Construction method and application of induced pluripotent stem cells and cell lines with dual reporter genes

Abstract

The present application relates to the field of biomedicine technology, and in particular to an induced pluripotent stem cell with dual reporter genes. The induced pluripotent stem cell with dual reporter genes comprises an endogenous COL2A1 locus and a COL10A1 locus in its genome, wherein the COL2A1 locus comprises a first reporter gene and an endogenous COL2A1 gene, and the COL10A1 locus comprises a second reporter gene and an endogenous COL10A1 gene, wherein the first reporter gene and the second reporter gene encode different proteins, respectively. The induced pluripotent stem cell with dual reporter genes has the ability to differentiate into chondrocytes and further induce the generation of hypertrophic cartilage organoids. The cell line of the above-mentioned cells can be used to construct human hypertrophic cartilage organoids in vitro, to track cartilage differentiation and hypertrophy in real time in vitro, and can be used in the fields of disease modeling, drug screening, tissue transplantation, etc.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a method for constructing and applying an induced pluripotent stem cell and a cell line having dual reporter genes. Specifically, the application is the application of the cell line of the induced pluripotent stem cell having dual reporter genes in screening osteoarthritis intervention drugs. Background Art

[0002] Osteoarthritis (OA), also known as degenerative joint disease, osteophyte, and osteoarthritis, is a group of heterogeneous diseases that cause joint symptoms and signs due to the destruction of articular cartilage integrity and lesions of the subchondral bone plate at the joint margin. Chondrocytes are the only cell type in cartilage. Studies have shown that phenotypic changes of chondrocytes (such as cell hypertrophy and matrix calcification) can lead to the occurrence of osteoarthritis cartilage degeneration, and proliferative chondrocytes (ProCs), prehypertrophic chondrocytes (preHTCs), and hypertrophic chondrocytes (HTCs) are closely related to this phenotype.

[0003] Articular cartilage is mainly composed of chondrocytes and extracellular matrix. Type II collagen (COL2A1) is the most important component of cartilage matrix and a specific marker of chondrocytes. COL2A1 can form a complex extracellular matrix scaffold together with other proteins and proteoglycans to withstand mechanical forces, maintain physiological balance, and provide anchoring sites for chondrocytes, extracellular matrix molecules and growth factors. Most human bones develop through the endochondral pathway, in which cartilage-forming chondrocytes proliferate and expand into hypertrophic chondrocytes, eventually undergoing apoptosis and being replaced by bone. Despite being in the terminal differentiation stage, hypertrophic chondrocytes are considered to be the main engine of bone growth. Abnormal chondrocyte hypertrophy has been found in many skeletal dysplasias and osteoarthritis. At the same time, as a specific marker of hypertrophic chondrocytes, the type X collagen gene (COL10A1) is also crucial for endochondral bone formation, because mutations and changes in COL10A1 expression are often accompanied by abnormal chondrocyte hypertrophy in many bone diseases.

[0004] Organoids are tissue-like 3D structures that can be generated from a variety of sources, including differentiation of pluripotent stem cells, fetal and adult primary tissues, and primary and metastatic tumors. In the expert consensus published in 2021, organoids are defined as self-organization of (pluripotent) stem cells, progenitor cells, and / or differentiated cells through cell-cell and cell-matrix interactions, thereby reproducing various aspects of natural tissue structure and function in vitro. Organoids can simulate the complexity of human organs and provide a good model for biological experimental research.

[0005] Compared with animal models, organoids differentiated from human stem cells have similar structural characteristics and genetic characteristics to human organs, and even have the same mutation direction. After going through the entire development process of the organ, it is possible to intuitively and conveniently study the process of organ development, maturation, and pathological changes, and are more valuable for reference in building human disease models. In addition, the rapid development of organoid chip technology, microplate fluorescence detection technology, stem cell gene editing technology, etc., has further promoted the use of organoids as a screening platform and evaluation model for drug development. With the help of organoid libraries of different tissues from multiple individuals, it is possible to quickly screen out drugs with good efficacy and high safety with high throughput, which not only reduces the cost of drug development, but also uses induced pluripotent stem cells (iPSCs) from the patient himself, taking into account the sensitivity of individual patients to drugs, and truly realizing precision medicine. In addition, the world is currently facing a shortage of transplant organs. Human organoids highly restore the structure and function of the original organs and have a stable genetic phenotype. Researchers can use iPSCs from patients themselves to culture organoids with low immune rejection reactions, which are expected to become a good source of autologous transplant organs and effectively avoid the immune rejection reactions caused by allogeneic / xenogeneic transplants. Summary of the invention

[0006] Based on this, this application addresses the research gap in the current verification scheme for cartilage differentiation schemes and provides a method for constructing cells and cell lines with dual reporter genes, which is used to clarify the differentiation state of cells, provide tools for real-time observation of chondrocyte differentiation, mechanism research of related diseases, screening of osteoarthritis intervention drugs and cell replacement therapy.

