

[0036] Cartilage-related diseases include type II collagen disorder, relapsing polychondritis, osteoarthritis, rheumatoid arthritis, achondroplasia, and the like.

[0037] Bone-related diseases include osteoarthritis, osteoporosis, osteomalacia, rheumatoid arthritis, femoral head necrosis, osteochondritis dissecans, osteogenesis imperfecta, and the like.

[0038] If the conditions for inducing differentiation of LBM from pluripotent stem cells such as iPS cells or ES cells are inappropriate, the quality of the LBM will be impaired. Therefore, it is desirable to check whether the LBM satisfies at least one, at least two, at least three, at least four, at least five or six conditions selected from the group consisting of CD44 positive, CD99 positive, CD140B positive, CD9 negative, CD49f negative and CD57 negative before inducing it into a chondrocyte precursor cell population or an osteoblast precursor cell population, and to perform quality control. Of the six types, CD44 positive, CD140B positive and CD49f negative, the three types CD44 positive, CD140B positive and CD49f negative are more important, and if at least one of these three types is satisfied, at least one of the remaining three types (CD99 positive, CD9 negative, CD57 negative) may also be satisfied. Furthermore, with regard to chondroprogenitor cells, it is preferable to perform a double quality control, that is, quality control based on the surface antigen of LBM and quality control based on the surface antigen of chondroprogenitor cells.

[0039] In preparing LBM, first, pluripotent stem cells can be induced to differentiate into lateral plate mesoderm cells.

[0040] A known method can be used to induce differentiation of mammalian, particularly human, pluripotent stem cells into lateral plate mesoderm cells. For example, a method similar to the method described in the literature: Loh et al., 2016, Cell, 451-467 can be adopted. Specifically, first, differentiation of mammalian, particularly human, pluripotent stem cells into primitive streak cells (mid-primitive streak) is induced, and then differentiation of the primitive streak cells into lateral plate mesoderm cells is induced.

[0041] Induction of differentiation into lateral plate mesoderm cells can be confirmed, for example, by detecting the expression of HAND1 protein, which is a specific marker for lateral plate mesoderm cells.

[0042] Next, the lateral plate mesoderm cells are cultured under an environment that activates the Wnt signal, and are induced to differentiate into LBM, and the LBM is further induced to differentiate to obtain CPCs. The CPCs quality-controlled according to the present invention can be cryopreserved and induced to become chondrocytes or cartilage tissue when necessary. hCPCs are particularly preferred as a transplant material for humans, since they can be induced to become human chondrocytes or human cartilage tissue.

[0043] The graft material of the present invention can be used for the treatment of bone or cartilage-related diseases, and is also suitable for use in plastic surgery or cosmetic surgery. For example, the shape of the nose is determined by the alar cartilage, nasal septum cartilage, lateral nasal cartilage, etc., and the shape of the ear is determined by the auricular cartilage. In cases of trauma such as traffic accidents, congenital nasal bone defects, microtia, etc., it is necessary to adjust or reconstruct the shape of the nose or ear, but these can be achieved by preparing and transplanting the LBM and CPC of the present invention or the cartilage cells and cartilage tissue derived therefrom in the desired shape. In cases of nose or ear reconstruction surgery and cosmetic surgery, silicone or patient-derived costal cartilage is used, but since silicone is a foreign body, foreign body reactions such as inflammation and immune reactions may occur, and the use of the patient's costal cartilage is invasive, but the cartilage obtained by the present invention can suppress such foreign body reactions and is non-invasive. Cartilage of the desired shape can be expected to have a cosmetic effect even when it is embedded in places other than the nose and ear where cartilage does not originally exist. For example, a cartilage plate or block can be prepared and carved into the desired shape to create a cartilage graft material of the desired shape.

[0044] In the following, a particularly preferred mammal, human, will be taken as an example for explanation. When hLBM is induced to become hCPCs under the following inappropriate conditions, a CPC population is obtained in which there is a peak of cells that do not satisfy all the conditions of CD90 positivity and CD140B positivity and a peak of cells that satisfy all the conditions (double peak), or a CPC population consisting of a single peak of cells that do not satisfy all the conditions of CD90 positivity and CD140B positivity is obtained (Figure 61). Even if these CPC populations are induced to differentiate into chondrocytes or cartilage tissue, high-quality chondrocytes and cartilage tissue suitable for transplantation cannot be obtained. In order to induce human limb bud mesenchymal cells (hLBM) into hCPCs, if they are cultured under confluent conditions even once during the subculture process, a peak of cells that do not meet all the conditions of being CD90-positive and CD140B-positive will appear, resulting in hCPCs that are not suitable for transplantation. In order to obtain hCPCs that satisfy both the conditions of being CD90-positive and CD140B-positive, it is preferable to perform passage culture before the cells become confluent.

[0045] As shown in Figure 61, an hCPC population (PRRX1 negative) consisting of a single peak of cells that do not satisfy at least one condition selected from the group consisting of CD90 positive and CD140B positive is unsuitable for induction into chondrocytes or cartilage tissue. However, if a double peak of cells that satisfy at least one condition selected from the group consisting of CD90 positive and CD140B positive (PRRX1 positive) and cells that do not satisfy the conditions (PRRX1 negative) is observed, as the subculture continues, the entire population will converge into a cell population composed of cells that do not satisfy all of the conditions of CD90 positive and CD140B positive. Therefore, the culture can be stopped at the point when a peak of cells that do not satisfy at least one condition selected from the group consisting of CD90 positive and CD140B positive is confirmed. At the end of the culture, hCPCs can be induced to good chondrocytes and cartilage tissues usable for transplantation to such an extent that no peaks due to cells not satisfying all the conditions of CD90 positivity and CD140B positivity can be confirmed, preferably the ratio of cells not satisfying all of at least one condition selected from the group consisting of CD90 positivity and CD140B positivity is 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, and particularly hCPCs that are substantially free of such inappropriate cells. Furthermore, such good hCPCs are themselves useful as transplantation materials for the treatment of cartilage defects and cartilage-related diseases including osteoarthritis.

[0046] As shown in Figure 68(b), LBM (PRRX1 negative) consisting of a single peak of cells that do not satisfy all of the conditions of CD44 positivity, CD99 positivity, CD140B positivity, CD9 negativity, CD49f negativity, and CD57 negativity is inappropriate for induction into PRRX1 positive hCPCs (= hCPCs that satisfy all of the conditions of CD90 positivity and CD140B positivity) or osteoblast progenitor cells. Furthermore, at the end of the culture, to the extent that no peaks due to cells not satisfying all of the conditions of CD44-positive, CD99-positive, CD140B-positive, CD9-negative, CD49f-negative, and CD57-negative, particularly CD44-positive, CD140B-positive, and CD49f-negative, can be confirmed, preferably the proportion of cells not satisfying all of the conditions of CD44-positive, CD99-positive, CD140B-positive, CD9-negative, CD49f-negative, and CD57-negative, particularly CD44-positive, CD140B-positive, and CD49f-negative is 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, and particularly LBM that is substantially free of such inappropriate cells, is capable of inducing hCPCs and osteoprogenitor cells sufficient to be induced into good chondrocytes, cartilage tissue that can be used for transplantation.

[0047] A particularly preferred embodiment of the present invention is a two-stage quality control method in which CPCs are prepared using LBM that has been shown to be of good quality by the quality control method of the present invention, and the quality control method of the present invention is then performed on the CPCs. By performing the two-stage quality control method for LBM and CPCs, the quality of the CPCs and the chondrocytes and cartilage tissues finally obtained can be further improved.

[0048] CPCs can be used as transplant materials not only by themselves but also by being induced to become chondrocytes or cartilage tissue to serve as transplant materials. Therefore, quality control of CPCs alone, or two-stage quality control of LBM and CPCs, is important in the preparation of transplant materials.

[0049] CPC may be either a two-dimensional culture or a three-dimensional culture in a mass. When a CPC cell population is directly injected into an affected area as a cell suspension (transplant material), a plate culture is preferably used, and a mass of cartilage tissue can be obtained by inducing differentiation of the mass of CPC. The cell suspension of CPC can be injected into an affected area having cartilage such as a painful knee or meniscus, for example, in the same manner as a hyaluronic acid injection solution, and unlike hyaluronic acid, the cause of pain can be eliminated by regenerating the cartilage.

[0050] As shown in Figure 67, in the case of a single-peak cell population in which hCPCs are uniform and the three surface antigens are within the range of the defined criteria, good chondrocytes / cartilage tissue are induced, whereas in the case of chondrocytes / cartilage tissue induced from hCPCs with two or more peaks (double peaks) having a peak that satisfies and a peak that does not satisfy at least one selected from the group consisting of CD90 positivity and CD140B positivity, the chondrocytes / cartilage tissue have defects and are therefore unsuitable as a transplant material. Furthermore, in the case of single-peak hCPCs in which the three surface antigens are outside the range of the defined criteria, substantially no chondrocytes / cartilage tissue can be obtained.

