An iPSC-derived chondrogenic mesenchymal precursor cell and culture medium composition and method for its preparation

Abstract

This invention relates to the field of stem cell and cartilage tissue engineering technology, and particularly to an iPSC-derived chondrogenic mesenchymal progenitor cell and its culture medium composition and preparation method. The culture medium composition and preparation method provided by this invention can directionally differentiate iPSCs into chondrogenic mesenchymal progenitor cells with highly efficient in vitro expansion capabilities. These MPCs differ from traditional MSCs, exhibiting low or negative expression of CD73 and CD44, possessing stable chondrogenic ability, and can form anti-hypertrophic cartilage tissue. Their chondrogenic ability is significantly superior to that of traditional MSCs, exhibiting advantages such as high spheroidization rate, structural stability, and distinct hyaline cartilage characteristics. They can be used for the large-scale preparation of anti-hypertrophic hyaline cartilage-like tissue for applications in articular cartilage defects, osteochondral injuries, early intervention in osteoarthritis, cartilage disease modeling, drug development, and drug screening.

Description

Technical Field

[0001] This invention relates to the field of stem cell and cartilage tissue engineering technology, and more particularly to an iPSC-derived chondrogenic mesenchymal precursor cell and the culture medium combination and method for its preparation. Background Technology

[0002] Articular cartilage is a highly specialized connective tissue covering the surface of the bone ends of synovial joints. It is mainly composed of a small number of chondrocytes and their secreted extracellular matrix, with type II collagen, proteoglycans and water as the main components. This tissue undertakes functions such as low-friction sliding, weight-bearing cushioning and joint stability. However, due to the lack of blood vessels, lymphatic vessels and nerve distribution, and the limited proliferation and migration capacity of mature chondrocytes, its endogenous repair capacity after injury is extremely weak[1]. Trauma, sports injury, degenerative changes and inflammatory states can all lead to cartilage defects or cartilage matrix degradation, which can further develop into chronic joint diseases such as osteoarthritis. Epidemiological studies show that in 2020, about 595 million people worldwide suffered from osteoarthritis, accounting for about 7.6% of the global population, a significant increase compared to 1990, indicating that there is a great clinical and social need for cartilage degeneration and joint repair[2].

[0003] Currently, clinical techniques for repairing articular cartilage damage include conservative treatment, microfractures, osteochondral transplantation, autologous chondrocyte transplantation, and matrix-induced autologous chondrocyte transplantation. Microfractures recruit bone marrow-derived cells into the defect area by destroying subchondral bone, but the resulting repair tissue is usually closer to fibrocartilage, and its mechanical properties and long-term durability are difficult to be equated with natural hyaline cartilage [3,4]. Autologous chondrocyte transplantation and its improved techniques can improve focal cartilage defects to some extent, but it requires obtaining the patient's own cartilage tissue, expanding it in vitro, and then reimplanting it, which has problems such as donor site damage, long individualized production cycle, dedifferentiation during cell expansion, batch-to-batch variability, and high cost [5]. Therefore, developing a scalable, quality-controllable cell source with strong hyaline cartilage-like matrix formation ability and long-term stability remains an important technical requirement in the field of cartilage regenerative medicine.

[0004] Mesenchymal stem / stromal cells are one of the most widely used seed cells in cartilage tissue engineering. The definition of human MSCs proposed by the International Cell Therapy Association includes: adhering growth under standard culture conditions, expressing CD105, CD73 and CD90, not expressing CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR, and having the ability to differentiate into osteogenic, adipogenic and chondrogenic triads in vitro [6]. However, this definition is mainly used to standardize the minimum identification criteria for cultured MSC-like cells, and is not equivalent to proving that the cell population is a real cartilage precursor cell in vivo, nor can it guarantee its ability to form stable hyaline cartilage tissue. Numerous studies have shown that MSCs from different sources have significant differences in chondrogenic potential, extracellular matrix deposition capacity, immunomodulatory capacity and hypertrophic risk, and their surface marker expression also changes with source, culture conditions and expansion passage.

[0005] A key bottleneck in the use of MSCs for cartilage regeneration is the tendency for hypertrophy and intrachondral osteogenic processes after chondrogenesis. Typical hypertrophy-related indicators include COL10A1, RUNX2, MMP13, ALPL, IBSP, and VEGFA. Studies by Pelttari et al. have shown that human bone marrow MSCs can undergo a stage similar to chondrocyte hypertrophy differentiation during in vitro chondrogenesis, and the chondrocyte-like tissues formed can undergo calcification and vascular invasion after ectopic transplantation [7]. Subsequent reviews have also pointed out that hypertrophy of MSC-derived cartilage engineered tissues is an important problem restricting their transformation into stable hyaline cartilage repair [8,9]. Therefore, ideal chondrogenic cells should not only have chondrogenic differentiation ability, but should also be able to maintain low COL10A1, low RUNX2, low MMP13, and low mineralization tendency under long-term culture, inflammatory stimulation, osteogenic / hypertrophy induction, or in vivo microenvironment, thereby forming hypertrophy-resistant hyaline cartilage-like tissues.

[0006] From the perspective of developmental biology, chondrogenesis is not simply one of the three lineages of MSCs, but rather a continuous process involving mesenchymal cell recruitment, aggregation, chondrogenic precursor cell formation, chondrocyte differentiation, and chondrocyte maturation. SOX9 is a core transcription factor in chondrogenesis, which can promote chondrogenic lineage, cell survival, and the expression of cartilage matrix genes such as COL2A1 and ACAN, and to a certain extent inhibit hypertrophic cartilage-related programs

[10] . During development, different types of cartilage have different fates: some chondrocytes enter the growth plate-like proliferation, hypertrophy, mineralization, and endochondral osteogenic pathway; while others form permanent articular cartilage, maintaining non-hypertrophic, low-mineralization, and lubricating functions for a long time. It can be seen that the cells required for cartilage repair should be as close as possible to the state of "permanent articular cartilage precursor" or "stable chondrogenic precursor", rather than simply relying on traditional MSC surface markers or three-lineage differentiation criteria for screening.

[0007] Induced pluripotent stem cells (iPSCs) possess self-renewal and theoretically unlimited expansion capabilities. They can be differentiated into chondrocytes, chondrocyte progenitor cells, or chondrocyte-like microtissues, thus being considered an important cell source for cartilage tissue engineering and disease modeling. Previous studies have reported that human iPSCs can be induced to form cells with high cartilage matrix formation capacity under specific induction systems, resulting in hyaline cartilage-like tissues or cartilage granules [11,12]. For example, Yamashita et al. established a method for generating scaffold-free hyaline cartilage-like tissues from human iPSCs

[12] ; Diederichs et al. reported a chondrogenic protocol for human iPSCs without using hypertrophic BMP-induced chondrogenesis, which produced cartilage-engineered tissues with high proteoglycan deposition and hypertrophic marker expression levels close to those of adult articular chondrocytes

[11] . These studies suggest that iPSC-derived cells have the potential to overcome the limitations of adult chondrocyte expansion and the instability of traditional MSC-derived chondrogenesis.

[0008] However, iPSCs still face problems such as heterogeneity, non-target cell residues, batch-to-batch differences, inconsistent maturity, and insufficient control of hypertrophy during differentiation into the chondrogenic lineage. In order to improve chondrogenic efficiency and uniformity, some studies have attempted to obtain iPSC-derived chondrogenic precursor cells through developmental stepwise induction and surface marker sorting. For example, Dicks et al. used the COL2A1 reporter system and single-cell analysis to identify a subpopulation of hiPSC-derived chondrogenic precursor cells that were CD146-positive, CD166-positive, PDGFRβ-positive, and CD45-negative. This subpopulation showed strong chondrogenic potential and more uniform matrix generation capacity

[13] . Rodríguez Ruiz et al. compared the new cartilage produced by hiPSCs via the MSC-like route and the chondrogenic precursor route. The results showed that neo-cartilage obtained via the chondrogenic precursor cell route was closer to human primary articular chondrocyte-derived tissue, while hiMSC-derived cartilage showed higher COL1A1 and hypertrophy-related characteristics

[14] . These results suggest that whether iPSC-derived cells first enter the appropriate cartilage precursor state may be a greater determinant of final cartilage tissue quality than whether they conform to the traditional MSC phenotype.

[0009] In recent years, the concept of developmental articular cartilage precursors has been continuously deepened. GDF5-positive cells are considered to be associated with the interarticular region and permanent cartilage formation. Pothiawala et al. reported that human pluripotent stem cell-derived mesoderm progeny can form two types of chondrogenic mesenchymal cells: SOX9-positive and GDF5-positive. Among them, GDF5-positive cells tend to form permanent chondrogenic cells with high PRG4 expression and low COL10A1 expression, while SOX9-positive cells are more likely to form cartilage with hypertrophy and mineralization tendency

[15] . PRG4 / lubricin is an important molecule for articular cartilage surface and synovial joint homeostasis, and PRG4-positive surface cells are also considered to contain precursor cell populations involved in articular cartilage formation and repair

[16] . Therefore, establishing stable cartilage precursor cells and their chondrocytes based on characteristics such as GDF5, PRG4, COL2A1, ACAN, and low COL10A1 / RUNX2 / MMP13 is an important direction in the field of cartilage regeneration.

[0010] Three-dimensional culture, microcluster culture, pellet culture, spheroid and cartilage particle systems can simulate the cell aggregation and matrix deposition process in chondrogenesis and are often used to evaluate chondrogenic function and construct transplantable cartilage-like tissues. Recent studies have shown that expandable human iPSC-derived limb bud-like mesenchymal cells can form uniform cartilage particles through cell self-aggregation. The particles express hyaline cartilage markers such as type II collagen and Aggrecan, but lack hypertrophy markers such as type X collagen, suggesting that there is an important translational potential between expandable intermediate precursor cells and scalable three-dimensional cartilage particle preparation

[17] . At the same time, published patent literature has also focused on chondrocytes, cartilage precursors, cartilage-like tissues, pellets or cartilage implants generated from pluripotent stem cells [18,19]. However, current technologies primarily focus on conventional MSC-like cells, CD73 / CD90 / CD105-positive iMSCs, CD146 / CD166 / PDGFRβ-positive chondrocyte precursors, GDF5-positive chondrocyte precursors, or terminal chondrocyte-like cells. However, MSC chondrogenesis has inherent drawbacks: high heterogeneity in MSC origin, unstable chondrogenic efficiency, decreased chondrogenic potential after expansion, and a tendency to exhibit elevated levels of COL10A1, RUNX2, MMP13, and ALPL, indicating hypertrophy and the risk of intrachondral osteogenic formation. Existing iPSC chondrogenic pathways suffer from highly heterogeneous, limited differentiation populations, a lack of clearly defined functional subpopulations, difficulty in simultaneously achieving expansion and chondrogenic capacity, and challenges in obtaining long-term stable hyaline cartilage-like tissue. For iPSC-derived mesenchymal / chondrocyte precursor cells that do not conform to classic MSC surface markers but can still expand efficiently, stably form chondroid globules, and possess hypertrophy resistance, there is still a lack of adequate definition and systematic development.