[0007] The technical solution of this application includes the following contents:

[0008] An induced pluripotent stem cell with dual reporter genes, comprising a first reporter gene and an endogenous COL2A1 gene in the COL2A1 locus of its genome, and a second reporter gene and an endogenous COL10A1 gene in the COL10A1 locus, wherein the first reporter gene and the second reporter gene respectively encode proteins capable of generating different reporter signals;

[0009] The induced pluripotent stem cells with dual reporter genes have the ability to differentiate into chondrocytes and further induce the generation of hypertrophic cartilage organoids.

[0010] Further, the first reporter gene can be co-expressed with the endogenous COL2A1 gene, and the second reporter gene can be co-expressed with the endogenous COL10A1 gene;

[0011] The first reporter gene is under the control of the regulatory element of the COL2A1 gene locus, and the first reporter gene is located downstream of the stop codon of the COL2A1 gene;

[0012] The second reporter gene is under the control of the regulatory element of the COL10A1 gene locus, and the second reporter gene is located downstream of the stop codon of the COL10A1 gene.

[0013] Furthermore, the first reporter gene encodes a first fluorescent protein, and the second reporter gene encodes a second fluorescent protein;

[0014] The colors of the fluorescence emission lights of the first fluorescent protein and the second fluorescent protein are different.

[0015] A method for constructing a cell line of induced pluripotent stem cells with dual reporter genes as described in any of the above technical solutions comprises integrating the first reporter gene and the second reporter gene into the COL2A1 locus and the COL10A1 locus of the induced pluripotent stem cells to be genetically modified, respectively.

[0016] Furthermore, the first reporter gene and the second reporter gene are integrated into the genome of the pluripotent stem cell to be genetically modified through homologous recombination;

[0017] The homologous recombination is mediated by the CRISPR / Cas system.

[0018] Furthermore, the method comprises the following steps:

[0019] (1) introducing a first donor DNA molecule into the induced pluripotent stem cell to be genetically modified, wherein the first donor DNA molecule comprises a first exogenous sequence flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm provide homologous recombination between the first donor DNA molecule and the COL2A1 locus of the induced pluripotent stem cell to be genetically modified, and the first exogenous sequence comprises a first reporter gene; providing a Cas9 protein and a first gRNA targeting the COL2A1 gene in the induced pluripotent stem cell to be genetically modified;

[0020] (2) introducing a second donor DNA molecule into the induced pluripotent stem cell to be genetically modified, wherein the second donor DNA molecule comprises a second exogenous sequence flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm provide homologous recombination between the second donor DNA molecule and the COL10A1 locus of the induced pluripotent stem cell to be genetically modified, and the second exogenous sequence comprises a second reporter gene; providing a Cas9 protein and a second gRNA targeting the COL10A1 gene in the induced pluripotent stem cell to be genetically modified;

[0021] Steps (1) to (2) may be performed in any order.

[0022] Further, the first gRNA comprises the sequence shown in SEQ ID NO: 1;

[0023] The second gRNA comprises the sequence shown in SEQ ID NO: 2.

[0024] Further, the 5' homology arm and the 3' homology arm flanked by the first exogenous sequence have the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively;

[0025] The 5' homology arm and the 3' homology arm flanked by the second exogenous sequence have the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0026] A use of the induced pluripotent stem cells with dual reporter genes described in any of the above technical solutions in screening osteoarthritis intervention drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 COL2A1 mCherry COL10A1 eGFP Schematic diagram of the construction and functional verification of hEPSC cell lines.

[0029] Figure 2 COL2A1 mCherry COL10A1 eGFP Structural identification results of hEPSC cell line induced to differentiate into hypertrophic cartilage organoids.

[0030] Figure 3 COL2A1 mCherry COL10A1 eGFP Results of mRNA level detection, Safranin O staining and cryosection fluorescence detection during the induction of differentiation of hEPSC cell lines.