[0051] As shown in Figure 69, in the case of a single-peak cell population in which the LBM is uniform and the six surface antigens are within the range of the specified criteria, good hCPCs are induced, whereas in the case of an LBM with two or more peaks (double peak) having a peak that satisfies and a peak that does not satisfy at least one selected from the group consisting of CD44 positive, CD99 positive, CD140B positive, CD9 negative, CD49f negative, and CD57 negative, particularly at least one selected from the group consisting of CD44 positive, CD140B positive, and CD49f negative, the chondrocytes / cartilage tissue induced therefrom have defects and are unsuitable as a transplant material. Furthermore, in the case of a single-peak LBM in which the six surface antigens are outside the range of the specified criteria, substantially no chondrocytes / cartilage tissue can be obtained.

[0052] In one embodiment of the present invention, CPCs can be differentiated into cartilage-like tissue and used as a graft material for transplantation into patients in need of cartilage transplantation.

[0053] In the method for inducing differentiation of CPCs selected by the quality control method of the present invention into chondrocytes, the period of culture in an environment that activates Wnt signals is not particularly limited as long as it does not impair the effects of the present invention. For example, it can be about 24 hours to 12 days, or about 4 days to 8 days. If necessary, the medium can be replaced. The culture conditions are preferably in accordance with conventional methods.

[0054] In the culture, subculture can be performed as necessary. When subculture is performed, the cells are collected before or immediately after reaching a confluent state, and the cells are seeded in a new medium. In the culture of the present invention, the medium can also be appropriately replaced.

[0055] The medium used in the present invention is not particularly limited. In a preferred embodiment, a known cartilage induction medium can be used. Examples of the cartilage induction medium include known compositions containing L-ascorbic acid, ITS, GDF5, BMP4, etc. If necessary, components such as antibacterial agents such as streptomycin and penicillin, Non-Essential Amino Acids (NEAA), and ROCK inhibitors (e.g., Y-27362) can be added.

[0056] Thus, chondrocytes or cartilage tissue are produced.

[0057] Whether chondrocytes have been obtained can be confirmed, for example, by positive Safranin O staining or positive Alcian Blue staining.

[0058] The LBM of the present invention can be used as a raw material for inducing the differentiation of functional cells and tissues into which PRRX1-positive cells (e.g., limb buds) differentiate in vivo. Examples of functional cells and tissues into which such cells differentiate include bone, fibroblasts (e.g., dermal fibroblasts), joints, cartilage, tendons, ligaments, and the like.

[0059] In a preferred embodiment of the LBM or its differentiated cells of the present invention, the LBM or its differentiated cells, chondroprogenitor cells and RUNX2-positive osteoprogenitor cells, can be cryopreserved. The cryopreserved LBM, chondroprogenitor cells, or RUNX2-positive osteoprogenitor cells can be propagated by subculture after thawing. 2. Method for inducing differentiation of pluripotent stem cells into LBM The above-mentioned LBM of the present invention can be produced by combining a step of inducing differentiation of pluripotent stem cells into lateral plate mesoderm cells and a step of culturing the cells induced to differentiate by the above step in an environment that activates Wnt signaling. Method for inducing differentiation via lateral plate mesoderm cells pluripotent stem cells In the method for producing LBM of the present invention, the above-mentioned pluripotent stem cells can be used.

[0060] Lateral plate mesoderm cells In the method for producing LBM of the present invention, first, differentiation of pluripotent stem cells into lateral plate mesoderm cells is induced.

[0061] Differentiation of pluripotent stem cells into lateral plate mesoderm cells can be carried out as described above (see FIG. 73).

[0062] Induction of differentiation of lateral plate mesoderm cells can be confirmed, for example, by detecting the expression of HAND1 protein, which is a specific marker for lateral plate mesoderm cells.

[0063] Induction of differentiation from lateral plate mesoderm cells to the PRRX1-positive limb bud mesenchymal cell population of the present invention Next, the differentiation-induced lateral plate mesoderm cells are cultured under an environment that activates the Wnt signal, and are induced to differentiate into the PRRX1-positive LBM of the present invention. In order to reduce the influence of the previous culture environment, it is preferable to perform the culture under an environment that activates the Wnt signal after washing with PBS buffer or the like as appropriate. The differentiation induction from the lateral plate mesoderm cells to LBM is preferably performed in the absence of an FGF signal activator such as FGF2.

[0064] Lateral plate mesoderm cells differentiate into cardiac mesoderm when cultured in the presence of FGF signal activators such as FGF2 and BMP. Therefore, when inducing LBM from lateral plate mesoderm, conditions that activate Wnt signaling are used, but it is desirable not to add an effective amount of FGF signal activators such as FGF2 to the medium. Since lateral plate mesoderm cells are induced to differentiate into LBM in a medium free of FGF signal activators, the number of cells does not increase significantly at the time of induction into LBM.

[0065] One of the features of the present invention is that chondroprogenitor cells are obtained while increasing the cell number by culturing LBM in a Wnt signal activation environment, preferably in the presence of an FGF signal activator such as FGF2. Conventionally, it was thought that FGF signal activators could not be used on LBM because they promote differentiation into unintended cells such as cardiac mesoderm when applied to lateral plate mesoderm, but the present inventors have found that high-quality chondroprogenitor cells can be obtained by applying a Wnt signal activator and an FGF signal activator after induction into LBM.

[0066] Culture in an environment that activates Wnt signaling Lateral plate mesoderm cells can be cultured in an environment in which Wnt signaling is activated, for example, by culturing in the presence of an effective amount of a Wnt signaling activator. As described above, during this culture, the content of the FGF signaling activator is preferably less than the effective amount for promoting cell proliferation, and more preferably does not contain an FGF2 signaling activator.

[0067] A Wnt signal activator enhances signal transduction mediated by Wnt (particularly, the canonical Wnt pathway). Examples of the Wnt signal activator include GSK3β inhibitors and Wnt family proteins.

[0068] Examples of GSK3β inhibitors include CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), XAV939, LiCl, and the like.

[0069] When CHIR99021 is used as the Wnt signal activator, the amount added can be, for example, about 0.1 to 20 μM, preferably 1 to 10 μM.

[0070] Culture under conditions that suppress BMP signaling In a preferred embodiment of the present invention, the step of culturing differentiation-induced lateral plate mesoderm cells in an environment that activates Wnt signaling can be carried out in an environment that suppresses BMP signaling.

[0071] Culturing under an environment that suppresses BMP signaling can be carried out, for example, by culturing in the presence of an effective amount of a BMP signaling suppressor.

[0072] BMP signal inhibitors suppress (inhibit) signal transduction mediated by BMP. Examples of BMP signal inhibitors include LDN193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) or its salt (e.g., hydrochloride), DMH-1, etc.

[0073] When LDN193189 is used as the BMP signal suppressor, the amount added can be, for example, about 0.1 to 10 μM, preferably 0.2 to 5 μM.

[0074] Culture in an environment that suppresses TGFβ signaling In a preferred embodiment of the present invention, the step of culturing differentiation-induced lateral plate mesoderm cells under an environment that activates Wnt signaling can be further carried out under an environment that suppresses TGFβ signaling.

[0075] Culturing under an environment that suppresses TGFβ signaling can be performed, for example, by culturing in the presence of an effective amount of a TGFβ signaling suppressor.

[0076] TGFβ signal inhibitors suppress (inhibit) signal transduction mediated by TGFβ. Examples of TGFβ signal activators include A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide) and SB431542.

[0077] When A-83-01 is used as a TGFβ signal suppressor, the amount added can be, for example, about 0.1 to 10 μM, preferably 0.2 to 5 μM.

[0078] Culture in an environment that suppresses Hedgehog signaling In a preferred embodiment of the present invention, the step of culturing differentiation-induced lateral plate mesoderm cells under an environment that activates Wnt signaling is preferably further carried out under an environment that suppresses Hedgehog (HH) signaling.

[0079] Culturing under an environment that suppresses hedgehog signaling can be carried out, for example, by culturing in the presence of an effective amount of a hedgehog signaling inhibitor.

[0080] Hedgehog signal inhibitors suppress (inhibit) signal transduction mediated by hedgehog. Hedgehog signal activators include vismordegib, cyclopamine, and sonidegib.

[0081] When bismoldegib is used as the hedgehog signal inhibitor, the amount added can be, for example, about 10 nM to 1 μM, and preferably about 50 nM to 500 nM.