[0011] Although CD73 and CD44 are classic positive markers in the minimum identification criteria for MSCs, their biological functions are not equivalent to hyaline cartilage formation ability. CD73, also known as NT5E, is an extracellular 5'-nucleotidase that can catalyze the production of adenosine from extracellular AMP, and participates in purinergic signaling, inflammation regulation, tissue damage response and cell homeostasis regulation

[20] . CD44 is a hyaluronic acid receptor. CD73 / CD44 positivity more often represents traditional MSC-like identity or a specific metabolic / signaling state, rather than necessarily representing the optimal chondrogenic precursor state. On the contrary, basic developmental biology studies have shown that some cell populations that do not conform to the classic MSC surface markers of low expression or negative CD73 and low expression or negative CD44 may retain earlier, more developmental or more cartilage lineage-defined precursor attributes. If these cells simultaneously possess efficient expansion, three-dimensional aggregation, hyaline cartilage matrix deposition and anti-hypertrophy ability, they may be able to overcome the problems of chondrogenic instability of traditional MSCs and cartilage differentiation heterogeneity of iPSCs.

[0012] In summary, existing cartilage repair technologies still suffer from problems such as insufficient hyaline cartilage regeneration, limited cell sources, post-expansion phenotypic drift, hypertrophy of MSC-derived cartilage tissue, and iPSC differentiation heterogeneity. Although pluripotent stem cell-derived cartilage precursors and cartilage granules have shown great application potential, existing technologies suffer from long differentiation cycles and high difficulty in process and quality control. Therefore, a new, scalable iPSC-derived chondrogenic precursor cell and its three-dimensional cartilage sphere preparation system is needed. This system should possess both efficient in vitro expansion capabilities and the ability to stably form anti-hypertrophic hyaline cartilage-like tissue rich in COL2A1, ACAN, and GAG, and low in COL10A1, RUNX2, MMP13, and mineralization indices. This tissue could then be used in fields such as articular cartilage defects, osteochondral injuries, early intervention in osteoarthritis, cartilage disease modeling, drug development, and drug screening. Summary of the Invention

[0013] This invention provides iPSC-derived chondrogenic mesenchymal precursor cells (MPC), a culture medium combination, and a method for their preparation.

[0014] Specifically, the present invention provides the following technical solutions.

[0015] In a first aspect, the present invention provides a culture medium composition for preparing iPSC-derived chondrogenic mesenchymal precursor cells, the culture medium composition comprising: The first induction medium consists of a basic differentiation medium and the following added components: 20-30 ng / mL Activin A, 1.5-3 μM CHIR99021 and 20-50 ng / mL bFGF; The second induction medium consists of a basic differentiation medium and the following added components: 1-5 μM A8301, 1-2 μM C59 and 250-500 nM LDN193189; And, the third induction medium, which consists of the differentiation basal medium and the following added components: 5-10 nM MSAG21K and 1-2 μM C59; The differentiation basal culture medium includes a basic culture medium and the following added components: insulin, transferrin, sodium selenite, B27, NEAA and β-mercaptoethanol; The concentration of the added components is based on the volume of the basal culture medium.

[0016] This invention aims to prepare scalable, stable, chondrogenic, and anti-hypertrophic iPSC-derived chondrogenic mesenchymal precursor cells. Specifically, chondrogenic mesenchymal precursor cells are prepared by using iPSCs as the starting cells and through staged induction. By selecting, combining, and screening the components added to the induction medium used in each induction stage, this invention found that using the above-mentioned first, second, and third induction media to induce iPSCs in stages yields chondrogenic mesenchymal precursor cells with a cell phenotype distinct from traditional MSCs. These cells exhibit low or negative expression of CD73 and CD44, high expression of COL2A1, SOX9, and ACAN, negative expression of OCT4 and NANOG, and low expression of COL1A1 and COL3A1. Furthermore, they possess good in vitro continuous expansion capacity and passage phenotypic stability, strong chondrogenic ability, and the ability to form stable anti-hypertrophic cartilage tissue with high spheroidization rate, stable structure, and obvious hyaline cartilage characteristics. The combined effect of the added components in the above-mentioned induction culture medium, and the synergistic effect of the added components in the induction culture medium at each stage, enables a good connection between the induction stages, resulting in chondrogenic mesenchymal precursor cells with the above-mentioned performance advantages.

[0017] Preferably, the differentiation basal culture medium contains the following components at concentrations based on the volume of the basal culture medium: penicillin-streptomycin 1-2% v / v, insulin-transferrin-selenium additive 1-2% v / v, B27 1-2% v / v, NEAA 1-2% v / v, and β-mercaptoethanol 90-120 μmol / L.

[0018] Preferably, the culture medium combination further includes MPC cell culture medium for the expansion of iPSC-derived chondrogenic mesenchymal precursor cells, comprising a basal medium and the following additives: 3%-5% v / v KnockOut serum substitute, 1-3 μM CHIR99021, 5-10 μM A8301 and 20-40 ng / mL EGF.

[0019] After induction using the three induction media described above, the induced MPCs can be amplified using the MPC cell culture medium. This invention screened and optimized the added components in the MPC cell culture medium, finding that the addition of CHIR99021, A8301, and EGF significantly promoted the formation of anti-hypertrophy and hyaline cartilage properties in MPCs. Removing these added components led to a significant increase in the expression of hypertrophy markers in MPCs, and the cartilage microtissue prepared from MPCs was more prone to forming fibrotic connective tissue.

[0020] In this invention, the basal culture medium is selected from one or more of DMEM, α-MEM, IMEM, and DMEM / F12 culture medium.

[0021] The basal culture media used for differentiation basal culture medium and MPC cell culture medium can be independently selected from one or more of DMEM high glucose medium, α-MEM, IMEM, and DMEM / F12 medium.

[0022] Preferably, the basal medium of the differentiation basal medium is DMEM high glucose medium.

[0023] Preferably, the basal culture medium for the MPC cell culture medium is DMEM / F12 medium.

[0024] In this invention, the iPSC is preferably a human iPSC.

[0025] Secondly, the present invention provides any of the following applications of the culture medium combination described above: (1) Preparation of iPSC-derived chondrogenic mesenchymal precursor cells; (2) Preparation of chondrocyte spheroids; (3) Preparation of cartilage microtissue.

[0026] iPSCs can be induced using the culture medium combination described in the first aspect to prepare iPSC-derived chondrogenic mesenchymal precursor cells, which can then be used to further prepare chondrocyte spheres and cartilage microtissues.

[0027] Thirdly, the present invention provides a method for preparing iPSC-derived chondrogenic mesenchymal precursor cells, the method comprising: using iPSCs as starting cells, and performing staged induction culture using the culture medium combination described in the first aspect.

[0028] Preferably, the method includes: performing a first-stage induction culture, a second-stage induction culture, and a third-stage induction culture sequentially using a first induction medium, a second induction medium, and a third induction medium.

[0029] The first stage of induction culture can induce iPSCs to enter the mesodermal stage, the second stage of induction culture can induce the formation of the mesenchymal precursor state, and the third stage of induction culture can obtain an MPC cell population with low or negative expression of CD73 and CD44.

[0030] Preferably, the induction culture time for the first stage, the second stage, and the third stage is 24-30 h.

[0031] Preferably, after the third stage of induction culture is completed, the method further includes amplifying the induced MPCs using MPC cell culture medium.

[0032] After expansion using MPC cell culture medium, MPC cells maintained low or negative expression of CD73 and CD44, as well as the ability to form chondrocytes and resist hypertrophy.

[0033] Fourthly, this invention provides a cell population of iPSC-derived chondrogenic mesenchymal precursor cells. After expansion through P8-P15 generations, the proportion of CD73-positive cells is no higher than 50%, the proportion of CD44-positive cells is no higher than 50%, the proportion of CD90-positive cells is no lower than 95%, and the proportion of CD105-positive cells is no lower than 80%. Furthermore, CD19, CD31, CD34, CD45, HLA-DR, and CD11B are expressed at low levels or are negative. The cell population expresses SOX9 and COL2A1 and has the ability to form cartilage microtissues. The cell population was prepared using the method described in the third aspect.

[0034] Preferably, the proportion of CD73-positive cells in the iPSC-derived chondrogenic mesenchymal precursor cell population is not higher than 40%.

[0035] Preferably, the proportion of CD44-positive cells in the iPSC-derived chondrogenic mesenchymal precursor cell population is not higher than 40%, more preferably not higher than 30%.

[0036] The iPSC-derived chondrogenic mesenchymal precursor cells provided by this invention have the following phenotypic characteristics: low expression or negative CD73, low expression or negative CD44; further preferably, high expression of COL2A1 and SOX9, low expression of COL1A1 and COL3A1, negative OCT4, and negative NANOG.

[0037] The iPSC-derived chondrogenic mesenchymal precursor cells can be continuously expanded in vitro (8-12 generations), have high survival rate and proliferation capacity, have stable chondrogenic capacity, and can form stable cartilage microtissues.

[0038] The iPSC-derived chondrogenic mesenchymal precursor cells highly express SOX9, COL2A1, and ACAN during chondrogenesis, and lowly express COL10A1.

[0039] Fifthly, the present invention provides a cartilage sphere prepared using a cell population of iPSC-derived chondrogenic mesenchymal precursor cells as described in the fourth aspect.

[0040] The cartilage globules have the following histological characteristics: positive for Safranin O and Alcian Blue, high expression of COL2A1, ACAN, and COMP, anti-hypertrophy characteristics, and low expression of COL10A1, RUNX2, MMP13, and ALPL; preferably, they have no obvious mineralization.

[0041] The cartilage spheres have the following structural characteristics: they are intact, do not easily disintegrate, and are stable during long-term culture.

[0042] In a sixth aspect, the present invention provides a cartilage microtissue prepared from a cell population of iPSC-derived chondrogenic mesenchymal precursor cells as described in the fourth aspect. The cartilage microtissue is positive for Alcian Blue, Safranin O-Fix Green and / or Toluidine Blue, expresses SOX9, COL2A1 and / or ACAN, and has low expression of COL10A1, RUNX2, MMP13 and / or ALPL.

[0043] The preparation of cartilage microtissue using iPSC-derived chondrogenic mesenchymal precursor cells of the present invention can be carried out using commonly used chondrocyte microtissue culture media.

[0044] Preferably, the chondrocyte microtissue culture medium comprises DMEM high glucose medium and the following additives: 0.5-1.5% KnockOut serum substitute, 0.5-1.5% insulin-transferrin-selenium additive, 0.5-1.5 mM sodium pyruvate, 80-120 μg / mL penicillin-streptomycin, 0.05-0.2 mmol / L sodium ascorbate, 0.05-0.2 μmol / L dexamethasone, 30-50 μg / mL proline, 15-25 ng / mL TGF-β3 and 15-25 ng / mL BMP2.

[0045] In a seventh aspect, the present invention provides any one of the following applications of the cell population of iPSC-derived chondrogenic mesenchymal precursor cells described in the fourth aspect, the chondrocytes described in the fifth aspect, or the cartilage microtissues described in the sixth aspect: (1) Prepare drugs or tissue-engineered products for treating articular cartilage defects, osteochondral injuries or osteoarthritis; (2) Construction of in vitro models of cartilage diseases; (3) Screening of drugs for the treatment of cartilage diseases.

[0046] The treatment of osteoarthritis includes early treatment of osteoarthritis or cartilage protection.