[0031] Figure 4 To construct COL2A1 mCherry COL10A1 eGFPSchematic representation of the plasmids used in the hEPSC cell line process. DETAILED DESCRIPTION

[0032] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature described or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0033] Therefore, it is intended that the present application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present application are disclosed in or are apparent from the following detailed description. It will be appreciated by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0034] In one aspect of the present application, an induced pluripotent stem cell with dual reporter genes is provided, wherein the COL2A1 locus of the genome of the induced pluripotent stem cell comprises a first reporter gene and an endogenous COL2A1 gene, and the COL10A1 locus comprises a second reporter gene and an endogenous COL10A1 gene, wherein the first reporter gene and the second reporter gene respectively encode proteins capable of generating different reporter signals;

[0035] Furthermore, induced pluripotent stem cells with dual reporter genes have the ability to differentiate into chondrocytes and further induce the formation of hypertrophic cartilage organoids;

[0036] Preferably, COL2A1 is continuously expressed during the formation of hypertrophic cartilage organoids, and COL10A1 is expressed during the hypertrophy induction stage.

[0037] In one embodiment, the first reporter gene can be co-expressed with the endogenous COL2A1 gene, and the second reporter gene can be co-expressed with the endogenous COL10A1 gene;

[0038] The first reporter gene is under the control of the regulatory element of the COL2A1 gene locus, preferably, the first reporter gene is located downstream of the stop codon of the COL2A1 gene;

[0039] The second reporter gene is under the control of the regulatory element of the COL10A1 gene locus. Preferably, the second reporter gene is located downstream of the stop codon of the COL10A1 gene.

[0040] In one embodiment, the first reporter gene encodes a first fluorescent protein, and the second reporter gene encodes a second fluorescent protein, wherein the first fluorescent protein is different from the second fluorescent protein;

[0041] Preferably, the colors of the fluorescent emission lights of the first fluorescent protein and the second fluorescent protein are different.

[0042] In one embodiment, the first fluorescent protein and the second fluorescent protein are each independently selected from a green fluorescent protein (e.g., GFP, eGFP, or sfGFP), a blue fluorescent protein (e.g., BFP, eBFP, or T-Sapphire), a cyan fluorescent protein (e.g., CyPet, CFP, or CerμLean), a yellow fluorescent protein (e.g., mCitrine, Venus, YPet, YFP, or eYFP), an orange or red fluorescent protein (e.g., mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, or mKO).

[0043] In one embodiment, the first fluorescent protein and the second fluorescent protein are detectably different; further, the first fluorescent protein and the second fluorescent protein exhibit emission spectra that differ by at least 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, or preferably 80 nm or more.

[0044] In one embodiment, the 5' end of the first reporter gene and the 5' end of the second reporter gene are flanked by a nucleic acid encoding a self-cleaving peptide or a nucleic acid encoding an internal ribosome entry site (IRES). Preferably, the self-cleaving peptide is a 2A peptide, such as T2A.

[0045] In one embodiment, the 5' end of the first reporter gene is flanked by a nucleic acid encoding a self-cleaving peptide, and the 5' end of the second reporter gene is flanked by a nucleic acid encoding an internal ribosome entry site (IRES). Preferably, the self-cleaving peptide is a 2A peptide, such as T2A.

[0046] In one embodiment, the first reporter gene is selected from a gene encoding a red fluorescent protein (e.g., mPlum, mCherry, tdTomato, mStrawberry, J-Red, or DsRed), and the second reporter gene is selected from a gene encoding a green fluorescent protein (e.g., GFP, eGFP, or sfGFP); or, the first reporter gene is selected from a gene encoding a green fluorescent protein (e.g., GFP, eGFP, or sfGFP), and the second reporter gene is selected from a gene encoding a red fluorescent protein (e.g., mPlum, mCherry, tdTomato, mStrawberry, J-Red, or DsRed);

[0047] Preferably, the first reporter gene and the second reporter gene are selected from genes encoding mCherry or eGFP.

[0048] In another aspect of the present application, a method for constructing a cell line of induced pluripotent stem cells with dual reporter genes as described in any of the above technical solutions is provided, which comprises integrating a first reporter gene and a second reporter gene into the endogenous COL2A1 locus and COL10A1 locus of the induced pluripotent stem cells to be genetically modified, respectively; the first reporter gene and the second reporter gene are as defined in the previous aspect of the present application.