[0082] In method (A) of the present invention, from the viewpoint of the efficiency of inducing the desired LBM of the present invention or its differentiated cells, it is preferable to perform the culture in an environment that activates Wnt signaling and suppresses BMP signaling, more preferably in an environment that activates Wnt signaling and suppresses BMP signaling, which suppresses TGFβ signaling and / or hedgehog signaling, and even more preferably in an environment that activates Wnt signaling, suppresses BMP signaling, suppresses TGFβ signaling, and suppresses hedgehog signaling.

[0083] The differentiation of lateral plate mesoderm cells into LBM can be induced in an environment that activates Wnt signaling, and further in the presence of one, two or three inhibitors selected from the group consisting of a TGFβ signaling inhibitor, a BMP signaling inhibitor and a hedgehog signaling inhibitor, and it is more preferable to perform the differentiation in the presence of a TGFβ signaling inhibitor, a BMP signaling inhibitor, a TGFβ signaling inhibitor and a hedgehog signaling inhibitor.

[0084] culture The culture can be carried out in a suitable container for storing the cells and the medium. Suitable culture methods include, but are not limited to, a culture method under conditions of about 37° C. and a carbon dioxide concentration of about 5%. Culture under the above conditions can be carried out, for example, by using a known CO 2 Temperature and CO using an incubator 2 This can be done while controlling the concentration.

[0085] The culture can be performed by two-dimensional cell culture (plate culture). Two-dimensional cell culture can be performed by coating the container as necessary to promote cell adhesion.

[0086] The period of culture in an environment that activates Wnt signals is not particularly limited as long as it does not impair the effects of the present invention. For example, it can be about 6 hours to 4 days, preferably about 1 day to 3 days, and more preferably about 2 days (about 48 hours). If necessary, the medium can be replaced. The culture conditions are preferably in accordance with conventional methods.

[0087] In the culture, subculture can be performed as necessary. When subculture is performed, the cells are collected before or immediately after reaching a confluent state, and the cells are seeded in a new medium. In the culture of the present invention, the medium can also be replaced as appropriate. If the culture is continued after reaching a confluent state, the quality of the LBM will decrease, and there is a high possibility that the conditions for quality control will not be met.

[0088] Culture medium It is preferable to use serum-free media such as CDM2 basal medium, IMDM medium, and F12 medium. Differentiation induction and maintenance culture are possible without containing animal-derived components. In addition, components such as antibiotics such as streptomycin and penicillin, non-essential amino acids (NEAA), and ROCK inhibitors (e.g., Y-27632) can be added.

[0089] Thus, LBM derived from the pluripotent stem cells of the present invention and positive for PRRX1 protein is produced.

[0090] It is believed that each PRRX1 protein-positive LBM cell produced by the present invention has properties equivalent to those of limb bud cells differentiated from lateral plate mesoderm cells in vivo. The LBM of the present invention is a homogeneous cell population, but LBM cells in vivo coexist with various cells, and exist as a population containing cells that do not satisfy one or both of the conditions of PRRX1 protein positivity and CD44 positivity, CD99 positivity, CD140B positivity, CD9 negativity, CD49f negativity, and CD57 negativity (particularly CD44 positivity, CD140B positivity, and CD49f negativity), so the limb bud mesenchymal cell population (LBM) of the present invention is novel.

[0091] The LBM of the present invention is homogeneous and of high quality and can be induced into chondrocytes and osteoprogenitor cells. The chondrocytes and osteoprogenitor cells themselves, or further the osteoblasts, chondrocytes, bone tissue, and cartilage tissue obtained therefrom, are excellent transplant materials.

[0092] 3. Differentiation into osteoblast precursor cells The LBM of the present invention can be used as a raw material cell for inducing differentiation into, for example, RUNX2-positive osteoprogenitor cells (hereinafter sometimes referred to as "RUNX2-positive cells").

[0093] RUNX2-positive cells refer to cells that can differentiate and develop into osteoblasts and further into bone tissue. RUNX2-positive cells and osteoblasts derived therefrom can be used as transplantation materials to be transplanted into patients who require bone regeneration.

[0094] RUNX2-positive cells can be obtained by a method including a step of culturing the LBM of the present invention in an environment that does not activate Wnt signals.

[0095] Culture in an environment that does not activate Wnt signaling Specifically, the culture in an environment that does not activate the Wnt signal can be performed by culture using a medium that does not substantially contain a Wnt signal activator. When a medium containing a Wnt signal activator is used immediately before the step of culturing in an environment that does not contain the Wnt signal activator, it is preferable to appropriately wash with a PBS buffer solution or the like to remove the Wnt signal activator, and then culture in an environment that does not contain the Wnt signal activator. When such washing is performed, even if a trace amount of a Wnt signal activator is contained in the medium, the Wnt signal is not activated, and therefore this is included in the "environment that does not activate the Wnt signal".

[0096] Examples of the Wnt signal activator include those mentioned above.

[0097] Culture under conditions that suppress BMP signaling In a preferred embodiment of the present invention for inducing differentiation into RUNX2-positive cells, LBM is cultured in a serum-free medium under an environment that suppresses BMP signaling.

[0098] Culturing under an environment that suppresses BMP signaling can be carried out, for example, by culturing in the presence of an effective amount of a BMP signaling suppressor.

[0099] Examples of the BMP signal inhibitor include those mentioned above.

[0100] When LDN193189 is used as the BMP signal suppressor, the amount added can be, for example, about 0.1 to 1 μM, preferably 0.2 to 5 μM.

[0101] Culture in an environment that activates TGFβ signaling In a preferred embodiment of the present invention for inducing differentiation into RUNX2-positive cells, the step of culturing in a serum-free medium can be carried out under an environment that further activates TGFβ signaling.

[0102] Culturing under an environment that activates TGFβ signaling can be carried out, for example, by culturing in the presence of an effective amount of a TGFβ signaling activator.

[0103] The TGFβ signal activator enhances signal transduction mediated by TGFβ. Examples of the TGFβ signal activator include TGFβ1 protein.

[0104] When TGFβ1 protein is used as the TGFβ signal activator, the amount added can be, for example, about 0.1 to 10 μM, preferably 0.2 to 5 μM.

[0105] Culture in an environment that activates Hedgehog signaling In a preferred embodiment of the present invention for inducing differentiation into RUNX2-positive cells, the step of culturing in a serum-free medium is preferably carried out under an environment that further activates hedgehog (HH) signaling.

[0106] Culturing under an environment that activates hedgehog signaling can be carried out, for example, by culturing in the presence of an effective amount of a hedgehog signaling activator.

[0107] The hedgehog signal activator enhances the signal transduction mediated by hedgehog. Examples of the hedgehog signal activator include hedgehog agonists such as 21K.

[0108] When 21K is used as the hedgehog signal activator, the amount added can be, for example, about 1 to 100 nM, preferably about 1 to 10 nM.

[0109] culture The culture can be carried out in a suitable container for storing the cells and the medium. Suitable culture methods include, but are not limited to, a culture method under conditions of about 37° C. and a carbon dioxide concentration of about 5%. Culture under the above conditions can be carried out, for example, by using a known CO 2 Temperature and CO using an incubator 2 This can be done while controlling the concentration.

[0110] In the method for inducing differentiation into RUNX2-positive cells, the culture can be performed by two-dimensional cell culture (plate culture). The two-dimensional cell culture can be performed by coating the culture equipment as necessary to promote cell adhesion.

[0111] In the method of inducing differentiation into RUNX2-positive cells, the period of culture in an environment that does not activate Wnt signals is not particularly limited as long as it does not impair the effects of the present invention. For example, the period can be about 1 to 20 days, preferably about 3 to 15 days, and more preferably about 5 to 10 days. If necessary, the medium can be replaced. The culture conditions are preferably in accordance with conventional methods.

[0112] In the culture, subculture can be performed as necessary. When subculture is performed, the cells are collected before or immediately after reaching a confluent state, and the cells are seeded in a new medium. In the culture of the present invention, the medium can also be appropriately replaced.

[0113] Culture medium In the present invention, it is preferable to use serum-free media such as CDM2 basal medium, IMDM medium, and F12 medium. Differentiation induction and maintenance culture are possible without containing animal-derived components. In addition, components such as antibiotics such as streptomycin and penicillin, non-essential amino acids (NEAA), and ROCK inhibitors (e.g., Y-27362) can be added.

[0114] Thus, RUNX2-positive cells are produced.

[0115] The fact that RUNX2-positive osteoblast precursor cells have been obtained can be confirmed, for example, by positive Alizarin Red staining after treating the RUNX2-positive osteoblast precursor cells with an osteoblast differentiation-inducing medium.

[0116] 4. Differentiation of LBM into chondrocyte precursor cells The LBM of the present invention can be used, for example, as a source cell for inducing differentiation into a population of chondroprogenitor cells.