[0047] The cell populations or cartilage microtissues described in this invention can be used as active ingredients in the preparation of cell therapy drugs or tissue engineering products. As an example, the cell populations or cartilage microtissues can be applied to cartilage defect sites; alternatively, the cell populations or cartilage microtissues can be combined with biocompatible scaffold materials (such as hydrogels, collagen, polylactic-co-glycolic acid copolymer (PLGA), etc.) to form tissue-engineered constructs, which are then surgically implanted into the cartilage defect area. After implantation, they can further differentiate into mature hyaline chondrocytes in the defect microenvironment, secrete cartilage-specific matrix, repair and reconstruct tissue with a structure and function similar to natural hyaline cartilage, and integrate with the host cartilage tissue.

[0048] The cell populations or cartilage microtissues described in this invention can also be used to construct in vitro human cartilage disease models. As an example, the cell populations or cartilage microtissues can be cultured in vitro and treated with specific physical stimuli or chemical / biological factors to simulate the pathological microenvironment of diseases such as osteoarthritis, thereby inducing disease-related phenotypes, such as excessive degradation of the cartilage matrix, apoptosis, and inflammatory responses.

[0049] Based on the in vitro disease model constructed above, the cell population or micro-tissue of the present invention can also be used to establish a drug screening platform. As an example, candidate drugs to be screened (such as small molecule compounds, antibodies, peptides, etc.) are applied to the model, and the efficacy of the candidate drugs is evaluated by detecting key biological indicators.

[0050] The beneficial effects of this invention include at least the following: the culture medium combination and preparation method provided by this invention can directionally differentiate iPSCs into chondrogenic mesenchymal precursor cells with efficient in vitro expansion capabilities. These MPCs differ from traditional MSCs, exhibiting low or negative expression of CD73 and CD44, possessing stable chondrogenic ability, and can form anti-hypertrophic cartilage tissue. They exhibit low expression of COL10A1, RUNX2, MMP13, and ALPL, and their chondrogenic ability is significantly superior to traditional CD73 / CD90 / CD44-positive MSCs. They possess advantages such as high spheroidization rate, structural stability, and distinct hyaline cartilage characteristics. This allows for the large-scale preparation of anti-hypertrophic hyaline cartilage-like tissue that stably forms COL2A1, ACAN, and GAG, and has low COL10A1, RUNX2, MMP13, and mineralization indices. This tissue can then be used for articular cartilage defects, osteochondral injuries, early intervention in osteoarthritis, cartilage disease modeling, drug development, and drug screening, potentially overcoming the problems of traditional MSC chondrogenicity and iPSC cartilage differentiation. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 This is a schematic diagram of the overall process of human induced pluripotent stem cells (hiPSCs) differentiating into cartilage mesenchymal precursor cells (MPCs) and further forming cartilage microtissue in Experiment Example 1 of the present invention, as well as microscopic images of cells at the main differentiation stages.

[0053] Figure 2 The results are single-cell transcriptome analysis of the MPC and iMSC-derived cell populations in Experiment Examples 2 and 4 of this invention.

[0054] Figure 3 The results of COL2A1 immunofluorescence detection in Experiment Examples 2 and 4 of this invention are shown.

[0055] Figure 4 This refers to the expression of key cellular attribute genes during the continuous in vitro amplification of MPC in Experimental Example 3 of this invention.

[0056] Figure 5 This represents the total number of live cells harvested by MPC under different passages and inoculation densities in Experimental Example 3 of this invention.

[0057] Figure 6 This is a comparison of the in vitro ability of MPC and iMSC to form cartilage microtissue in Experimental Example 5 of the present invention.

[0058] Figure 7 SO staining of MPC chondrocytes and expression of hyaline cartilage-related genes in Experimental Example 6 of this invention.

[0059] Figure 8 This refers to the in vivo colonization and cartilage matrix maintenance capacity of MPC-derived cartilage microtissue after subcutaneous transplantation in Experimental Example 7 of this invention.

[0060] Figure 9 This is the in-situ repair effect of MPC-derived cartilage microtissue in a rat knee cartilage defect model, as shown in Experimental Example 8 of this invention.

[0061] Figure 10 This is a gross observation and histological staining of MPC-derived cartilage microtissue in an in situ repair model of cartilage defects in a rabbit knee joint, as shown in Experimental Example 9 of this invention, one month after surgery.

[0062] Figure 11The results of anti-human lamin A (Lamin A) antibody detection are shown in Experimental Example 9 of this invention, which describes the in situ repair of MPC-derived cartilage microtissue in a rabbit knee joint cartilage defect model.

[0063] Figure 12 This is a long-term observation result of the in situ repair of MPC-derived cartilage microtissue in a rabbit knee joint cartilage defect model in Experiment Example 9 of this invention.

[0064] Figure 13 This invention demonstrates the biomechanical property recovery effect of MPC-derived cartilage microtissue in the in-situ repair of a rabbit knee joint cartilage defect model after 6 months.

[0065] Figure 14 This is the result of 6 months of in-situ repair of MPC-derived cartilage microtissue in a porcine knee joint cartilage defect model, as shown in Experimental Example 11 of this invention.

[0066] Figure 15 The results of Safranin O-Fixed Green staining and COL2 and COL10 immunostaining in the normal control group, ACLT+Sham group, ACLT+Defect group and ACLT+MPCCS group in Experiment Example 12 of this invention are shown.

[0067] Figure 16 The results are as follows: OARSI score (a), elastic modulus of repaired tissue (b, e), and histological, immunohistochemical and Micro-CT (c), and synovitis score (d) of each group in Experiment Example 12 of this invention at 2 and 8 weeks postoperatively.

[0068] Figure 17 and Figure 18 MPC-derived cartilage microtissue cultured in the MPC cell culture medium in Experimental Example 15 of this invention ( Figure 17 ) and MPC cell-derived cartilage microtissue cultured in the same medium as previously reported precursor cells from other tissues ( Figure 18 The results of histological staining were obtained 3 months after subcutaneous transplantation.

[0069] Figure 19 The results of COLII-based immunofluorescence staining analysis are shown for the MPC-A and MPC-B groups after changing the concentration of key differentiation factors in Experiment Example 16 of this invention.

[0070] Figure 20 This is the morphological observation result of the chondrogenic sphere differentiation experiment conducted in Experiment Example 16 of the present invention after changing the concentration of the key differentiation factor.

[0071] Figure 21The results of hyaline cartilage property key gene expression detection in the chondrogenic sphere differentiation experiment after changing the concentration of key differentiation factors in Experiment Example 16 of this invention are shown. In the figure of relative normalized expression levels, 1-23 are Y106-SPARC, Y108-MIA, Y10-ACAN, Y12-PRG4, Y13-COL10A1, Y16-RUNX2, Y17-COL1A1, Y25-COL1A2, Y2-NANOG, Y57-CRTL1, Y58-MAGP2, Y66-CD44, Y67-UCMA, Y68-MATN3, Y71-EPYC, Y73-MATN4, Y75-CILP, Y77-COL9A1, Y78-COL11A1, Y7-SOX9, Y8-COL2A1, Y96-CRTAC1, and Y9-COL11A2, respectively. Detailed Implementation

[0072] The abbreviations involved in this invention are explained as follows: iPSC, induced pluripotent stem cell; hiPSC, human induced pluripotent stem cell; MPC, mesenchymal progenitor cell; iMSC, iPSC-derived mesenchymal stromal cell, is a mesenchymal stromal cell derived from human induced pluripotent stem cells (also referred to as mesenchymal stem cells in this article). MPCCS, MPC-derived cartilage spheroids, are chondrocyte spheroids derived from mesenchymal precursor cells.

[0073] ACLT, Anterior Cruciate Ligament Transection.

[0074] NEAA, Non-Essential Amino Acids.

[0075] In a specific embodiment of the present invention, a human induced pluripotent stem cell-derived MPC that is low in CD73 or negative and low in CD44 or negative is provided, as well as the preparation, identification and application of anti-hypertrophic hyaline cartilage microtissue formed from the MPC.

[0076] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0077] The human induced pluripotent stem cells used in the following examples are BC-hiPSC-ME-20, which was deposited on May 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Human Induced Pluripotent Stem Cells hiPSC, with accession number CGMCC No. 46536. It has been disclosed in Chinese Patent CN120366195B.

[0078] In the following examples, penicillin-streptomycin, insulin-transferrin-selenium additive, B27, and NEAA all used their respective commercially available stock solutions.

[0079] Example 1 This embodiment provides a culture medium composition for inducing human induced pluripotent stem cells to differentiate into MPCs, the composition of which is as follows: The first induction medium, used for the first stage of induction culture, is a basic differentiation medium supplemented with the following added components: 30 ng / mL Activin A, 3 μM CHIR99021 and 20 ng / mL bFGF, with the concentration of the added components based on the volume of DMEM high glucose medium; The second induction medium, used for the second stage of induction culture, is a basic differentiation medium supplemented with the following added components: 1 μM A8301, 1 μM C59, 250 nM LDN193189, with the concentration of the added components based on the volume of DMEM high-glucose medium. In addition, the third induction medium, used for the third stage of induction culture, is a basic differentiation medium supplemented with the following additives: 5 nM SAG21K and 1 μM C59, with the concentration of the additives based on the volume of DMEM high-glucose medium. The differentiation basal medium consisted of DMEM high-glucose medium and the following additives: 1% penicillin-streptomycin, 1% insulin-transferrin-selenium additive, 2% B27, 1% NEAA, and 90 μmol / L β-mercaptoethanol. Except for β-mercaptoethanol, which is expressed as a final concentration, the amounts of penicillin-streptomycin, insulin-transferrin-selenium additive, B27, and NEAA are all volume percentages (v / v) relative to the DMEM high-glucose medium.

[0080] Example 2 This embodiment provides a culture medium composition for inducing human induced pluripotent stem cells to differentiate into MPCs, the composition of which is as follows: The first induction medium, used for the first stage of induction culture, is a basic differentiation medium supplemented with the following components: 20 ng / mL Activin A, 1.5 μM CHIR99021 and 50 ng / mL bFGF, with the concentration of the added components based on the volume of DMEM high glucose medium. The second induction medium, used for the second stage of induction culture, is a basic differentiation medium supplemented with the following added components: 5 μM A8301, 2 μM C59, 500 nM LDN193189, with the concentration of the added components based on the volume of DMEM high-glucose medium. In addition, the third induction medium, used for the third stage of induction culture, is a basic differentiation medium supplemented with the following added components: 10 nM SAG21K and 2 μM C59, with the concentration of the added components based on the volume of DMEM high glucose medium; The differentiation basal medium consisted of DMEM high-glucose medium and the following additives: 2% penicillin-streptomycin, 2% insulin-transferrin-selenium additive, 1% B27, 2% NEAA and 120 μmol / L β-mercaptoethanol. The concentration of the additives was based on the volume of DMEM high-glucose medium.

[0081] Example 3 This embodiment provides an MPC cell culture medium for the expansion of MPC cells, which supplements DMEM / F12 medium with the following additives: 5% v / v KnockOut serum substitute, 3 μM CHIR99021, 10 μM A8301 and 20 ng / mL EGF; except for CHIR99021, A8301 and EGF expressed as final concentrations, KnockOut serum substitute is a volume percentage (v / v) relative to DMEM / F12 medium.