[0049] In one embodiment, the first reporter gene and the second reporter gene are integrated into the genome of the cell by homologous recombination (e.g., double-strand break-mediated homologous recombination). Optionally, homologous recombination is mediated by ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), or CRISPR (clustered regularly interspaced short palindromic repeats) / Cas system. Preferably, homologous recombination is mediated by CRISPR / Cas system (e.g., CRISPR / Cas9 system).

[0050] Furthermore, the construction method comprises the following steps:

[0051] (1) introducing a first donor DNA molecule into a pluripotent stem cell to be genetically modified, wherein the first donor DNA molecule comprises a first exogenous sequence flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm provide homologous recombination between the first donor DNA molecule and the COL2A1 locus of the pluripotent stem cell to be genetically modified, and the first exogenous sequence comprises a first reporter gene; providing a Cas9 protein and a first gRNA targeting the COL2A1 gene in the pluripotent stem cell to be genetically modified;

[0052] (2) introducing a second donor DNA molecule into the pluripotent stem cell to be genetically modified, wherein the second donor DNA molecule comprises a second exogenous sequence flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm provide homologous recombination between the second donor DNA molecule and the COL10A1 locus of the pluripotent stem cell to be genetically modified, and the second exogenous sequence comprises a second reporter gene; providing a Cas9 protein and a second gRNA targeting the COL10A1 gene in the pluripotent stem cell to be genetically modified;

[0053] Steps (1) to (2) may be performed in any order.

[0054] Further, the first gRNA includes the sequence shown in SEQ ID NO:1, and the second gRNA includes the sequence shown in SEQ ID NO:2.

[0055] Furthermore, the 5' homology arm and the 3' homology arm in step (1) have homology with the upstream sequence of the stop codon and the downstream sequence of the stop codon of the COL2A1 locus, respectively; the 5' homology arm and the 3' homology arm in step (2) have homology with the upstream sequence of the stop codon and the downstream sequence of the stop codon of the COL10A1 locus, respectively.

[0056] Furthermore, the 5' homology arm and 3' homology arm flanked by the first exogenous sequence have sequences as shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; the 5' homology arm and 3' homology arm flanked by the second exogenous sequence have sequences as shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0057] In one embodiment, the first exogenous sequence further comprises a nucleic acid sequence encoding a self-cleaving peptide (e.g., a 2A peptide, such as P2A or T2A) adjacent to the 5' end of the first reporter gene; and / or, the second exogenous sequence further comprises a nucleic acid sequence of an internal ribosome entry site (IRES) adjacent to the 5' end of the second reporter gene.

[0058] In one embodiment, the first exogenous sequence further comprises a positive selection marker expression cassette located downstream of the first reporter gene, and the second exogenous sequence further comprises a positive selection marker expression cassette located downstream of the second reporter gene; wherein the positive selection marker expression cassette comprises a positive selection marker gene; preferably, the positive selection marker gene is an antibiotic resistance gene; preferably, the antibiotic resistance gene is selected from Neo (neomycin resistance gene), Puro (puromycin resistance gene), Hygro (hygromycin resistance gene), Blast (blasticidin resistance gene), Zeo (bleomycin resistance gene).

[0059] In another aspect of the present application, there is provided the use of induced pluripotent stem cells with dual reporter genes as described in any of the above technical solutions in real-time monitoring of chondrogenesis and hypertrophy.

[0060] Chondrogenesis and hypertrophy is a complex process affected by multiple factors, which is of great significance to articular cartilage regeneration and maintenance of cartilage health. Currently, no verification scheme for cartilage induction differentiation scheme has been reported. In order to better verify these differentiation schemes and observe the cell differentiation state in real time, the pluripotent stem cells with dual reporter genes of the present application contain modified endogenous COL2A1 locus and COL10A1 locus in their genome, and the modified endogenous COL2A1 locus and COL10A1 locus contain the first reporter gene and the second reporter gene respectively, which can be used to track the process of cartilage differentiation and hypertrophy degeneration in real time in vitro, providing a powerful tool for the verification of cartilage induction differentiation scheme.

[0061] The construction method of the present application combines and improves the existing cartilage differentiation scheme to construct a cell line of pluripotent stem cells with dual reporter genes, which was successfully cultured in vitro to induce hypertrophic cartilage organoids, proving that the reporter gene can accurately reflect the expression of the corresponding gene, and realizes the real-time dynamic observation of the process of chondrocyte differentiation and hypertrophy in vitro, providing an efficient and reliable tool for the next step of high-throughput drug screening.