[0117] The chondroprogenitor cell population and the chondrocyte cell population can be used as graft material for transplantation into a patient in need of a cartilage transplant.

[0118] A chondroprogenitor cell population can be obtained by a method comprising the step of culturing the LBM of the present invention in an environment that activates Wnt signaling, preferably further in the presence of an FGF signaling activator such as FGF2.

[0119] Culture in an environment that activates Wnt signaling Culturing under an environment that activates Wnt signaling can be carried out, for example, by culturing in the presence of an effective amount of a Wnt signaling activator.

[0120] A Wnt signal activator enhances signal transduction mediated by Wnt (particularly, the canonical Wnt pathway). Examples of the Wnt signal activator include GSK3β inhibitors and Wnt family proteins.

[0121] GSK3β inhibitors include those mentioned above.

[0122] When CHIR99021 is used as the Wnt signal activator, the amount added can be, for example, about 0.1 to 20 μM, preferably 1 to 10 μM.

[0123] Culture in the presence of FGF signal activators The differentiation of LBM into chondrocyte precursor cells requires FGF signal activators.

[0124] The FGF signal activator not only induces differentiation into chondroprogenitor cells, but also contributes to an increase in cell number, making it possible to obtain large amounts of chondroprogenitor cells.

[0125] The FGF signal activator includes FGF2, and is preferably FGF2.

[0126] It is preferable that the differentiation of chondroprogenitor cells into chondrocytes is performed in multiple stages. Specifically, (i) culturing the cells under an environment that activates Wnt signaling and an environment that activates FGF signaling; (ii) culturing in the presence of an FGF signal activator (preferably in the absence of a Wnt signal activator); (iii) culturing preferably in the absence of a Wnt signal activator and in the absence of an FGF signal activator; It is preferable to carry out the above three steps.

[0127] When differentiation is induced in the above three stages, steps (i) and (ii) can be performed by two-dimensional cell culture (plate culture) or three-dimensional culture. Two-dimensional cell culture can be performed by coating the culture equipment as necessary to promote cell adhesion. Step (iii) is preferably performed by three-dimensional culture. In addition, between each stage of culture, it is preferable to appropriately wash the culture medium with PBS buffer solution or the like before culturing in order to reduce the influence of the previous culture environment.

[0128] culture The culture can be carried out in a suitable container for storing the cells and the medium. Suitable culture methods include, but are not limited to, a culture method under conditions of about 37° C. and a carbon dioxide concentration of about 5%. Culture under the above conditions can be carried out, for example, by using a known CO 2 Temperature and CO using an incubator 2 This can be done while controlling the concentration.

[0129] In the method of the present invention for inducing differentiation of chondroprogenitor cells, (i) the period of culture in an environment that activates Wnt signaling is not particularly limited as long as it does not impair the effects of the present invention. For example, it can be about 24 hours to 12 days, or about 4 days to 8 days. (ii) The period of culture in the presence of an FGF signaling activator is not particularly limited as long as it does not impair the effects of the present invention. (iii) The period during which the culture is carried out preferably in the absence of a Wnt signal activator and an FGF signal activator is not particularly limited as long as the effect of the present invention is not impaired. In the culture of (i) to (iii), the medium can be replaced as necessary. The culture conditions are preferably in accordance with conventional methods.

[0130] In the culture, subculture can be performed as necessary. When subculture is performed, the cells are collected before or immediately after reaching a confluent state, and the cells are seeded in a new medium. In the culture of the present invention, the medium can also be appropriately replaced.

[0131] Culture medium The medium used in the method of the present invention is not particularly limited. In a preferred embodiment, a known cartilage induction medium can be used. Examples of the cartilage induction medium include known compositions containing L-ascorbic acid, ITS, GDF5, BMP4, etc. If necessary, components such as antibacterial agents such as streptomycin and penicillin, Non-Essential Amino Acids (NEAA), and ROCK inhibitors (e.g., Y-27362) can be added.

[0132] Thus, chondrocytes are produced.

[0133] Whether chondrocytes have been obtained can be confirmed, for example, by positive Safranin O staining or positive Alcian Blue staining.

[0134] The media used in the kit of the present invention are shown below. (i) Culture medium for inducing pluripotent stem cells into primitive streak cells Known media can be used, for example, the medium described in the literature: Loh et al., 2016, Cell, 451-467. (ii) Medium for inducing primitive streak cells to lateral plate mesoderm cells Known media can be used, for example, the medium described in the literature: Loh et al., 2016, Cell, 451-467. (iii) Culture medium for inducing limb bud mesenchymal cells from lateral plate mesoderm cells An example of such a medium is the above serum-free medium supplemented with a Wnt signal activator. This medium does not contain FGF signal activators such as FGF2. (iv) Culture medium for inducing chondrocyte progenitor cells from limb bud mesenchymal cells A medium containing an FGF signal activator such as FGF2 and a Wnt signal activator in addition to the above serum-free medium can be used. (v) Medium for inducing chondrocytes from chondroprogenitor cells For the induction of differentiation from chondroprogenitor cells to chondrocytes, it is preferable to use at least one of the following media (a) to (c), more preferably containing all of the media (a) to (c), or containing the media (a) and (c). (a) A medium in which a Wnt signal activator and an FGF signal activator were added to the above serum-free medium. The presence of the Wnt signal activator allows the cells to be induced into clumps of chondroprogenitor cells. When cultured in this medium, the cells have the properties of chondroprogenitor cells. (b) A medium obtained by adding an FGF signal activator to the above serum-free medium. This medium preferably does not contain a Wnt signal activator. By culturing in this medium, chondroprogenitor cells can be induced to differentiate into chondrocytes. (c) The above-mentioned serum-free medium may be used without adding a Wnt signal activator or an FGF signal activator, and preferably, BMP4 may be added to the medium. The medium may contain either one of the above (b) and (c), or may contain both. (vi) Culture medium for inducing RUNX2-positive osteoprogenitor cells from limb bud mesenchymal cells An example of such a serum-free medium is one that does not contain a Wnt signal activator. EXAMPLES

[0135] The present invention will now be described in more detail with reference to examples.

[0136] FIG. 1 shows an outline of the differentiation induction protocol of the present invention.

[0137] From the limb bud mesenchymal cell population, it is possible to induce further differentiation of cells such as chondrocyte precursor cells and RUNX2-positive osteoprogenitor cells into osteoblasts, chondrocytes, tendon ligament cells, and dermal fibroblasts, which are mainly cells of the bone and joint tissue that make up the limbs.

[0138] Reference Example 1: Generation of PRRX1 reporter human iPS cells Using the targeting vector and CRISPR / Cas9 system shown in Figure 2, the fluorescent protein tdTomato was knocked into the PRRX1 locus using 414C2 cells, an iPS cell line, as the parent line. This resulted in the creation of PRRX1 reporter iPS cells that could visualize PRRX1 expression in iPS cells with the tdTomato fluorescent protein.

[0139] Example 1: Induction of differentiation into PRRX1-positive cells via the lateral plate mesoderm lineage (Induction of lateral plate mesoderm from iPS cells via the primitive streak) According to the protocol shown in FIG. 3, each of the iPS cell lines (414C2, PRRX1 reporter cell line) was induced to differentiate into the mid-primitive streak (day 1) and then into the lateral plate mesoderm (day 2).

[0140] First, the undifferentiated PRRX1 reporter iPS cells obtained in Reference Example 1 were seeded and cultured in a normal iPSC medium for 72 hours (hr) (day 0).

[0141] Next, after washing once with PBS, the cells were cultured for 24 hours in a medium having the following composition in order to induce differentiation into primitive streaks. Primitive streak differentiation induction medium CDM2 basal medium 30 ng / ml Activin A 40 ng / ml BMP4 6 μM CHIR99021 20 ng / ml FGF2 100 nM PIK90 10 μM Y-27632 Next, after washing once with PBS, the cells were cultured for 24 hours in a medium having the following composition in order to induce differentiation into lateral plate mesoderm. Primitive streak differentiation induction medium CDM2 basal medium 1 μM A-83-01 30 ng / ml BMP4 1 μM C59 10 μM Y-27632 The induced cells were immunostained for HAND1 (a marker for lateral plate mesoderm) and CDX2 (a marker for paraxial mesoderm). The results are shown in Figures 4 and 5. It was confirmed that the induced lateral plate mesoderm cells were HAND1 positive and CDX2 negative.

[0142] (Investigation of conditions for inducing differentiation of PRRX1-positive cells) Lateral plate mesoderm cells (day 2) carrying the induced PRRX1 reporter as described above were washed once with PBS and then cultured for 48 hours in CDM2 basal medium containing activators and / or inhibitors of various signals (16 combinations) shown in Figure 7 (Figure 6).