[0082] Example 4 This embodiment provides an MPC cell culture medium for the expansion of MPC cells, which is a DMEM / F12 medium supplemented with the following additives: 3% v / v KnockOut serum substitute, 1 μM CHIR99021, 5 μM A8301, 40 ng / mLEGF. Except for CHIR99021, A8301 and EGF, which are expressed as final concentrations, KnockOut serum substitute is a volume percentage (v / v) relative to DMEM / F12 medium.

[0083] Experimental Example 1: Induction of human induced pluripotent stem cells into MPCs and iMSCs The culture medium combination described in Example 1 was used to induce the differentiation of human induced pluripotent stem cells into MPCs. The specific methods and results are as follows.

[0084] 1. Starting cells: Human induced pluripotent stem cells were used as the starting cells.

[0085] 2. Culture container coating The culture vessels were coated with recombinant human vitronectin. Taking a 6-well plate as an example, take a 15 mL centrifuge tube, add 8.9 mL of DPBS, then add 100 μL of Vitronectin solution, mix well, add 1 mL of coating solution to each well, and incubate at 37°C for at least 1 h before use.

[0086] 3. iPSC resuscitation and vaccination After rapidly thawing the frozen human iPSCs in a 37°C water bath, they were transferred to 15 mL centrifuge tubes containing 9 mL of mTeSR1 medium and centrifuged at 300 × g for 5 min. The supernatant was discarded, and the cells were resuspended in iPSC resuscitation medium and counted.

[0087] The iPSC resuscitation medium was mTeSR1 medium supplemented with 1% penicillin-streptomycin and 10 μmol / L Y-27632.

[0088] Discard the Vitronectin coating solution from the culture plate and add human iPSCs at approximately 1 × 10⁻⁶. 4 cells / cm 2 The Vitronectin-coated 6-well plates were seeded at a density of [missing information], gently shaken, and then incubated at 37°C in a 5% CO2 incubator.

[0089] 4. First stage induction culture Discard the iPSC resuscitation medium and replace it with the first induction medium, and culture at 37℃ and 5% CO2 for 24 h.

[0090] 5. Second stage induction culture Discard the first induction medium and replace it with the second induction medium, and incubate at 37℃ and 5% CO2 for 24 h.

[0091] 6. Third stage induction culture Discard the second induction medium and replace it with the third induction medium, and incubate at 37℃ and 5% CO2 for 24 hours.

[0092] 7. MPC cell harvesting and P0 generation culture After the third stage of induction, discard the culture supernatant, add DPBS to gently wash the cell surface, and then discard the DPBS. Add an appropriate amount of TrypLE and incubate at 37°C for about 5 minutes. After the cells detach, add an equal volume of MPC cell culture medium to stop the digestion, and gently pipette to prepare a single-cell suspension.

[0093] The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 300 × g for 5 min. After discarding the supernatant, the cells were resuspended in the MPC cell culture medium of Example 3 and counted.

[0094] Based on the counting results, the cells were divided into groups of approximately 3 × 10⁻⁶. 4 cells / cm 2 The cells were seeded at a density of 100% into T75 cell culture flasks and cultured at 37°C and 5% CO2 for 48 h. This generation was designated as P0, which is the P0 generation MPC.

[0095] 8. MPCs continue to differentiate into iMSC-like cell states. MPC at approximately 3×10 4 cells / cm 2 Single cells were seeded into T75 cell culture flasks at a density of [specific value], and the culture medium was replaced with commercial MSC passage medium. After passage for more than 5 generations, iMSC cells with CD markers were obtained.

[0096] 9. Results A schematic diagram of the differentiation process and morphological characteristics of MPC are shown below. Figure 1As shown, hiPSCs, after directional induction, yielded MPCs, which could further differentiate into MPC-derived cartilage microtissue. Simultaneously, MPCs could continue to transform into iMSC-like cell states and form iMSC-derived cartilage microtissue. Microscopic images revealed that hiPSCs, MPCs, and iMSCs exhibited different cell morphologies in adherent culture. The cartilage microtissue formed by MPCs was larger and more structurally complete than that derived from iMSCs. After the aforementioned induction, iPSCs gradually transformed from a typical clonal morphology to a uniformly adherent MPC-like morphology, appearing as short spindle-shaped cells with a diameter of approximately 12 μm (compared to approximately 18 μm for conventional MSCs). This MPC population could further expand and form three-dimensional cartilage spheres or cartilage microtissue.

[0097] Experimental Example 2: Phenotypic Identification of MPC and iMSC The MPCs and iMSCs prepared in Experiment 1 were used for phenotypic identification by flow cytometry and single-cell sequencing, respectively.

[0098] 1. Preparation of cell suspension MPC cells in good growth condition were digested with TrypLE recombinase, and 20 μL of cell suspension was mixed with an equal volume of ViaStain AOPI staining solution. Cells were then counted using a cell counter.

[0099] Based on the live cell concentration, take a sample containing 2×10⁻⁶ cells. 6 The cell suspension of each live cell was placed in a new 15 mL sterile centrifuge tube and centrifuged at 400×g for 5 min. After discarding the supernatant, the cell suspension was resuspended in 1 mL of cell staining buffer and set aside for later use.

[0100] iMSC cell suspensions from the same source were prepared using the same method as a control.

[0101] 2. Flow cytometry antibody staining Mix the MPC and iMSC cell suspensions separately, and aliquot each type of cell into flow cytometry tubes at a rate of 100 μL / tube, with approximately 2 × 10⁶ cells per tube. 5 indivual.

[0102] The detection antibodies included PE-CD105, PE-CD73, FITC-CD44, and FITC-CD90, with corresponding PE or FITC isotype controls. 1 μL of the corresponding detection antibody or isotype control antibody was added to each tube, resulting in a reaction volume of 100 μL. The samples were incubated at 4°C in the dark for 30 min. After incubation, 2 mL of cell staining buffer was added to each tube, mixed well, and centrifuged at 400×g for 5 min, discarding the supernatant. This washing process was repeated twice. Subsequently, each tube was resuspended in 0.3 mL of cell staining buffer.

[0103] Before flow cytometry analysis, add 10 μL of 7-AAD live / dead staining solution to each tube, incubate at room temperature in the dark for 10 min, and then perform flow cytometry analysis.

[0104] 3. Flow cytometry detection and gating strategy Sample parameters were recorded using the FSC and SSC channels. The 7-AAD+FITC or 7-AAD+PE channel was selected for detection based on the antibody fluorescence type. Samples were loaded at a low or medium speed, and at least 10,000 events were collected within gate 1.

[0105] The gating strategy is as follows: GATE1: Use FSC / SSC scatter plots to delineate cell morphological groups and exclude cell debris and impurities; GATE2: Within GATE1, use FSC-A / FSC-H to delineate single cell populations and exclude adherent cell clumps; GATE3: Delineate the 7-AAD negative cell population within GATE2 and define it as a viable cell population; GATE4: Observe the FITC or PE channel within GATE3 and record the positive rate of the target marker.

[0106] The negative range, fluorescence channel voltage, and positive gate location were determined using isotype controls, with the positive rate of isotype controls controlled below 0.5%. Results were analyzed using FlowJo software.

[0107] 4. Result Determination The flow cytometry (FACS) results of MPC are shown in Table 1. In this representative sample, the proportions of CD73, CD105, CD44, and CD90 positive cells in MPC were 39.3%, 99.3%, 21.6%, and 100.0%, respectively; while the proportions of CD73, CD105, CD44, and CD90 positive cells in the same batch of iMSCs were 100.0%, 100.0%, 100.0%, and 99.8%, respectively. Therefore, compared with iMSCs, the CD73 and CD44 positivity rates of MPCs were significantly lower, while CD90 and CD105 expression remained high, suggesting that MPCs are different from traditional CD73- and CD44-positive iMSCs or MSC cell populations.

[0108] Table 1

[0109] 5. Single-cell sequencing sample preparation Take 20 μL of cell suspension, mix it with an equal volume of ViaStain AOPI staining solution, and count the cells using a cell counter. Based on the viable cell concentration, take a sample containing 2 × 10⁻⁶ cells. 6A suspension of live cells was placed in a 1.5 mL sterile centrifuge tube and centrifuged at 400 × g for 5 min. After discarding the supernatant, the cells were resuspended in 1 mL of MEM basal medium and placed on ice for single-cell transcriptome sequencing.

[0110] 6. Single-cell transcriptome analysis Single-cell transcriptional library assembly, sequencing, quality control, dimensionality reduction, clustering, and differential gene analysis were performed on MPC and iMSC.

[0111] The expression levels of hyaline cartilage-related genes (e.g., COL2A1, SOX9), fibrocartilage or mesenchymal-related genes COL1A1, ENG, NT5E, and hypertrophic cartilage-related gene RUNX2 were detected.

[0112] UMAP dimensionality reduction results show that ( Figure 2 MPCs and iMSCs form distinct cell populations. Gene expression distribution showed that the MPC population had higher expression of hyaline cartilage-related markers COL2A1, SOX9, and ACAN, while the iMSC population had higher expression of mesenchymal or fibrocartilage-related markers such as COL1A1, ENG, and NT5E.

[0113] Further immunofluorescence assays were performed to detect COL2A1 protein expression, with DAPI used for nuclear counterstaining. Results showed ( Figure 3 The COL2A1 signal in the MPC group was significantly stronger than that in the iMSC group, suggesting that MPC has a stronger tendency to differentiate into hyaline cartilage, while iMSC is more likely to be fibrocartilaginous or traditional MSC-like.

[0114] The above results indicate that the MPC obtained in this invention is different from traditional CD73-positive and CD44-positive iMSCs or MSCs, but is a population of MPC cells with specific cartilage properties derived from human iPSCs.

[0115] Experimental Example 3: In vitro serial amplification and phenotypic stability identification of MPC The MPC cells induced from differentiation in Example 1 were continuously passaged using the MPC cell culture medium from Example 3, and their phenotypic stability was tested. The specific methods and results are as follows.

[0116] 1. MPC cells were passaged every 2 days in MPC cell culture medium, using TrypLE digestion during passage. After digestion, the cells were centrifuged at 300×g for 5 min, resuspended in MPC cell culture medium, and then centrifuged at approximately 3×10⁻⁶ cells / day. 4 cells / cm 2 Re-inoculate at the desired density. Continuously subculture to P8, P12, P15 or higher generations.

[0117] 2. Detection of Amplification Capacity of MPC at Different Generations Cell seeding density, number of harvested cells, viability, and expansion fold were recorded at different passages (P5, P7, P8, P9, P10, P11, P12, and P13) of MPC.

[0118] 3. MPC phenotypic stability testing MPCs from generations P8, P12, and P15 were collected, and the expression of markers such as CD19, CD31, CD34, CD44, CD45, CD90, HLA-DR, CD73, CD105, and CD11B was detected by flow cytometry.