[0062] For some sequence information involved in this article, please refer to Table 1, and for other sequence information, please refer to the examples.

[0063] Table 1

[0064]

[0065] Example 1

[0066] 1.1 Materials

[0067] 1.1.1 Main reagents and materials

[0068] The specific information of the cell culture and differentiation induction reagents used in this experiment is shown in Table 2, the specific information of the reagents used for molecular cloning is shown in Table 3, the specific information of the antibodies used in the experiment is shown in Table 4, and the main instruments used in the experiment are shown in Table 5.

[0069] Table 2

[0070]

[0071] Table 3

[0072]

[0073] Table 4

[0074]

[0075] Table 5

[0076]

[0077] 1.1.2 Cell lines

[0078] The cell lines M1 hEPSC and STO feeder cells used were from the laboratory of Professor Gao Xuefei, School of Basic Medical Sciences, Southern Medical University. All experiments were approved by the Biomedical Ethics Committee of Southern Medical University.

[0079] 1.2 Methods

[0080] 1.2.1 Human Expanded Pluripotent Stem Cell Culture

[0081] 1.2.1.1 Preparation of feeder cells

[0082] Feeder layer cells are used for the maintenance and culture of hEPSCs. The preparation method includes the following steps: resuscitate the pre-frozen STOfeeder cell line in a 15 cm culture dish pretreated with 0.1% gelatin, and culture it in M10 culture medium (90% DMEM high glucose medium + 10% FBS + 1% penicillin-streptomycin (P / S)) without changing the medium. When the cell density reaches 95%, expand it to a 15 cm culture dish pretreated with gelatin at a ratio of 1:16. Continue to culture with M10 for 4 to 5 days without changing the medium. When the cell density reaches about 90%, add mitomycin (10 μg / mL) for 2 hours, discard the culture medium, wash with PBS, digest with 0.125% trypsin, add serum to stop digestion when the cells are detached from the bottom of the dish, collect the cell suspension by blowing, centrifuge at 3000rpm for 5 minutes, and discard the supernatant. According to 1×10 6 The cells were cryopreserved (cryopreservation solution: 90% FBS+10% DMSO), and after programmed cooling, they were transferred to a liquid nitrogen tank for storage and were recorded as feeder cells.

[0083] 1.2.1.2 Human expanded pluripotent stem cell culture

[0084] (1) Culture medium configuration

[0085] M1 hEPSC cell line was used and maintained and cultured with EPSCM medium. EPSCM medium is a serum-free medium based on N2B27. Taking the preparation of 500 mL of medium as an example, the components include: 482.5 mL DMEM / F-12 medium, 2.5 mL N2 supplement, 5 mL B27 supplement, 5 mL 1× glutamine penicillin-streptomycin, 5 mL 1× NEAA, 0.1 mM 2-mercaptoethanol 5 mL, 5 μM XAV939, 1 μM CHIR99021, 0.1 μM A-419259, 50 μg / mL vitamin C and 10 ng / mL LIF.

[0086] (2) Cell recovery, culture and cryopreservation

[0087] Take out the feeder cells prepared earlier, quickly put them in a 37℃ water bath to melt, add an equal volume of M10 culture medium and transfer them into a centrifuge tube, centrifuge at 3000rpm for 4min, discard the supernatant, resuspend them with M10, and evenly distribute them into a 12-well culture plate pretreated with gelatin. Wait 2~3 days for the feeder cells to expand and evenly cover the bottom of the dish before use.

[0088] Take out the frozen M1 hEPSCs, revive them in the same way as above, resuspend them in subculture medium (EPSCM medium + 5% fetal bovine serum (FBS) + 10μM Y27632), and inoculate them on feeder well plates with good growth status. The inoculation cell density is 6×10 3 / well. After the cells adhered for 24 hours, the culture medium was replaced with EPSCM medium, and then the culture medium was continuously used, and the medium was changed every day. When the cell clones grew up and the density was about 90%, the cells were subcultured or frozen. When expanding, the culture medium was discarded from the cells, washed with PBS twice, and digested with 0.05% trypsin for about 4 minutes. Observe under a microscope. After the cells in the cloned clumps shrink and become round and gaps appear, the digestion was terminated with M10, the cells were gently blown, centrifuged at 3000rpm for 5 minutes, the supernatant was discarded, and the subculture medium was used to resuspend. Depending on the needs of subsequent experiments, it was usually transferred to the feeder plate with good growth status at a ratio of 1:2~15. After the cells adhered for 24 hours, the EPSCM medium was replaced. When freezing, the cells were digested and centrifuged, resuspended with freezing medium (90% FBS+10%DMSO+10μM Y27632), and transferred to liquid nitrogen for storage after program cooling.