[0143] The reagents used are as follows: CHIR(CHIR99021, GSK3β inhibitor) C59 (Wnt-C59, PORCN inhibitor) BMP4(Bone morphogenetic Protein 4) LDN (LDN193189, ALK2 / ALK3 inhibitor) TGFβ1(Tumor growth factorβ1) A8301 (A83-01, ALK5 inhibitor) 21K(Hedgehog agonist) Vismodegib (Hedgehog inhibitor) After 48 hours of culture (day 4), the expression of PRRX1 in the cells was observed and evaluated by observing the expression of the reporter tdTomato under a fluorescent microscope.

[0144] The results are shown in Figure 8. Particularly high tdTomato expression was observed in combinations 1, 2, 3, 4, 5, 6, 7, and 8.

[0145] RNA was collected from the cells after 48 hours of culture (day 4), and the expression of PRRX1 was evaluated by qPCR (quantitative RT-PCR).

[0146] The results are shown in Figure 9. Particularly high expression of PRRX1 was observed in combinations 1, 2, 3, 4, 5, 6, 7, and 8.

[0147] In addition, RNA was collected from the cells after 48 hours of culture (day 4), and the expression of HAND1, a marker of the lateral plate mesoderm, was measured by qPCR (quantitative RT-PCR).

[0148] The results are shown in Figure 10. In combinations 1, 2, 3, and 4, HAND1 expression was increased compared to day 2, but in combinations 5, 6, 7, and 8, HAND1 expression was decreased.

[0149] In addition, RNA was collected from the cells after 48 hours of culture (day 4), and the expression of ISL-1, a marker of the lateral plate mesoderm, was measured by qPCR (quantitative RT-PCR).

[0150] The results are shown in Figure 11. In combinations 5, 6, 7, and 8, ISL1 expression was decreased compared to day 2.

[0151] In addition, RNA was collected from the cells after 48 hours of culture (day 4), and the expression of FOXF1, a marker of the lateral plate mesoderm, was measured by qPCR (quantitative RT-PCR).

[0152] The results are shown in Figure 12. In combinations 5, 6, 7, and 8, FOXF1 expression was decreased compared to day 2.

[0153] From the above results, Combination 8 was selected as a particularly effective condition for inducing PRRX1-positive limb bud mesenchymal cells from lateral plate mesoderm cells (day 2) (FIG. 13).

[0154] (Evaluation of differentiation induction efficiency) The efficiency of induction into tdTomato-positive cells in the above differentiation induction process was evaluated by flow cytometry. The results are shown in Figure 14. The efficiency of induction into tdTomato-positive cells on Day 4 was 98% or higher.

[0155] Furthermore, immunostaining was performed using PRRX1 antibody on the cells on Day 4. The results are shown in Figure 15. All of the PRRX1-positive cells were tdTomato-positive.

[0156] (CD antigen expression profile) The expression of CD antigens in tdTomato-positive cells in the above differentiation induction step was evaluated using a CD antibody array (product name: BD Lyoplate Screening Panels, manufactured by BD).

[0157] The results are shown in Figures 16 to 18. On Day 4, tdTomato-positive cells were 1) CD24 positive, CD29 positive, CD44 positive, CD46 positive, CD55 positive, CD58 positive, CD90 positive, CD140A positive, CD140B positive (Figure 16), 2) SSEA1 negative, SSEA3 negative, SSEA4 positive, TRA-1-60 negative, TRA-1-81 negative ( FIG. 17 ), and 3) CD73 negative, CD90 positive, CD105 negative, and CD166 negative (Figure 18).

[0158] Specifically, tdTomato-positive cells at Day 4 were: 1) substantially homogeneous, 2) expression of most ES cell markers was reduced during the differentiation induction process up to Day 4, and 3) expression of human mesenchymal stem cell (MSC) markers was also low.

[0159] (Study on human iPS cells (1383D2 line)) A human iPS cell line (1383D2 line) was treated with the differentiation induction protocol established for the PRRX1 reporter cell line. The protocol is shown in FIG.

[0160] FIG. 20 shows phase contrast microscopic images at each time point, and FIG. 21 shows the PRRX1 mRNA expression level at each time point. In differentiation induction using the 1383D2 strain, an increase in PRRX1 expression was also observed at day 4. FIG. 22 shows the mRNA expression levels of lateral plate mesoderm markers (HAND1, ISL1, FOXF1) at each time point. In differentiation induction using the 1383D2 strain, the mRNA of each lateral plate mesoderm marker was significantly reduced. FIG. 23 shows the results of immunostaining using a PRRX1 antibody on days 0 and 4. Most of the induced cells at day 4 were PRRX1 positive.

[0161] (Study on human ES cells (SEES4, SEES5, SEES6, SEES7)) Fig. 24 shows phase contrast microscopy images at each time point when human ES cell lines (SEES4, SEES5, SEES6, SEES7) were treated with the differentiation induction protocol established for the PRRX1 reporter cell line. Fig. 25 shows the mRNA expression levels of PRRX1 and mesodermal markers (HAND1, ISL1, FOXF1) at each time point. When differentiation was induced from day 2 to day 4, PRRX1 expression increased and the mRNA of each lateral plate mesoderm marker significantly decreased. Fig. 26 shows the results of immunostaining using a PRRX1 antibody on day 4. Most of the cells induced at day 4 were PRRX1-positive limb bud mesenchymal cells.

[0162] Example 2: Induction of differentiation into PRRX1-positive cells via the paraxial mesoderm lineage (Induction of lateral plate mesoderm from iPS cells via the primitive streak) According to the protocol shown in FIG. 27, each of the iPS cell lines (414C2, PRRX1 reporter cell line) was induced to develop into the primitive streak (anterior-primitive streak, day 1) and the paraxial mesoderm (day 2).

[0163] First, the undifferentiated PRRX1 reporter iPS cells obtained in Reference Example 1 were seeded and cultured in a normal iPSC medium for 72 hours (hr) (day 0).

[0164] Next, the cells were washed once with PBS, and then cultured for 24 hours in a medium having the following composition to induce differentiation into paraxial mesoderm. Primitive streak differentiation induction medium CDM2 basal medium 30 ng / ml Activin A 4 μM CHIR99021 20 ng / ml FGF2 100 nM PIK90 Next, after washing once with PBS, the cells were cultured for 24 hours in a medium having the following composition in order to induce differentiation into paraxial mesoderm. Paraxial mesoderm differentiation induction medium CDM2 basal medium 1 μM A-83-01 3 μM CHIR99021 250 nM LDN-193189 20 ng / ml FGF2 The induced cells were immunostained for HAND1 (a marker for lateral plate mesoderm) and CDX2 (a marker for paraxial mesoderm). The results are shown in Figures 28 and 29. It was confirmed that the induced paraxial mesoderm cells were HAND1 negative and CDX2 positive.

[0165] (Investigation of conditions for inducing differentiation of PRRX1-positive cells) The above-mentioned induced PRRX1 reporter-containing paraxial mesoderm cells (day 2) were washed once with PBS and then cultured for 48 hours in CDM2 basal medium containing various signal activators and / or inhibitors (six combinations) shown in Figure 30.

[0166] The reagents used are as follows: CHIR(CHIR99021, GSK3β inhibitor) BMP4 (Bone morphogenetic Protein 4) DAPT (γ-Secretase inhibitor) PD0325901 (MEK inhibitor) After 48 hours of culture (day 4), the expression of PRRX1 in the cells was evaluated by observing the expression of the reporter tdTomato under a fluorescent microscope.

[0167] The results are shown in Figure 31. Particularly high tdTomato expression was observed in the four combinations: CHIR / BMP4, CHIR / BMP4, CHIR / BMP4 / DAPT, CHIR / BMP4 / PD, and CHIR / BMP4 / DAPT / PD.

[0168] Treatment with each of the four types of treatment, CHIR / BMP4, CHIR / BMP4 / DAPT, CHIR / BMP4 / PD, and CHIR / BMP4 / DAPT / PD, was performed, and the induction efficiency of tdTomato-positive cells was examined 48 hours later (day 4) by flow cytometry. The results are shown in Figure 32. The induction efficiency of tdTomato-positive cells was found to be CHIR / BMP4 (61%), CHIR / BMP4 / DAPT (75%), CHIR / BMP4 / PD (91%), and CHIR / BMP4 / DAPT / PD (96%).