[0119] 4. Results The expression of cartilage-related, mesenchymal-related, and cell attribute-related genes in different samples and MPC generations are as follows: Figure 4 As shown in the figure, the relative expression levels of SOX9 in MPC P5, P9, and P12 were 155.02, 112.38, and 102.39, respectively, all higher than the 19.64 of iMSC P12; the relative expression levels of COL2A1 in MPC P5, P9, and P12 were 65.99, 684.32, and 397.54, respectively, all higher than the 3.61 of iMSC P12. The total number of live cells harvested at different seeding densities is shown in the figure. Figure 5 As shown: During P5~P13, 4×10^4, 5×10^4 and 6×10^4 cells / cm 2 The total number of representative viable cells harvested under inoculation conditions was approximately 2.89 × 10^6 to 7.41 × 10^6, indicating that MPCs can continuously expand. Flow cytometry results are shown in Table 2. The proportions of CD73-positive cells in P8, P12, and P15 generations of MPCs were 17.40%, 7.86%, and 10.20%, respectively; the proportions of CD44-positive cells were 20.20%, 13.30%, and 0.95%, respectively; the proportions of CD90-positive cells were 100%, 99.80%, and 87.50%, respectively; and the proportions of CD105-positive cells were 99.40%, 98.20%, and 99.60%, respectively. CD19, CD31, CD34, CD45, HLA-DR, and CD11B all maintained low expression or were negative.

[0120] Table 2

[0121] The above results indicate that MPCs have the ability to continuously expand in vitro and maintain phenotypic stability during continuous expansion. P8, P12 and P15 generation cells still maintain the characteristics of low expression or negative expression of CD73 and CD44, while not expressing or expressing low expression of one or more of CD19, CD31, CD34, CD45, HLA-DR and CD11B.

[0122] 5. Conclusion The above results indicate that the human iPSC-derived MPCs prepared in this invention have the ability to continuously expand in vitro and exhibit phenotypic stability, and can be used as stable starting cells for subsequent cartilage microtissue preparation.

[0123] Experiment Example 4: Single-cell transcriptome and hyaline cartilage tendency analysis of MPC and iMSC-derived cartilage microtissues 1. Preparation of cartilage microtissue samples Cartilage microtissues were prepared using MPC and iMSC, respectively. The preparation methods for cartilage microtissues are described in Experiment 5.

[0124] 2. Preparation of cartilage micro-tissue digestion solution Type I collagenase and Type II collagenase were weighed separately and prepared into 0.2% (w / v) solutions of Type I collagenase and Type II collagenase, respectively, using MEM basal medium. The collagenase solutions were sterilized by filtration through a 0.22 μm filter membrane and stored at 4°C.

[0125] When using, mix 0.2% type I collagenase solution and 0.2% type II collagenase solution at a volume ratio of 1:1 to obtain cartilage micro-tissue digestion solution.

[0126] 3. Cartilage micro-tissue digestion Cartilage microtissue samples from MPC and iMSC sources were collected, old culture media were discarded, and the samples were washed twice with PBS buffer. 2 mL of cartilage microtissue digestion solution was added to each sample, and digestion was performed at 37°C and 5% CO2. During digestion, the samples were gently pipetted and mixed every 20 minutes until the cartilage microtissue was fully dissociated into a single-cell suspension.

[0127] 4. Single-cell sequencing sample preparation Take 20 μL of cell suspension, mix it with an equal volume of ViaStain AOPI staining solution, and count the cells using a cell counter. Based on the viable cell concentration, take a sample containing 2 × 10⁻⁶ cells. 6 A suspension of live cells was placed in a 1.5 mL sterile centrifuge tube and centrifuged at 400 × g for 5 min. After discarding the supernatant, the cells were resuspended in 1 mL of MEM basal medium and placed on ice for single-cell transcriptome sequencing.

[0128] 5. Single-cell transcriptome analysis Single-cell transcription modeling, sequencing, quality control, dimensionality reduction, clustering, and differential gene analysis were performed on cartilage microtissues derived from MPC and iMSC.

[0129] Genes related to hyaline cartilage, including SOX9, COL2A1, ACAN, and COMP, were detected. Genes related to fibrocartilage or mesenchyme were detected, including COL1A1, COL3A1, ENG, and NT5E. Genes related to hypertrophic cartilage were detected, including COL10A1, RUNX2, MMP13, and ALPL.

[0130] 6. COL2A1 Immunofluorescence Validation MPC and iMSC samples were fixed separately, immunofluorescence staining was performed using COL2A1 antibody, and nuclear counterstaining was performed using DAPI.

[0131] 7. Results Single-cell transcriptome analysis showed that ( Figure 2 MPC-derived cell populations show a tendency to differentiate into hyaline cartilage, with high expression of hyaline cartilage-related genes such as COL2A1, SOX9, and ACAN. In contrast, iMSC-derived cell populations are more likely to be in a fibrocartilaginous or conventional mesenchymal cell state, with high expression of genes such as COL1A1, ENG, and NT5E.

[0132] Immunofluorescence assay showed ( Figure 3 The COL2A1 signal in the MPC group was significantly stronger than that in the iMSC group.

[0133] 8. Conclusion MPCs exhibit a stronger tendency to differentiate into hyaline cartilage compared to iMSCs, and also show higher COL2A1 expression at the protein level.

[0134] Experimental Example 5: Preparation of Cartilage Microtissue from MPCs Derived from Human iPSCs 1. Preparation of P8 generation MPCs: Take MPCs continuously amplified to the P8 generation (amplified using the MPC cell culture medium from Example 3), and digest them into a single-cell suspension using TrypLE. After centrifugation, resuspend in MPC cell culture medium, and take samples for counting and later use.

[0135] 2. Elplasia 12K Flask preprocessing Cartilage microtissue was prepared using Corning Elplasia 12K Flask. The 12K culture flask was placed upright, and 10 mL of 35% sterile ethanol solution was added. The sides and edges of the flask were gently rotated and tapped to ensure the wetting agent completely wetted the micropore surface, which was then aspirated. Next, 15 mL of sterile water was added to the 12K culture flask. The flask was then placed horizontally, and the mixture was gently rotated to ensure the water completely covered the micropore surface, which was then aspirated. This step was repeated twice. The second batch of sterile water was discarded before cell inoculation, keeping the culture flask moist.

[0136] 3. Cell inoculation Based on the cell count results, take approximately 6 × 10⁶ cells. 7 One P8 generation MPC cell was placed in a 50 mL centrifuge tube and brought to a final volume of 25 mL with MPC cell culture medium. The mixture was thoroughly mixed. The sterile water washing solution in the 12K culture flask was discarded. The 12K culture flask was then held upright, and 25 mL of cell suspension was added to the bottom of the flask. The flask was then placed horizontally and gently shaken to ensure even distribution of cells in the microwells. The flask was incubated at 37°C with 5% CO2 for 72 h to allow the cells to aggregate and form MPC cell microtissues.

[0137] 4. Cartilage microtissue induction culture medium After culturing for 72 h, discard as much of the culture supernatant as possible, and slowly add 25 mL of room temperature chondrocyte microtissue culture medium. This is recorded as 3D0d.

[0138] The chondrocyte microtissue culture medium includes DMEM high glucose medium and the following added components: 1% KnockOut serum substitute, 1% insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 μg / mL penicillin-streptomycin, 0.1 mmol / L sodium ascorbate, 0.1 μmol / L dexamethasone, 40 μg / mL proline, 20 ng / mL TGF-β3 and 20 ng / mL BMP2.

[0139] 5. Static culture and dynamic culture Starting from 3D0d, fresh cartilage microtissue culture medium was replaced every 2-3 days, and the microtissue diameter was photographed and recorded.

[0140] At day 3 (d7), the culture supernatant in the 12K culture flask was aspirated, and 20 mL of cartilage microtissue culture medium was added. The bottom of the flask was gently tapped to detach the microtissue. The microtissue in the culture flask was collected into a 50 mL centrifuge tube. Another 20 mL of cartilage microtissue culture medium was added to wash the bottom of the flask, and the washings were collected into the same centrifuge tube. The microtissue suspension in the centrifuge tube was mixed well, and the microtissue was transferred to 10 cm culture dishes at a 1:5 ratio. Cartilage microtissue culture medium was added to each dish to a final volume of 25 mL. The culture dishes were placed on a horizontal shaker in a 37°C, 5% CO2 incubator at 60 rpm and cultured for day 3 (d21).

[0141] 6. Results The results are as follows Figure 6 As shown in the bright-field images, during the culture periods of 0, 6, and 14 days, MPCs were able to form cartilage microtissues with regular morphology (round or near-round, relatively uniform in size), dense structure, and large volume. In contrast, iMSC-derived cartilage microtissues were smaller in volume and had weaker morphological stability. Compared with iMSC or MSC-derived cartilage microtissues, MPC-derived cartilage microtissues were larger in volume, more structurally complete, and had better morphological stability.

[0142] Experimental Example 6: Histological staining and hyaline cartilage property identification of MPC-derived cartilage microtissue 1. Cartilage microtissue fixation MPC-derived cartilage microtissue samples were collected and washed twice with deionized water or PBS. The samples were then placed in 3–5 volumes of 4% tissue cell fixative and fixed at room temperature for at least 48 h.

[0143] 2. Paraffin embedding and sectioning After fixation, the cartilage microtissue sample was placed in a tissue embedding cassette and rinsed with running water for at least 30 minutes to remove the fixative.

[0144] The samples were then subjected to gradient dehydration, clearing, and paraffin embedding. They were sequentially treated with 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, xylene, paraffin-xylene, and paraffin. After paraffin embedding, paraffin blocks containing cartilage microtissue were obtained. Serial sections were prepared using a paraffin microtome, with a section thickness of approximately 5 μm. After being placed on glass slides, the sections were dried in a 60°C oven for at least 4 hours.

[0145] 3. HE staining After dewaxing and rehydration, paraffin sections were stained with hematoxylin and eosin to observe the overall structure, cell morphology, and cell distribution of cartilage microtissue.

[0146] 4. Alcian Blue (SO) staining After dewaxing and rehydration, the sections were treated with Alcian acidification solution, followed by staining with Alcian Blue for approximately 30 minutes, and counterstained with nuclear solid red. Alcian Blue staining was used to detect acidic glycosaminoglycan deposition.

[0147] 5. Safranin O-Fast Green Staining After dewaxing and rehydration, the sections were stained with Weigert hematoxylin, differentiated by acid, stained with Fast Green, and stained with Safranin O. Safranin O-Fast Green staining was used to detect chondroitin proteoglycan matrix deposition.

[0148] 6. Toluidine blue staining After dewaxing and rehydration, the sections were stained with toluidine blue for about 30 minutes to observe the metachromaticity of the cartilage matrix and the deposition of proteoglycans.

[0149] 7. Determination of the properties of transparent cartilage If MPC-derived cartilage microtissue exhibits one or more of the following characteristics, it is determined to have hyaline cartilage-like properties: (1) HE staining showed that the micro-tissue structure was dense and the cells were evenly distributed; (2) Alcian blue staining positive; (3) Safranin O was positive in Safranin O-Fast Green staining; (4) Positive toluidine blue staining or obvious metachromaticity; (5) The cartilage matrix is ​​deposited uniformly; (6) Express SOX9, COL2A1 and / or ACAN; (7) Positive expression of COLII protein.

[0150] 8. Determination of resistance to hypertrophication Further examination was conducted to detect the expression of COL10A1, RUNX2, MMP13, and / or ALPL. If the expression of COL10A1, RUNX2, MMP13, and / or ALPL in the MPC-derived cartilage microtissue was low, and the expression of COLX protein was low or negative, it was determined that it had anti-chondrogenesis characteristics.