[0089] 1.2.2 Gene Editing of Human Expanded Pluripotent Stem Cells

[0090] 1.2.2.1 Construction of COL2A1-mCherry fluorescent reporter gene vector

[0091] (1) Skeleton and fragment construction

[0092] A gene sequence with homology to the upstream sequence of the start codon and the downstream sequence of the stop codon of the COL2A1 locus was designed, and the sequence information was: 508-bp 5'arm, Chr12: 47973410-47973917; 501-bp 3'arm, Chr12: 47972872-47973372; a donor plasmid with the above sequence characteristics was synthesized (synthesized by Anshengda Life Science Technology Co., Ltd.), and the Mlu I / Bgl II enzyme digestion (plasmid 10μg, enzyme 2μL each, buffer H 5μL, 0.1% BSA 5μL, H2O filled to 50μL) was performed overnight to recover the COL2A1-5' arm 3' arm skeleton.

[0093] Take the PB-TRE-T2A-H2B-mCherry plasmid (see Figure 4 Middle C) The T2A-H2B-mCherry fragment was recovered by digestion with Mlu I / Xho I (10 μg of plasmid, 2 μL of each enzyme, 5 μL of buffer H, and H2O to 50 μL) for more than 4 h.

[0094] Take TOP-puro plasmid (see Figure 4 Middle E) The Loxp-EF1α-PuroR-Loxp fragment was recovered by digestion with Xho I / BamH I (10 μg of plasmid, 2 μL of each enzyme, 5 μL of buffer H, and H2O to 50 μL) for more than 4 h.

[0095] (2) Agarose gel electrophoresis and DNA recovery

[0096] The above enzyme digestion products were first subjected to electrophoresis: 1.5% agarose recovery gel was prepared, 250 bp DNA Marker was added to the first loading well, 1 μL of Loading Buffer was added to the PCR product, mixed and added to the loading well, and exposed with an agarose gel imager after 20 minutes of electrophoresis at 180V. The size of the PCR target fragment was compared according to the distance between the upstream and downstream primers. Under the UV gel excision exposure instrument, the band of the target gene corresponding to the size was cut with a blade and weighed in a clean 1.5mL centrifuge tube. A common agarose gel electrophoresis recovery kit (Tiangen DP209-03) was used for recovery. According to the standard of adding 100 μL of PN for every 0.1g, PN was added according to the actual weight of the target gene band, and placed in a metal bath at 60°C for heating, and the centrifuge tube was constantly turned upside down during the period. After the agarose gel was completely dissolved, it was placed at room temperature to cool. Place the adsorption column in the recovery kit into the collection tube, add 500 μL of balancing solution BL to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube for use. Add the above-mentioned cooled agarose gel solution to the balanced adsorption column, place it at room temperature for 5 minutes, centrifuge it at 12,000 rpm for 1 minute, and discard the waste liquid in the collection tube. Add 600 μL of rinsing solution PW to the adsorption column, centrifuge it at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, put the adsorption column back into the collection tube, add PW again and repeat the above steps. Finally, put the adsorption column back into the collection tube, centrifuge it at 12,000 rpm for 2 minutes, spin off the rinsing solution, open the lid and let it stand at room temperature for 5 minutes to allow the rinsing solution to dry thoroughly. Transfer the adsorption column to a 1.5 mL autoclaved centrifuge tube, add 30 μL of ddH2O preheated at 60°C, let stand at room temperature for 10 minutes to allow the DNA to fully dissolve, centrifuge at 12000 rpm for 2 minutes, collect the recovered product solution into a centrifuge tube, and measure the concentration of the recovered product using an IMPLEN ultra-micro spectrophotometer.

[0097] (3) Skeleton and fragment connection transformation

[0098] The recovered skeleton and each fragment were connected overnight using T4 ligase. 50 μL of competent cells DH5α were melted on ice, and the above-mentioned ligation product was added and gently mixed. It was placed on ice for 30 minutes, and then placed in a 42°C water bath for heat shock for 90 seconds and immediately placed on ice. After ice bathing for 2 minutes, 600 μL of LB medium was added and shaken and cultured in a 37°C shaker for 1 hour. Centrifuge at 4000 rpm and 4°C for 5 minutes, discard the supernatant, and evenly spread the transformation product on the LB solid medium containing ampicillin antibiotics and invert it in a 37°C incubator for overnight culture.