[0169] Four types of treatments were performed: CHIR / BMP4, CHIR / BMP4 / DAPT, CHIR / BMP4 / PD, and CHIR / BMP4 / DAPT / PD. After 24 hours (day 3) or 48 hours (day 4), RNA was collected from each, and the expression of PRRX1, paraxial mesoderm marker (CDX2), and somite markers (MEOX1, PARAXIS) was measured by qPCR. The results are shown in Figure 33. Although PRRX1 expression was increased in all combinations, the combination of CHIR / BMP4 / PD showed low expression levels of both paraxial mesoderm markers and somite markers.

[0170] Example 3: Induction of differentiation of limb bud mesenchymal cells into RUNX2-positive osteoprogenitor cells Limb bud mesenchymal cells were obtained according to the method described in Example 1. Next, the obtained cells were treated with activators or inhibitors of various signals shown in Figure 34 to investigate the conditions for inducing them into RUNX2-positive osteoprogenitor cells (16 combinations). CHIR(CHIR99021, GSK3b inhibitor) C59 (Wnt-C59, PORCN inhibitor) BMP4 (Bone morphogenetic Protein 4) LDN (LDN193189, ALK2 / ALK3 inhibitor) TGFβ1 (Tumor growth factor β1) A8301 (A83-01, ALK5 inhibitor) 21K(Hedgehog agonist) Vismodegib (Hedgehog inhibitor) Limb bud mesenchyme cells (day 4, limb bud mesenchyme) were treated with 16 different combinations, and 48 hours later (day 6), RNA was extracted from each treatment and the expression of RUNX2 (a transcription factor essential for osteoblast differentiation) was measured by qPCR.

[0171] The results are shown in Figure 35. High expression of RUNX2 was observed in combinations 9, 10, 11, 12, 13, 14, 15, and 16. Among these, combination 13 in particular showed higher RUNX2 expression than the other groups.

[0172] From the above results, combination 13 was selected as a particularly effective condition for inducing RUNX2-positive osteoprogenitor cells from limb bud mesenchymal cells (FIG. 36).

[0173] RNA was collected over time from limb bud mesenchymal cells (day 4) to day 12, and the expression of various genes (PRRX1, RUNX2, SP7, COL1A1, bone sialoprotein (BSP)) was measured by qPCR.

[0174] The results are shown in Figure 37. It was revealed that by inducing limb bud mesenchymal cells to become RUNX2-positive osteoblast progenitor cells, the expression of PRRX1 decreased over time, while the expression of RUNX2 and other osteoblast differentiation markers (SP7, COL1A1, BSP) increased.

[0175] The 414C2, PRRX1 reporter cell line, 1383D2, SEES4, and SEES7 lines were each induced to develop into limb bud mesenchyme cells (day 4), and then treated with Combination 13. RNA was collected 8 days later (day 12), and RUNX2 expression was measured by qPCR.

[0176] The results are shown in Figure 38. It was revealed that RUNX2 expression was significantly increased by combination 13 treatment in limb bud mesenchymal cells derived from all cell lines.

[0177] Immunostaining of cells using RUNX2 antibody was performed on days 4 and 12 for the PRRX1 reporter cell line, 1383D2 line, and SEES4 line.

[0178] The results are shown in Figure 39. No RUNX2 expression was observed on Day 4, but on Day 12, most of the cells were RUNX2 positive.

[0179] Furthermore, flow cytometry analysis using RUNX2 antibody was performed on day 12 for each of the 414C2 strain, the PRRX1 reporter cell line, the SEES4 strain, and the SEES7 strain.

[0180] The results are shown in Figure 40. 95% or more of the induced cells in each line were RUNX2 positive.

[0181] The PRRX1 reporter cell line was induced to differentiate into limb bud mesenchyme cells (day 4), then treated with Combination 13 and cultured for 8 days. To examine whether the cells at day 12 had the ability to differentiate into osteoblasts, the cells were cultured in osteoblast induction medium for 12 days and then stained with Alizarin Red.

[0182] The results are shown in Figure 41. Alizarin red staining was significantly increased in the induced cells.

[0183] Example 4: Method for inducing hCPCs by passage of LBM using stem medium 3 (hCPC medium) and method for subculture of hCPCs using stem medium 3 Using the PRRX1 reporter cell line obtained in Reference Example 1, lateral plate mesoderm-derived PRRX1-positive cells (day 4, limb bud mesenchyme) were prepared according to the differentiation induction conditions of Example 1. Conditions for the method of expanding and culturing the cells were examined.

[0184] Using Accutase, PRRX1-positive cells derived from the lateral plate mesoderm were collected on day 4, and the collected cells were subcultured in culture media of various compositions (stem medium 1, 2, 3). As a result, good results were obtained when Y-27632 was added to stem medium 3 (CDM2 basal medium + CHIR + A8301 + EGF + FGF).

[0185] The protocol is shown in FIG. 42, and phase contrast images under each condition are shown in FIG.

[0186] We also investigated the type of dish coating. Comparing the cell counts one week after subculture, the results were as follows: Non-coat = Gelatin < iMatrix-511 silk = Geltrex = Fibronectin. We selected coating with iMatrix-511, which is clinical grade and was the same as that used for differentiation induction, and used it in the following experiment (Figure 44).

[0187] Phase contrast microscopic images taken at each passage number when subculture was performed under the above conditions are shown in Figure 45. The limb bud mesenchymal cells were subcultured while maintaining their morphology.

[0188] Figure 46 shows the flow cytometry analysis results and growth curves of tdTomato at each passage number during subculture. It was revealed that subculture was possible while maintaining the tdTomato (PRRX1) positive state. Example 5: Properties of hCPCs derived from limb bud mesenchymal cells (proliferation, karyotype, storage in liquid nitrogen) Human chondrocyte precursor cells (CPCs) were generated from iPSC lines derived from healthy subjects (414C2, 1383D2, PRRX1 reporter, HPS1042, HPS1043) or human ES cell lines (SEES4, SEES6, SEES7).

[0189] Human LBM was prepared from iPSC lines derived from healthy subjects (414C2, 1383D2, HPS1042, HPS1043), a PRRX1 reporter iPSC line (Reporter), or a human ES cell line (SEES6), and human chondroprogenitor cells (hCPCs) were further prepared from the human LBM. The number of cells when hCPCs were prepared from human LBM is shown in Figure 47. It was revealed that hCPCs have a doubling time of approximately 2 to 3 days.

[0190] In addition, hCPCs (PN3) were generated from human iPS cells (414C2), and the karyotypes of human iPS cells (414C2) (Figure 48(a)) and hCPCs (PN3) (Figure 48(b)) were examined. Figure 48 revealed that the karyotype did not change during the process of inducing iPS cells to hCPCs.

[0191] Furthermore, it was revealed that the subcultured limb bud mesenchymal cells could be stored in liquid nitrogen in a STEMCELL BANKER, and that the stored cells could be thawed, replated, and subcultured (Figure 49). Example 6: Comparison of the expression levels of RUNX2 (a marker gene for osteoblast precursor cells) when LBM (day 4) or hCPCs were cultured in a RUNX2-positive osteoblast precursor cell differentiation-inducing medium.

[0192] Limb bud mesenchymal cells (LBM) (Day 4) and hCPCs were treated with Combination 13 (RUNX2-positive osteoprogenitor cell differentiation-inducing medium: composition is shown in Figure 50), and RNA was collected over time. As a result, RUNX2 expression increased over time in LBM (Day 4), but no such change was observed in hCPCs (Figure 50). hCPCs were expanded LBM (Day 4), but had a different differentiation induction ability from RUNX2-positive osteoprogenitor cells. hCPCs did not have the ability to differentiate into RUNX2-positive osteoprogenitor cells, and were oriented toward cartilage differentiation, making it clear that hCPCs and LBM (Day 4) are different entities. ·RUNX2 measurement conditions RNA was collected from the cells cultured for each number of days, converted to cDNA, and the mRNA expression of RUNX2 and ACTB (internal standard) was measured by real-time PCR (the primers used are shown in Table 1). After measurement, the expression level of RUNX2 was corrected by the expression level of ACTB.

[0193] [Table 1]

[0194] Example 7 For iPS (day 0), lateral plate mesoderm cells (day 2), LBM (day 4), hCPCs, and hCPCs treated with Step 1 medium and Step 2 medium shown in Figure 51(b), RNA was collected and the expression of chondrocyte markers (SOX5, SOX6, SOX9, COL2A1, ACAN) was examined. As a result, it was revealed that the chondrocyte markers were significantly increased in cells treated with Step 2 medium (Figure 51(d)).