[0151] 9. Results SO staining results of MPC chondroid globules and hyaline cartilage-related gene expression are as follows: Figure 7As shown in the figure. Histological staining revealed that MPC-derived cartilage microtissue exhibited more pronounced cartilage matrix deposition. Single-cell transcriptomic or gene expression analysis further showed that MPC-derived cartilage microtissue was enriched with hyaline cartilage-related markers such as SOX9, COL2A1, and ACAN, while hypertrophic cartilage-related markers such as COL10A1 were expressed at lower levels. These results indicate that MPC-derived cartilage microtissue possesses hyaline cartilage-like properties and an anti-hypertrophic tendency. The results also suggest that MPC-derived cartilage microtissue is rich in glycosaminoglycans and proteoglycan matrices, possesses hyaline cartilage-like properties, and exhibits a low tendency to hypertrophicate.

[0152] Experimental Example 7: In vivo colonization and survival ability of subcutaneous transplantation of MPC-derived cartilage microtissue 1. Transplant preparation MPC-derived cartilage microtissue was prepared according to the method in Experimental Example 5, with iMSC-derived cartilage microtissue used as a control.

[0153] 2. Subcutaneous transplantation Immunodeficient mice or nude mice were selected as recipient animals, and MPC-derived cartilage microtissue was transplanted into the subcutaneous region of the back of the experimental animals. iMSC-derived cartilage microtissue was used as a control group.

[0154] 3. Obtain materials Approximately two months after transplantation, tissue samples were taken to observe the general morphology, number, and tissue integrity of the grafts.

[0155] 4. Histological examination The removed grafts were fixed with 4% histocellular fixative, embedded in paraffin, and sectioned. The sections were then stained with hematoxylin and eosin (HE), alicin blue, safranin O-fast green, and / or toluidine blue.

[0156] 5. Results A comparison of the in vivo survival and colonization ability of MPC-derived cartilage microtissue and iMSC-derived cartilage microtissue after subcutaneous transplantation, as follows: Figure 8 As shown in the figure. Approximately two months post-transplantation, gross observation revealed well-defined subcutaneous grafts in the MPC group, while the iMSC group showed weaker colonization ability, with some samples exhibiting small grafts or no obvious grafts. Histological staining results showed that MPC-derived grafts, after staining with HE, alcine blue, safranin O-Fixed green, and toluidine blue, exhibited cartilage-like tissue structure and positive staining for cartilage matrix (rich in cartilage matrix components), suggesting that they can maintain cartilage matrix components such as glycosaminoglycans and proteoglycans in vivo. These results indicate that MPC-derived cartilage microtissues have good in vivo colonization, survival, and cartilage-like matrix maintenance capabilities.

[0157] 6. Conclusion MPC-derived cartilage microtissues exhibit good in vivo colonization, survival, and cartilage matrix maintenance capabilities.

[0158] Experimental Example 8: MPC-derived cartilage microtissue for in situ repair of cartilage defects in rat knee joints 1. Model Establishment A rat model of knee joint cartilage defect was established.

[0159] 2. Cartilage microtissue transplantation The MPC-derived cartilage microtissue obtained in Experiment 5 was transplanted to the cartilage defect site. iMSC-derived cartilage microtissue was used as a control. If necessary, fibrin glue, collagen gel, hyaluronic acid gel, or other biocompatible materials could be used to assist in fixing the cartilage microtissue.

[0160] 3. Material collection time Samples were taken at 1, 2 and 8 weeks postoperatively for histological and immunohistochemical testing.

[0161] 4. Preparation of animal tissue samples Animal joint tissue samples were collected and washed twice with deionized water or PBS. The samples were then placed in 3–5 volumes of 4% cell fixative and fixed at room temperature for at least 72 h. After fixation, the fixative was discarded, and the samples were washed twice with deionized water. 3–5 volumes of EDTA decalcification solution were added, and decalcification was carried out at 37°C for at least 48 h. After decalcification, the samples were washed twice with deionized water, and the tissue to be tested was cut for subsequent embedding and staining.

[0162] 5. Paraffin sections and staining Animal joint tissues were dehydrated, cleared, permeabilized in paraffin, and embedded in paraffin to obtain sections approximately 5 μm thick. After dewaxing and rehydration, the sections were stained with hematoxylin and eosin (HE) and safranin O-fast green. Further immunohistochemistry or immunofluorescence was used to detect the expression of COLII and COLX.

[0163] 6. Results The results are as follows Figure 9 As shown in the figure. Histological staining results at 1, 2, and 8 weeks post-surgery showed that the defect area in the MPC group gradually formed repair tissue rich in cartilage matrix, with positive Safranin O staining, indicating proteoglycan deposition (positive COLII expression, low COLX expression). Compared with the iMSC group, the repair area in the MPC group was more fully filled, and the cartilage matrix was more significantly preserved. Immunohistochemical results showed that the repair tissue in the MPC group was positive for COLII expression, while COLX expression was low, suggesting that the repair tissue maintained hyaline cartilage properties and had a low tendency to hypertrophy. In contrast, the iMSC-derived cartilage microtissue control group showed weaker repair tissue formation, poorer hyaline cartilage properties, and may be accompanied by fibrosis or hypertrophy-related changes.

[0164] 7. Conclusion MPC-derived cartilage microtissue can promote in situ repair in a rat knee cartilage defect model and maintain hyaline cartilage properties and anti-hypertrophy characteristics.

[0165] Experimental Example 9: MPC-derived cartilage microtissue for repairing cartilage defects in rabbit knee joints and humanization tracking 1. Model establishment and porting A rabbit knee joint cartilage defect model was established, and MPC-derived cartilage microtissue was transplanted into the defect area. Three control groups were established: a defect control group, a Sham group, and an iMSC-derived cartilage microtissue control group. The Sham group consisted of joint exposure and the same surgical procedure, but without cartilage defect preparation or cartilage microtissue implantation; the defect control group underwent the same cartilage defect preparation but without cartilage microtissue implantation.

[0166] 2. Postoperative check-ups at 1, 3, and 6 months Postoperative samples were taken at corresponding time points for gross observation, histological staining, and immunohistochemical testing.

[0167] 3. Histological examination HE staining was used to observe the repaired tissue structure and the integrity of the joint surface.

[0168] Safranin O-Fast Green staining was used to detect proteoglycan matrix deposition.

[0169] COLII immunostaining was used to detect hyaline cartilage-associated matrix formation.

[0170] COLX immunostaining was used to detect characteristics related to hypertrophic cartilage.

[0171] 4. Human-to-human tracing Anti-human Lamin A antibody was used to detect human cell signals in the repair tissue to determine whether the repair tissue contained or originated from transplanted human iPSC-derived MPC cartilage microtissue.

[0172] 5. Results One month post-surgery, gross observation and histological staining showed ( Figure 10 MPC-derived cartilage microtissue promoted the formation of cartilage-like repair tissue in the defect area, was positive for Safranin O staining, showed positive COLII expression, but low COLX expression; iMSC-derived cartilage microtissue showed fibrous tissue formation and / or calcification. Anti-human Lamin A detection showed ( Figure 11 The presence of human cell-related signals in the repaired tissue suggests that transplanted human iPSC-derived MPC cartilage microtissue participated in the repair process. Long-term observation showed ( Figure 12The MPC group showed better defect filling and joint surface repair at 1, 3 and 6 months postoperatively. Histological staining and scoring results at 6 months postoperatively showed that the repair effect of the MPC group was better than that of the iMSC group and the model control group.

[0173] 6. Conclusion MPC-derived cartilage microtissue can participate in the repair of cartilage defects in rabbit knee joints, forming hyaline cartilage-like repair tissue, and has a low tendency to hypertrophy.

[0174] Experimental Example 10: MPC-derived cartilage microtissue for long-term repair and biomechanical property restoration of cartilage defects in rabbit knee joints. 1. Long-term repair and observation A rabbit knee joint cartilage defect model was established according to Experiment Example 9, and MPC-derived cartilage microtissue was transplanted. Gross observations were conducted at 1 month, 3 months, and 6 months postoperatively to assess the filling of the defect area, the continuity of the joint surface, and the integrity of the repaired tissue.

[0175] 2. Histological examination 6 months postoperatively Six months post-surgery, tissue samples were harvested for HE staining and Safranin O-Fixed Green staining. HE staining was used to observe the morphology, thickness, integration with surrounding tissues, and changes in the subchondral bone region of the repaired tissue. Safranin O-Fixed Green staining was used to detect chondroitin matrix deposition in the repaired tissue. A histological scoring system was further used to evaluate the repair outcome.

[0176] 3. Preparation of biomechanical testing samples Rabbit knee joint tissue samples were collected and washed twice with 3-5 times the volume of PBS buffer to remove surface residues. The samples were then fixed onto the nanoindenter platform using Vetbond Tissue Adhesive to ensure stable sample positioning during the testing process.

[0177] 4. Elastic modulus testing The elastic modulus of the test samples was tested using a nanoindenter or other equipment suitable for cartilage tissue mechanical testing. At least 5 test points were selected for each sample, and at least 5 tests were performed at each point. The elastic modulus values ​​at each point were recorded, and an elastic modulus distribution map or thermogram was plotted.

[0178] The detection areas include normal cartilage areas, modeled damaged areas, iMSC-derived cartilage microtissue transplantation areas, and MPC-derived cartilage microtissue transplantation areas.

[0179] 5. Representative Results In a representative set of experiments, the elastic modulus of different test regions is as follows ( Figure 13The elastic modulus of the normal cartilage region was approximately 1.552 ± 0.292 MPa or 1.836 ± 0.193 MPa; the modeled damaged region was approximately 0.322 ± 0.122 MPa; the iMSC-derived cartilage microsphere transplanted region was approximately 0.134 ± 0.027 MPa; the MPC-derived cartilage microsphere transplanted region was approximately 1.489 ± 0.366 MPa; and the normal region of the same tissue was approximately 1.462 ± 0.180 MPa. Biomechanical testing results showed that the elastic modulus of the MPC-derived cartilage microtissue transplanted region was close to that of the normal cartilage region and significantly higher than that of the damaged region and the iMSC-derived cartilage microtissue transplanted region, suggesting that MPC-derived cartilage microtissue can promote the regeneration of mechanically functional hyaline cartilage-like tissue.

[0180] 6. Result Determination If the elastic modulus of the MPC-derived cartilage microtissue transplantation area recovers to more than 50% of that of the normal cartilage area, it is considered to have an effect on improving mechanical properties.

[0181] 7. Conclusion MPC-derived cartilage microtissue can not only promote the histological repair of cartilage defects in rabbit knee joints, but also promote the recovery of mechanical function in the repaired area, forming functional hyaline cartilage-like tissue with an elastic modulus close to that of normal cartilage.

[0182] Experimental Example 11: MPC-derived cartilage microtissue for the repair of cartilage defects in the pig knee joint 1. Model Establishment Establish a model of cartilage defects in the pig knee joint.

[0183] 2. Cartilage microtissue transplantation MPC-derived cartilage microtissue was transplanted to the defect area. A defect control group, a Sham group, and other cell control groups were established.

[0184] 3. General observation After 6 months, samples were taken to observe the filling of the defect area, the smoothness of the joint surface, and the integrity of the repaired tissue.