[0099] (4) Plasmid extraction

[0100] Pick a single colony on the solid culture medium into LB medium containing ampicillin antibiotics and shake it in a constant temperature shaker at 37℃ for 13-15 hours. Centrifuge the bacterial solution at 5000rpm for 10 minutes. Use Tiangen Plasmid Extraction Kit (DP103) to extract the plasmid. First, place the adsorption column in the collection tube and add 500μL of balancing solution BL to balance the adsorption column. Centrifuge at 12000rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Discard the supernatant of the centrifuged bacterial solution, add 250μL of solution P1 (add RNase) to resuspend the bacteria, and transfer to a 1.5mL centrifuge tube. Add 250μL of solution P2 to the centrifuge tube, gently turn it upside down several times to fully lyse the bacterial solution. At this time, the liquid inside the centrifuge tube gradually becomes viscous. Carefully open the centrifuge tube, add 350μL solution P3 to the inside, continue to flip 6~8 times, then a large amount of white precipitate will appear inside the centrifuge tube, centrifuge at 12000rpm for 10 minutes. Pour the supernatant after centrifugation into the balanced adsorption column, centrifuge at 12000rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Add 600μL rinse solution PW, centrifuge at 12000rpm for 1 minute, pour out the waste liquid in the collection tube. Repeat this step 2 times. Finally, idle at 12000rpm for 2 minutes. Transfer the adsorption column to a 1.5mL centrifuge tube, let it stand at room temperature for 5 minutes, and dry the rinse solution in the adsorption column. Add 50μL preheated PBS or ddH2O, let it stand at room temperature for 10 minutes, then centrifuge at 12000rpm for 2 minutes, elute the plasmid and collect it in the centrifuge tube, and use IMPLEN ultra-micro spectrophotometer to measure the concentration of the extracted plasmid.

[0101] (5) Plasmid identification

[0102] Select appropriate enzymes for enzyme digestion identification, which takes about 1 to 2 hours; use 1.5% agarose gel electrophoresis and then expose with an agarose gel imager, select plasmids that are cut into the correct size and number of fragments, and send them to the company for sequencing. The plasmid with the correct sequencing is the COL2A1-H2B-mCherry targeting vector, which will be used for subsequent cell electroporation.

[0103] 1.2.2.2 Construction of COL2A1 targeting gRNA

[0104] The gRNA sequence targeting COL2A1 was designed as: 5'-TTGTGTTGTTTCTGGGTTCAGG-3', and two forward and reverse primers were synthesized (the sticky ends of the primers were designed with BBSI restriction sites), and the primers were gradient annealed to form a double-stranded duplex, which was ligated with the pLenti-U6-gRNA-Kan plasmid constructed earlier in our laboratory (see Figure 4 The specific connection, transformation, plasmid extraction and identification methods are the same as those specified in "1.2.2.1".

[0105] 1.2.2.3 Construction of COL10A1-eGFP fluorescent reporter gene vector

[0106] (1) Skeleton and fragment construction

[0107] A gene sequence with homology to the upstream sequence of the start codon and the downstream sequence of the stop codon of the COL10A1 locus was designed, and the sequence information was: 500-bp 5' arm, Chr6: 116119879-116120378; 500-bp 3' arm, Chr6:116119353-116119852. A donor plasmid with the above sequence characteristics was synthesized (synthesized by Qingke Biotechnology Co., Ltd.), and digested with Mlu I / Bgl II (plasmid 10μg, enzyme 2μL each, buffer H 5μL, 0.1% BSA 5μL, H2O to 50μL) overnight, and the COL10A1-5' arm 3' arm skeleton was recovered.

[0108] Take the PB-TRE-IRES-H2B-eGFP plasmid (see Figure 4 Middle D) The IRES-H2B-eGFP fragment was recovered by digestion with Mlu I / Xho I (10 μg of plasmid, 2 μL of each enzyme, 5 μL of buffer H, and H2O to 50 μL) for more than 4 h.

[0109] Take TOP-puro plasmid (see Figure 4 Middle E) The Loxp-EF1α-PuroR-Loxp fragment was recovered by digestion with Xho I / BamH I (10 μg of plasmid, 2 μL of each enzyme, 5 μL of buffer H, and H2O to 50 μL) for more than 4 h.