[0195] Example 8: Staining of hyaline cartilage-like tissue Human chondroprogenitor cells (hCPCs) were prepared from a PRRX1 reporter iPSC line (Reporter) or a human ES cell line (SEES5). The cells were harvested from the culture dish and seeded at 100,000 cells / well per well of an ultra low U-bottom 96-well plate, and then aggregates were prepared on the wells by centrifugation. The cells were then treated according to the protocol shown in Figure 52(a), and sections were prepared from the hyaline cartilage tissue on the 42nd day of step 3 treatment (Figure 52(b)). They were stained with HE, Alcian Blue, and Safranin O, and further immunostained for chondrocyte markers (SOX9, COL2), hypertrophic chondrocyte marker (COLX), and fibrous cartilage marker (COL1) (Figure 52(c)). The results in Figure 52 revealed that hyaline cartilage tissue was obtained.

[0196] Example 9: Preparation of cartilage tissue using LBM or hCPCs Hyaline cartilage tissue was induced from LBM or hCPC under the conditions shown in Figure 53, and the results of Safranin O staining are shown in Figure 53. It was revealed that the induction efficiency into hyaline cartilage tissue derived from LBM differed from that derived from hCPC.

[0197] Example 10: Subcutaneous transplantation of hyaline cartilage-like tissue into NOD-SCID mice Human chondroprogenitor cells (hCPCs) were prepared from a healthy individual-derived iPSC line (414C2), a PRRX1 reporter iPSC line (Reporter), or a human ES cell line (SEES6). The cells were collected from a culture dish and seeded at 100,000 cells / well per well of an ultra low U-bottom 96-well plate, and then aggregates were prepared on the wells by centrifugation. The cells were then treated according to the protocol shown in Figure 54(a), and the aggregates on the 21st day of step 3 treatment were subcutaneously transplanted into NOD-SCID mice. Sections were prepared from the hyaline cartilage tissue obtained 8 weeks after transplantation (front side, center part, innermost part, Figure 54(b)), and stained with Safranin O (SafO), which stains the area where the cartilage matrix is ​​present red. As a result, a uniform hyaline cartilage tissue was formed without tumor formation (Figure 54(c)).

[0198] Example 11: Transplantation experiment of human-derived cartilage tissue mass into cartilage defect site of SCID rat Human chondroprogenitor cells (hCPCs) were prepared from the PRRX1 reporter iPSC line (Reporter). The cells were collected from a culture dish and seeded at 100,000 cells / well per well of an ultra low U-bottom 96-well plate, and then aggregates were prepared on the wells by centrifugation. The cells were then treated according to the protocol shown in FIG. 55(a), and the aggregates on the 21st day of step 3 treatment were stained with SafO and confirmed to be safranin positive. A hole was made in the knee articular cartilage of a SCID rat with a biopsy trephine (1 mm), and the cartilage tissue mass (above aggregates) was transplanted. Four weeks after transplantation, SafO staining, toluidine blue staining, and immunostaining using a human vimentin-specific antibody were performed. The engraftment of the human-derived cartilage tissue mass into the cartilage defect site of the SCID mouse was confirmed (FIG. 55(b)).

[0199] Example 12: Subrenal capsule transplantation of hCPC cells into NOD-SCID mice Human chondrocyte precursor cells (hCPCs) were generated from the PRRX1 reporter iPSC line. hCPC cells 1×10 5 hCPC cells were suspended in 10 μl of type I collagen gel and transplanted under the kidney capsule of NOD-SCID mice (Figure 56(a)). The results of staining (HE, Alcian Blue, Safranin O), SOX9 expression, and human nucleolus staining (human Nucleoli) after 8 weeks are shown in Figure 56(b). It was confirmed that cartilage tissue was formed at the transplant site simply by transplanting a cell suspension of hCPC into an adult.

[0200] Example 13: Preparation of cartilage plates Using quality-controlled human chondroprogenitor cells (Figure 57(a)), chondroprogenitor cell spheroids were formed using a dimple plate, which was then placed in a mold and cultured, successfully forming plate-shaped cartilage tissue that was larger than conventional cartilage tissue balls (Figure 57(b)).

[0201] The obtained plate-like cartilage had a size suitable for use in human transplantation.

[0202] Furthermore, the tissue formed after induction of chondrocyte differentiation in the mold was fixed and stained with Safranin O. As a result, it was confirmed that most of the tissues were morphologically hyaline cartilage-like and Safranin O-positive (Figure 58(a)). Furthermore, the plate-shaped cartilage tissue was transplanted subcutaneously into NOD-SCID mice. The tissues were removed 4 weeks after transplantation and stained with Safranin O. The results are shown in Figure 58(b). It was confirmed that the prepared cartilage plate was engrafted subcutaneously and that the hyaline cartilage tissue was maintained in the body without forming tumors.

[0203] Example 14: Differentiation induction from human LBM cells to hCPCs and quality control method for hCPCs The human LBM (day 4) cell population obtained in Example 1 was cultured under the following conditions to obtain hCPCs. Culture medium and conditions for hCPCs (i) PRRX1-positive single-peak hCPCs (Figure 61(a, b)) Using iMatrix511 as the coating agent, human LBM cells are suspended in the following medium and seeded onto a culture dish. hCPCs can be maintained in a PRRX1-positive state by subculturing the cells before they become subconfluent. ·hCPC medium CDM2 basal medium 1 μM A-83-01 3 μM CHIR99021 20 ng / ml FGF2 20 ng / ml EGF 10 μM Y-27632

[0204] (ii) PRRX1-positive and PRRX1-negative double peaks for hCPCs (Figure 61(a, b)) hCPC cells are cultured in a confluent state for one week and then subcultured to obtain a double-peak hCPC cell population. The double-peak hCPC cell population eventually becomes a PRRX1-negative hCPC cell population, which will be described later, and therefore cannot be maintained in culture.

[0205] (iii) PRRX1-negative single-peak hCPCs (Figure 61(a, b)) By continuously subculturing the above double-peak hCPCs, a PRRX1-negative hCPC single-peak cell population can be obtained.

[0206] (iv) Quality control of chondrocyte precursor cells (CPCs) and limb bud mesenchymal cells (LBM) An overview of the quality control technology for CPCs is shown in Figure 59. It has become clear that in order to stably supply chondrocytes / tissues from CPCs, it is important to expand and culture hCPCs under conditions that satisfy at least one condition selected from the group consisting of CD90 positivity and CD140B positivity. An overview of LBM quality control technology is shown in Figure 60. In order to supply CPCs with high cartilage tissue formation ability from LBM, it has become clear that it is important to induce CPCs from "LBM in a state that satisfies at least one condition selected from the group consisting of CD44-positive, CD99-positive, CD140B-positive, CD9-negative, CD49f-negative, and CD57-negative," and in particular, it is important to induce CPCs from "LBM in a state that satisfies at least one condition selected from the group consisting of CD44-positive, CD140B-positive, and CD49f-negative."

[0207] Example 15: Induction of cartilage tissue from quality-controlled hCPCs in one step Furthermore, cartilage tissue was induced from each hCPC under the following conditions, and the results of HE staining, Alcian Blue staining, and Safranin O staining are shown in Figure 61(c).

[0208] ·HE staining conditions After deparaffinization, the specimens were stained with Eosin for 1 minute, and then treated with hematoxylin for 20 minutes for nuclear staining.

[0209] ·Alcian Blue staining conditions, After deparaffinization, the specimens were treated with 1% Alcian Blue / 3% acetic acid for 20 minutes, washed with 3% acetic acid, and then treated with hematoxylin for 20 minutes for nuclear staining.

[0210] ·Safranin O staining conditions After deparaffinization, the sections were stained with Weigert's iron hematoxylin solution for 10 minutes, then stained with fast green solution for 5 minutes, washed with 1% acetic acid, and then stained with 0.1% Safranin O solution for 5 minutes.

[0211] Furthermore, for PRRX1-positive single-peak hCPCs and PRRX1-negative single-peak hCPCs, nodule formation was promoted by inducing chondrocyte differentiation, and the results of Alcian Blue staining are shown in FIG. 62. Furthermore, the obtained hCPCs (PRRX1-positive single peak, PRRX1-negative single peak, and PRRX1-positive and PRRX1-negative double peak) were analyzed by RNA sequencing, and mRNAs highly expressed in PRRX1-positive and PRRX1-negative were selected. The results are shown in Figures 63 to 66. Furthermore, the expression of CD antigens in human iPS cells (414C2), hCPCs (PRRX1 negative), and hCPCs (PRRX1 positive) was evaluated using antibodies against each CD antigen. The results are shown in Figure 67.

[0212] Example 16: Quality control method for human LBM Human iPS cells, 3x10 4 The cells are seeded at a density of 10 cells / 3.5 cm dish, and 3 days (Pluripotent 3 days) or 7 days (Pluripotent 7 days) after seeding, treatment to obtain LBM cells (described in Example 1) is performed.