[0185] 4. Histological and immunological testing Animal joint tissues were fixed, decalcified, embedded in paraffin, and sectioned before being stained with Safranin O-Fix Green to detect cartilage matrix deposition. Further immunostaining was used to detect COL2 and COL10 expression.

[0186] 5. Results The results are as follows Figure 14As shown in the figure. Gross observation revealed that after treatment with MPC-derived cartilage microtissue, the defect area was filled with cartilage-like tissue, improving the continuity of the joint surface. Histological staining showed that the repaired area was positive for Safranin O, indicating the deposition of cartilage proteoglycan matrix. Immunostaining showed that the repaired area was positive for COL2 expression, while COL10 expression was low, suggesting that MPC-derived cartilage microtissue can promote hyaline cartilage-like repair and maintain a low level of hypertrophy in large animal joint defect models.

[0187] 6. Conclusion MPC-derived cartilage microtissues exhibit cartilage repair capabilities in large animal (pig) articular cartilage defect models, and can form repair tissues with hyaline cartilage properties and low hypertrophy.

[0188] Experimental Example 12: MPC-derived cartilage microtissue used for osteoarthritis model treatment 1. Establishment of an osteoarthritis model A rat model of knee osteoarthritis was established, specifically using an anterior cruciate ligament transection (ACLT) combined with a focal articular cartilage defect model.

[0189] 2. Grouping and processing methods The study included a normal control group, an ACLT+Sham group, an ACLT+Defect group, and an ACLT+MPC cartilage microtissue treatment group. The normal control group did not receive ACLT or cartilage defect surgery; the ACLT+Sham group underwent only ACLT and joint exposure, without creating a focal cartilage defect or implanting microtissue; the ACLT+Defect group underwent ACLT and created a focal cartilage defect, but without implanting microtissue; and the ACLT+MPC cartilage microtissue treatment group underwent ACLT and created the same cartilage defect, then implanted MPC-derived cartilage microtissue into the defect area.

[0190] The procedure involves in-situ implantation to fill the cartilage defect; if necessary, a small amount of fibrin glue or other biocompatible materials are used to locally fix the cartilage micro-tissue.

[0191] 3. Detection Method After sampling, gross observation, HE staining, Safranin O-Fixed Green staining, COL2 immunostaining, COL10 immunostaining, Micro-CT examination, and histological scoring were performed.

[0192] 4. Results Histological staining showed ( Figure 15Compared with the model control group, the MPC-treated group showed better articular cartilage surface integrity and enhanced Safranin O staining, indicating increased cartilage matrix preservation or regeneration. Immunostaining showed increased COL2 expression and lower COL10 expression in the MPC-treated group, suggesting that it promotes hyaline cartilage-like repair and inhibits cartilage hypertrophy. Micro-CT and histological scoring results further showed ( Figure 16 MPC-derived cartilage microtissue improved cartilage degeneration and subchondral bone changes in an osteoarthritis model. These results support the use of MPC-derived cartilage microtissue for the treatment of osteoarthritis, cartilage degeneration, and articular cartilage injury.

[0193] 5. Conclusion MPC-derived cartilage microtissue can be used to prepare cell therapy products for treating osteoarthritis, cartilage degeneration, or articular cartilage damage.

[0194] In summary, this invention provides MPC cells derived from human induced pluripotent stem cells and their cartilage microtissue. The MPC cells have the following characteristics: (1) Derived from human iPSC; (2) It can be continuously amplified in vitro; (3) Low or negative CD73 expression; (4) Low or negative CD44 expression; (5) Unlike traditional CD73-positive and CD44-positive iMSCs or MSCs; (6) It has a tendency to differentiate into hyaline cartilage; (7) It can form three-dimensional cartilage micro-tissue; (8) The formed cartilage microtissue expresses SOX9, COL2A1, ACAN and / or COLII; (9) The cartilage microtissues formed showed low expression levels of COL10A1, RUNX2, MMP13, ALPL and / or COLX; (10) It has anti-chondrodysplasia characteristics; (11) It can colonize and maintain the cartilage matrix in the body; (12) It can promote the repair of articular cartilage defects in rats, rabbits and pigs; (13) It can improve cartilage degeneration in osteoarthritis models; (14) It can promote the restoration of biomechanical properties of repaired tissues.

[0195] Therefore, the human iPSC-derived, CD73-low or CD44-low or CD44-low MPCs and the cartilage microtissues formed therefrom provided by this invention can be used to prepare anti-hypertrophic hyaline cartilage repair products, articular cartilage defect repair products, cartilage tissue engineering products, and osteoarthritis treatment products.

[0196] Experiment Example 13: Effect of altered components in the culture medium for inducing differentiation of human induced pluripotent stem cells into MPCs on MPC performance. The following culture medium combination for inducing human induced pluripotent stem cells to differentiate into MPC was used as a control for inducing human induced pluripotent stem cells to differentiate into MPC.

[0197] Culture medium combination A: The culture medium combination for inducing differentiation of human induced pluripotent stem cells into MPCs in Example 1; Culture medium combination B: The culture medium combination for inducing differentiation of human induced pluripotent stem cells into MPC in Example 2; The composition of control culture medium combination C is as follows: The first induction medium, used for the first stage of induction culture, is a basic differentiation medium supplemented with the following additives: 50 ng / mL BMP4, 1.5 μM CHIR99021 and 50 ng / mL bFGF, with the concentration of the additives based on the volume of DMEM high-glucose medium. The second induction medium, used for the second stage of induction culture, is a basic differentiation medium supplemented with the following added components: 5 μM A8301, 2 μM C59, 500 nM LDN193189, with the concentration of the added components based on the volume of DMEM high-glucose medium. In addition, the third induction medium, used for the third stage of induction culture, is a basic differentiation medium supplemented with the following added components: 10 nM SAG21K and 2 μM C59, with the concentration of the added components based on the volume of DMEM high glucose medium; The composition of the differentiation basal culture medium is the same as in Example 2.

[0198] The composition of control culture medium combination D is as follows: The first induction medium, used for the first stage of induction culture, is a basic differentiation medium supplemented with the following added components: 20 ng / mL Activin A, 2 μM BIO and 50 ng / mL bFGF, with the concentration of the added components based on the volume of DMEM high glucose medium; The second induction medium, used for the second stage of induction culture, is a basic differentiation medium supplemented with the following added components: 5 μM A8301, 2 μM C59, 500 nM LDN193189, with the concentration of the added components based on the volume of DMEM high-glucose medium. In addition, the third induction medium, used for the third stage of induction culture, is a basic differentiation medium supplemented with the following added components: 10 nM SAG21K and 2 μM C59, with the concentration of the added components based on the volume of DMEM high glucose medium; The composition of the differentiation basal culture medium is the same as in Example 2.

[0199] The composition of control culture medium combination E is as follows: The first induction medium, used for the first stage of induction culture, is a basic differentiation medium supplemented with the following added components: 20 ng / mL Activin A, 1.5 μM CHIR99021, with the concentration of the added components based on the volume of DMEM high glucose medium; The second induction medium, used for the second stage of induction culture, is a basic differentiation medium supplemented with the following added components: 5 μM A8301, 2 μM C59, 500 nM LDN193189, with the concentration of the added components based on the volume of DMEM high-glucose medium. In addition, the third induction medium, used for the third stage of induction culture, is a basic differentiation medium supplemented with the following added components: 10 nM SAG21K and 2 μM C59, with the concentration of the added components based on the volume of DMEM high glucose medium; The composition of the differentiation basal culture medium is the same as in Example 2.

[0200] The composition of the control culture medium combination F is as follows: The first induction medium, used for the first stage of induction culture, is a basic differentiation medium supplemented with the following components: 20 ng / mL Activin A, 1.5 μM CHIR99021 and 50 ng / mL bFGF, with the concentration of the added components based on the volume of DMEM high glucose medium. The second induction medium, used for the second stage of induction culture, is a basic differentiation medium supplemented with the following added components: 10 μM SB431542, 2 μM C59, and 500 nM LDN193189. The concentration of the added components is based on the volume of DMEM high-glucose medium. In addition, the third induction medium, used for the third stage of induction culture, is a basic differentiation medium supplemented with the following added components: 10 nM SAG21K and 2 μM C59, with the concentration of the added components based on the volume of DMEM high glucose medium; The composition of the differentiation basal culture medium is the same as in Example 2.

[0201] The composition of the control culture medium combination G is as follows: The first induction medium, used for the first stage of induction culture, is a basic differentiation medium supplemented with the following components: 20 ng / mL Activin A, 1.5 μM CHIR99021 and 50 ng / mL bFGF, with the concentration of the added components based on the volume of DMEM high glucose medium. The second induction medium, used for the second stage of induction culture, is a basic differentiation medium supplemented with the following added components: 5 μM A8301, 2 μM C59, 500 nM LDN193189, with the concentration of the added components based on the volume of DMEM high-glucose medium. And, the third induction medium, used for the third stage of induction culture, is a basic differentiation medium supplemented with the following additives: 2 μM Purmorphamine and 2 μM C59, with the concentration of the additives based on the volume of DMEM high glucose medium; The composition of the differentiation basal culture medium is the same as in Example 2.

[0202] The composition of control culture medium combination H is as follows: The first induction medium, used for the first stage of induction culture, is a basic differentiation medium supplemented with the following components: 20 ng / mL Activin A, 1.5 μM CHIR99021 and 50 ng / mL bFGF, with the concentration of the added components based on the volume of DMEM high glucose medium. The second induction medium, used for the second stage of induction culture, is a basic differentiation medium supplemented with the following added components: 5 μM A8301, 2 μM C59, 500 nM LDN193189, with the concentration of the added components based on the volume of DMEM high-glucose medium. And, the third induction medium, used for the third stage of induction culture, is a basic differentiation medium supplemented with the following added components: 10 nM SAG21K, with the concentration of the added components based on the volume of DMEM high-glucose medium; The composition of the differentiation basal culture medium is the same as in Example 2.

[0203] Using the above-mentioned culture medium combinations, human induced pluripotent stem cells were induced to differentiate into MPCs according to the method described in Experiment 1. The induction culture time for each stage was 30 h. The cells obtained from the induced differentiation were analyzed by FACS. The proportions of CD11b, CD105, CD73, CD19, CD31, CD34, CD44, CD45, and CD90 positive cells are shown in Table 3. The results show that replacing or removing key factors at different stages of differentiation affects the expression of CD73 and CD44 in the differentiated MPCs, making them more similar to the properties of MSCs with high expression of CD73 and CD44. This property makes the formed cartilage have an inherent risk of hypertrophy and calcification [21,22].

[0204] Table 3

[0205] Experiment Example 14: Effects of altered components in MPC cell culture medium on the microtissue properties of MPC differentiated chondrocytes P0 MPCs were seeded in MPC cell culture medium with altered core components for maintaining MPC amplification, and then passaged to P8 cells for chondrocyte differentiation. The passage amplification method was the same as in Experiment 3.

[0206] Compared with the MPC cell culture medium in Example 3, the MPC cell culture medium used to maintain the core components of MPC amplification was modified by removing one or more added components, as detailed below: MPC complete culture medium, namely the MPC cell culture medium of Example 3; MPC-CHIR99021 refers to the MPC cell culture medium from which CHIR99021 has been removed in Example 3.