[0213] When treatment was started 3 days after seeding, LBM with high PRRX1 expression (PRRX1 positive, pluripotent 3 days) was obtained, whereas when treatment was started 7 days after seeding, LBM with low PRRX1 expression (PRRX1 negative, pluripotent 7 days) was obtained (Figure 68(a)-(c)). In other words, this shows that the state of human pluripotent stem cells at the start of induction is important in obtaining LBM (PRRX1 positive).

[0214] In the first passage to induce hCPCs from LBM (PRRX1 negative, pluripotent 7 days) [hCPC (pluripotent7 days, PN1)], a population with low PRRX1 expression was obtained. With increasing passage numbers (PN1→PN2→PN3→PN4→PN5), at the PN2 stage, a heterogeneous cell population of low and high PRRX1 expression was obtained [hCPC, (pluripotent7 days, PN2)], and at the final PN5 stage, a homogeneous population with high PRRX1 expression was obtained [hCPC, (pluripotent7 days, PN5)] (Figure 68(b)). When a heterogeneous cell population with low / high PRRX1 expression [hCPC (pluripotent 7 days, PN2)] or a homogeneous cell population with high PRRX1 expression [hCPC (pluripotent 7 days, PN5)] was induced to differentiate into cartilage, cartilage tissue that was uniformly stained with Alcian blue / Safranin O was formed from hCPC (pluripotent 7 days, PN5), whereas heterogeneous cartilage tissue that was sparsely stained with Alcian blue / Safranin O was formed from hCPC (pluripotent 7 days, PN2) (Figure 68(c)).

[0215] Furthermore, the expression of CD antigens was evaluated for human iPS cells (negative control), LBM (PRRX1 negative, 7 days), and hCPCs (PRRX1 positive, 3 days) using antibodies against each CD antigen. The results are shown in Figure 69.

[0216] Example 17: Type II collagen disorder disease model 1 hCPCs were prepared from iPSC lines (ACGII, HCG) derived from patients with type II collagen dysplasia-related diseases (Figure 70(a)). The expression of PRRX1 was immunostained for each hCPC (Figure 70(b)) and the growth curve (Figure 70(c)) was confirmed. Each was subjected to the above-mentioned treatment process under "plate culture" to induce chondrocyte differentiation, and cartilage matrix staining with Alcian blue or the measurement of the expression of various chondrocyte differentiation marker genes (qPCR) was performed. As a result, compared with hCPCs derived from healthy subjects (414C2), hCPCs derived from PRRX1 reporter iPSC lines (Reporter), and ES cell-derived hCPCs (SEES4, SEES5, SEES7), the hCPCs derived from patients with type II collagen dysplasia-related diseases (ACG-II-1, HCG-1) had a reduced ability to form a cartilage matrix positive for Alcian blue staining (Figure 70(d)), and the expression of various chondrocyte differentiation marker genes was reduced (Figure 70(e)). Cells derived from patients with type II collagen deficiency-related diseases have a reduced ability to differentiate into chondrocytes compared to cells derived from healthy individuals. By using hCPCs, we can reproduce the pathological condition of patients. By using this pathological model, we can search for drugs that are effective not only for type II collagen deficiency but also for a wide range of cartilage-related diseases.

[0217] Example 18: Type II collagen disorder disease model 2 hCPCs were prepared from iPSCs derived from healthy subjects (414C2) or from patients with type II collagen deficiency-related diseases (ACGII-1, HCG-1). The cells were harvested from culture dishes and seeded at 100,000 cells / well in an ultra low U-bottom 96-well plate, and then centrifuged to produce aggregates on the wells. The cells were then treated according to the protocol described above, and the aggregates finally obtained were observed under an electron microscope. As a result, a decrease in glycogen levels in chondrocytes was observed in cells derived from patients with type II collagen deficiency-related diseases compared to cells derived from healthy subjects. In addition, it was found that the normal orientation of ER observed in normal cells was not observed in cells derived from patients (Figure 71). A decrease in glycogen levels in chondrocytes was observed in cells derived from patients with type II collagen deficiency-related diseases compared to cells derived from healthy subjects. In addition, the normal orientation of ER observed in normal cells was not observed in cells derived from patients. The use of hCPCs derived from patient-derived iPS cells can recapitulate the patient's pathology.

[0218] Example 19: Osteogenesis imperfecta disease model iPSC lines derived from patients with osteogenesis imperfecta (OIO2-1, OIO8-12) or iPSC lines in which the COL1A1 mutations were reverted to normal by genome editing (various rescue lines; OIO2-1(res1), OIO8-12(res1)) were induced to become RUNX2-positive osteoprogenitor cells in plate culture, and then induced to differentiate into osteoblasts (Figure 72). We then performed bone matrix staining with Alizarin Red, measurement of Ca2+ accumulation and its ratio to total protein, and ALP activity. As a result, it was found that Alizarin Red stainability and various measured values ​​were significantly higher in the rescue lines (Figure 72). Compared to cells in which the mutations were reverted to normal, cells derived from patients with osteogenesis imperfecta have a reduced ability to differentiate into osteoblasts. By using a protocol for inducing differentiation of RUNX2-positive osteoprogenitor cells from PRRX1-positive limb bud mesenchymal cells, the pathology of patients with osteogenesis imperfecta can be reproduced in vitro. [Industrial Applicability]

[0219] The LBM, CPCs, and osteoprogenitor cells quality-controlled according to the present invention, as well as chondrocytes or cartilage tissue, osteoblasts, or bone tissue induced therefrom, can be preferably used for transplantation to treat bone- or cartilage-related diseases (such as knee osteoarthritis, meniscus injury, rheumatoid arthritis, and osteoporosis).

Claims

1. A human chondrocyte precursor cell population consisting of a single-peak cell population that satisfies the conditions of PRRX1 positivity, CD90 positivity, and CD140B positivity.

2. The human chondroprogenitor cell population according to claim 1, which is substantially free of cells that do not satisfy at least one condition selected from the group consisting of CD90 positive and CD140B positive.

3. The human chondrocyte precursor cell population according to claim 1 or 2, which is derived from a single-peak human limb bud mesenchymal cell population that satisfies the conditions of PRRX1 positivity, CD44 positivity, CD140B positivity and CD49f negativity.

4. The human chondrocyte precursor cell population according to claim 1 or 2, which is derived from a single-peak human limb bud mesenchymal cell population that satisfies the following conditions: PRRX1 positive, CD44 positive, CD140B positive, CD49f negative, CD99 positive, CD9 negative and CD57 negative.

5. The human chondrocyte precursor cell population according to claim 1 or 2, wherein the human chondrocyte precursor cell population is derived from a single-peak human limb bud mesenchymal cell population that satisfies the conditions of PRRX1 positivity, CD44 positivity, CD140B positivity and CD49f negativity, and the proportion of cells in the human limb bud mesenchymal cell population that do not satisfy all of the conditions of CD44 positivity, CD140B positivity and CD49f negativity is 5% or less.

6. The human chondrocyte precursor cell population according to claim 1 or 2, wherein the human chondrocyte precursor cell population is derived from a single-peak human limb bud mesenchymal cell population that satisfies the conditions of PRRX1 positivity, CD44 positivity, CD140B positivity and CD49f negativity, and the human limb bud mesenchymal cell population is substantially free of cells that do not satisfy all of the conditions of CD44 positivity, CD140B positivity and CD49f negativity.

7. The human chondroprogenitor cell population according to claim 1 or 2, which has been cryopreserved.

8. A method for quality control of a population of human chondroprogenitor cells, comprising the step of determining whether or not human chondroprogenitor cells satisfy the conditions of being CD90 positive and CD140B positive.

9. A kit for inducing a human pluripotent stem cell into the human chondrocyte precursor cell population according to any one of claims 1 to 7, comprising the following (i) to (iv): (i) Culture medium for inducing pluripotent stem cells into primitive streak cells (ii) Medium for inducing primitive streak cells to lateral plate mesoderm cells (iii) Culture medium for inducing limb bud mesenchymal cells from lateral plate mesoderm cells (iv) A medium for expanding limb bud mesenchymal cells and inducing them into a population of human chondrocyte precursor cells.

10. A transplant material comprising the human chondroprogenitor cell population according to any one of claims 1 to 7, or human chondrocytes or cartilage tissue differentiated from said human chondroprogenitor cell population.

11. A human cartilage-related disease model comprising a human chondrocyte precursor cell population induced from iPS cells derived from a patient with a cartilage-related disease and consisting of a single-peak cell population that satisfies the conditions of PRRX1 positivity, CD90 positivity and CD140B positivity, or human chondrocytes induced from the human chondrocyte precursor cell population.

12. A method for preparing a mammalian chondrocyte precursor cell population, comprising a step of culturing a limb bud mesenchymal cell population derived from mammalian lateral plate mesoderm cells and positive for PRRX1 protein in an environment that activates Wnt signaling.

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