[0207] MPC-A8301 refers to the MPC cell culture medium from which A8301 has been removed, as described in Example 3.

[0208] MPC-EGF, which is the MPC cell culture medium in Example 3 with EGF removed.

[0209] MPC-CHIR99021-A8301 refers to the MPC cell culture medium from which CHIR99021 and A8301 are removed.

[0210] MPC-CHIR99021-EGF, which is the MPC cell culture medium of Example 3 with CHIR99021 and EGF removed.

[0211] MPC-CHIR99021-A8301-EGF, which is the MPC cell culture medium of Example 3 with CHIR99021, A8301 and EGF removed.

[0212] The obtained P8 MPC cells were differentiated into chondroids using the same method as in Experiment 5, and differentiation lasted for 14 days. Chondroids from each group were collected, total RNA was extracted and reverse transcribed; real-time quantitative PCR was used to detect ACTB, COL2A1, ACAN, Versican, COL1A1, COL1A2, SOX9, COL10A1, CRTAC1, UCMA, CILP, and PRG4. Each sample was prepared in technical replicates, with ACTB used as an internal control; Ct values ​​were used for quality verification, and the relative expression levels were calculated using the 2^-ΔΔCt method.

[0213] The q-PCR results are shown in Table 4, and the specific expression levels (Ct values) are shown in Table 5. Changes in the core components of MPC maintenance amplification significantly affect the quality properties of the subsequently formed cartilage tissue. Removal of any single component increases the expression of the hypertrophy marker COL10A1 in the subsequently formed cartilage tissue, with the most severe impact observed in groups where CHIR99021 was removed (R16, R17, R18). This indicates that CHIR99021 plays a decisive role in cartilage differentiation within the MPC cell culture medium of this invention.

[0214] Table 4

[0215] Table 5

[0216] Experimental Example 15: Comparison of the ability of the MPC cell culture medium of the present invention with conventional expansion culture media of other tissue precursor cells to form cartilage tissue in vivo. 1. Transplant preparation MPC-derived cartilage microtissues (cartilage globules and membranes) were prepared according to the method in Example 5. Other tissue precursor cells were used as a control in a conventional expansion medium. The conventional expansion medium was DMEM high glucose medium supplemented with 3 μM CHIR, 0.5 μM MA8301 and 10 μM Y27632.

[0217] 2. Subcutaneous transplantation Immunodeficient mice or nude mice were selected as recipient animals. MPC-derived cartilage microtissues obtained by conventional expansion of other tissue precursor cells using the MPC cell culture medium of Example 3 of this invention and MPC-derived cartilage microtissues were transplanted subcutaneously into the back of the experimental animals.

[0218] 3. Obtain materials The tissue was harvested approximately 3 months after transplantation and subjected to histological examination.

[0219] 4. Histological examination The removed grafts were fixed with 4% histocellular fixative, embedded in paraffin, and sectioned. The sections were then stained with hematoxylin and eosin (HE), alicin blue, safranin O-fast green, and / or toluidine blue.

[0220] 5. Results The results are as follows Figure 17 and Figure 18As shown. A1063 corresponds to MPC-derived cartilage microtissue prepared after expansion using the MPC cell culture medium of Example 3 of this invention. Three months after subcutaneous transplantation, A1063 formed a clear graft. HE, Allicin Blue, Safranin O-Fixed Green, and Toluidine Blue staining showed cartilage-like tissue structure and positive cartilage matrix, suggesting that it maintains cartilage matrix components such as glycosaminoglycans and proteoglycans in vivo. Control samples prepared using conventional culture medium for expansion of other reported tissue precursor cells are labeled A1045 and A1054. After development, they only formed fibrotic connective tissue, and their histological morphology is shown in [Figure 3]. Figure 18 .

[0221] Experiment Example 16: The Effect of Changing the Concentration of Key Differentiation Factors on MPC Differentiation and Amplification MPC-A group: iPSC differentiation and MPC amplification were performed using the culture medium combination of Example 1 and Example 3, respectively; MPC-B group: iPSC differentiation and MPC amplification were performed using the culture medium combination of Example 2 and Example 4, respectively. The methods for induction of differentiation and amplification are described in Experiment 1.

[0222] Immunofluorescence staining based on COLII and FACS analysis based on SOX9 were performed on MPCs (P9) obtained from MPC-A and MPC-B groups. The results showed that changing the concentration of key factors in the culture system within the concentration range set in this invention did not affect the chondrogenic tendency of MPCs. Figure 19 Chondroid differentiation experiments were performed on the two groups of MPCs (P12), and the results showed no morphological difference. Figure 20 The expression of key genes for hyaline cartilage properties showed little difference. Figure 21 ).

[0223] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0224] References: [1] Sophia Fox AJ, Bedi A, Rodeo S A. The basic science of articularcartilage: structure, composition, and function[J]. Sports health, 2009, 1(6): 461-468. [2] Steinmetz J D, Culbreth G T, Haile L M, et al. Global, regional,and national burden of osteoarthritis, 1990-2020 and projections to 2050: asystematic analysis for the Global Burden of Disease Study 2021[J]. TheLancet Rheumatology, 2023, 5(9): e508-e522. [3] Brittberg M. Clinical articular cartilage repair—an up to datereview[J]. Annals of Joint, 2018, 3. [4] Song S J, Park C H. Microfracture for cartilage repair in theknee: current concepts and limitations of systematic reviews. Annals ofTranslational Medicine, 2019, 7(Suppl 3): S108. [5] Yue L, Lim R, Owens B D. Latest advances in chondrocyte-basedcartilage repair. Biomedicines, 2024, 12(6): 1367. [6] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria fordefining multipotent mesenchymal stromal cells. The International Society forCellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317. [7] Pelttari K, Winter A, Steck E, et al. Premature induction ofhypertrophy during in vitro chondrogenesis of human mesenchymal stem cellscorrelates with calcification and vascular invasion after ectopictransplantation in SCID mice. Arthritis&Rheumatism: Official Journal of theAmerican College of Rheumatology, 2006, 54(10): 3254-3266. [8] Chen S et al., Strategies to minimize hypertrophy in cartilageengineering and regenerative medicine, 2015. [9] Shigley C, Trivedi J, Meghani O, et al. Suppressing chondrocytehypertrophy to build better cartilage. Bioengineering, 2023, 10(6): 741.

[10] Lefebvre V, Dvir-Ginzberg M. SOX9 and the many facets of itsregulation in the chondrocyte lineage[J]. Connective tissue research, 2017,58(1): 2-14.

[11] Diederichs S, Klampfleuthner F A M, Moradi B, et al. Chondraldifferentiation of induced pluripotent stem cells without progression intothe endochondral pathway. Frontiers in Cell and Developmental Biology, 2019,7: 270.

[12] Yamashita A, Morioka M, Yahara Y, et al. Generation ofscaffoldless hyaline cartilaginous tissue from human iPSCs. Stem cellreports, 2015, 4(3): 404-418.

[13] Dicks A, Wu C L, Steward N, et al. Prospective isolation ofchondroprogenitors from human iPSCs based on cell surface markers identifiedusing a CRISPR-Cas9-generated reporter. Stem Cell Research&Therapy, 2020, 11(1): 66.

[14] Rodríguez Ruiz A, Dicks A, Tuerlings M, et al. Cartilage fromhuman-induced pluripotent stem cells: comparison with neo-cartilage fromchondrocytes and bone marrow mesenchymal stromal cells. Cell and TissueResearch, 2021, 386(2): 309-320.

[15] Pothiawala A, Sahbazoglu B E, Ang B K, et al. GDF5+chondroprogenitors derived from human pluripotent stem cells preferentiallyform permanent chondrocytes. Development, 2022, 149(11): dev196220.

[16] Saito T .The superficial zone of articular cartilage.Inflammation and Regeneration, 2022, 42(1):1-6.DOI:10.1186 / s41232-022-00202-0.

[17] Hanaki S , Yamada D, Takao T ,et al. Efficient Production ofChondrocyte Particles from Human iPSC-Derived Chondroprogenitors Using aPlate-Based Cell Self-Aggregation Technique. International Journal ofMolecular Sciences, 2024, 25(22).DOI:10.3390 / ijms252212063.

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Claims

1. A culture medium composition for preparing iPSC-derived chondrogenic mesenchymal precursor cells, characterized in that, The culture medium assembly includes: The first induction medium consists of a basic differentiation medium and the following added components: 20-30 ng / mL Activin A, 1.5-3 μM CHIR99021 and 20-50 ng / mL bFGF; The second induction medium consists of a basic differentiation medium and the following added components: 1-5 μM A8301, 1-2 μM MC59 and 250-500 nM LDN193189; In addition, the third induction medium, which consists of the differentiation basal medium and the following added components: 5-10 nM SAG21K and 1-2 μM C59; The differentiation basal culture medium includes a basic culture medium and the following added components: insulin, transferrin, sodium selenite, B27, NEAA and β-mercaptoethanol; The concentration of the added components is based on the volume of the basal culture medium.

2. The culture medium composition for preparing iPSC-derived chondrogenic mesenchymal precursor cells according to claim 1, characterized in that, The differentiation basal medium contains the following components at concentrations based on the volume of the basal medium: penicillin-streptomycin 1-2% v / v, insulin-transferrin-selenium additive 1-2% v / v, B27 1-2% v / v, NEAA 1-2% v / v, and β-mercaptoethanol 90-120 μmol / L.

3. The culture medium composition for preparing iPSC-derived chondrogenic mesenchymal precursor cells according to claim 1, characterized in that, The culture medium combination also includes MPC cell culture medium for the expansion of iPSC-derived chondrogenic mesenchymal precursor cells, comprising a basal medium and the following additives: 3%-5% v / v KnockOut serum substitute, 1-3 μM CHIR99021, 5-10 μM A8301 and 20-40 ng / mL EGF.

4. The culture medium composition for preparing iPSC-derived chondrogenic mesenchymal precursor cells according to any one of claims 1 to 3, characterized in that, The basal medium for differentiation was DMEM high-glucose medium; And / or, the basal medium for MPC cell culture is DMEM / F12 medium.

5. Any of the following applications of the culture medium combination according to any one of claims 1 to 4: (1) Preparation of iPSC-derived chondrogenic mesenchymal precursor cells; (2) Preparation of chondrocyte spheroids; (3) Preparation of cartilage microtissue.

6. A method for preparing iPSC-derived chondrogenic mesenchymal precursor cells, characterized in that, The method includes: using iPSCs as starting cells, and performing staged induction culture using the culture medium combination described in any one of claims 1 to 4.

7. The method for preparing iPSC-derived chondrogenic mesenchymal precursor cells according to claim 6, characterized in that, The method includes: sequentially using a first induction medium, a second induction medium, and a third induction medium to perform a first-stage induction culture, a second-stage induction culture, and a third-stage induction culture.

8. The method for preparing iPSC-derived chondrogenic mesenchymal precursor cells according to claim 7, characterized in that, The induction culture time for the first, second, and third stages is 24–30 h.

9. The method for preparing iPSC-derived chondrogenic mesenchymal precursor cells according to any one of claims 6 to 8, characterized in that, After the third stage of induction culture, the method further includes expanding the induced chondrogenic mesenchymal precursor cells using MPC cell culture medium.

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