[0110] (2) Agarose gel electrophoresis and DNA recovery

[0111] Agarose gel electrophoresis and DNA recovery methods are the same as those specified in "1.2.2.1".

[0112] (3) Skeleton and fragment connection transformation

[0113] The method for connecting and transforming the backbone and fragments is the same as specified in "1.2.2.1".

[0114] (4) Plasmid extraction

[0115] Plasmid extraction is the same as specified in “1.2.2.1”.

[0116] (5) Plasmid identification

[0117] Plasmid identification is the same as that specified in "1.2.2.1". The plasmid that is finally sequenced correctly is the COL10A1-eGFP targeting vector, which will be used for subsequent cell electroporation.

[0118] 1.2.2.4 Construction of COL10A1 targeting gRNA

[0119] The gRNA sequence targeting COL10A1 was designed as: 5'-TTTCTAGTTAGCAATCTTAAGG -3'. Two forward and reverse primers were synthesized (the sticky ends of the primers were designed with BBSI restriction sites), and the primers were gradient annealed to form a double-stranded duplex, which was ligated with the pLenti-U6-gRNA-Kan plasmid constructed earlier in our laboratory (see Figure 4 The specific connection, transformation, plasmid extraction and identification methods are the same as those specified in "1.2.2.1".

[0120] 1.2.2.5 Construction of COL2A1 mCherry COL10A1 eGFP hEPSC cell lines

[0121] M1 hEPSC cells were subjected to four rounds of gene editing, including COL2A1-H2B-mCherry knock-in, puro resistance knock-out, COL10A1-H2B-eGFP knock-in, and puro resistance knock-out, to construct COL2A1 mCherry COL10A1 eGFP The method for constructing hEPSC comprises the following steps:

[0122] (1) COL2A1-H2B-mCherry knock-in

[0123] When M1 hEPSCs reached 70% to 80% confluency, the medium was switched to hEPSC medium containing 5% FBS and the cells were cultured overnight. On the day of transfection (day 0), the cells were digested with 0.05% trypsin and separated into single cells by pipetting with M10. After centrifugation, 1 to 1.5 × 10 6 Each cell was resuspended in 100µL Opti-MEM (containing 3µg COL2A1-H2B-mCherry targeting vector, 1.5µg pCAG-Cas9-BSD plasmid and 1.5µg pLenti-U6-gRNA1 COL2A1 targeting gRNA expression vector) and electroporated using an Amaxa electroporator (Lonza) with program A-023. After electroporation, the subculture medium was added to blow and aspirate the cells from the electroporation cup and transfer them to a feeder plate in good condition. After the cells were cultured for 24 hours after attachment, the culture medium was aspirated and EPSCM culture medium containing 1μg / mL Puromycin was added to select target cells. The cell medium was changed every day. After 7-9 days of transfection, when a single clone grew up obviously, the single clones were picked out one by one under a microscope and subcultured and amplified separately.

[0124] (2) Cell genotype identification

[0125] Collect monoclonal cell precipitates and use the Bioteke Cell / Tissue Genomic DNA Rapid Extraction Kit to extract cell DNA. First, wash the cell precipitate with 1 mL of PBS, centrifuge at 12,000 rpm for 4 minutes, and discard the supernatant. Then add 300 μL of protein lysis buffer and boil it in a metal bath at 60°C for 3 to 4 hours. After cooling at room temperature, add 100 μL of protein precipitation buffer to the lysed suspension, mix it with a vortex shaker, quickly put it in an ice bath for 5 minutes, centrifuge it at 12,000 rpm for 5 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube. Add 300 μL of isopropanol and mix it upside down 6 to 9 times. Gradually, silk floccules are observed in the Ep tube. Centrifuge at 12,000 rpm for 1 minute to allow the floccules to sink to the bottom of the tube, and discard the supernatant. Add 500 μL of 70% ethanol solution, gently turn the centrifuge tube upside down, rinse the floccules, then centrifuge at 12,000 rpm for 2 minutes, and discard the rinse solution. Open the lid and air-dry the precipitate for 3-5 minutes, add 40 μL of ddH2O preheated at 60°C, and let it stand at room temperature for 15 minutes until the DNA is completely dissolved.

[0126] Genotype identification primers were designed at one end of the homology arm and H2B, the COL2A1 genotype identification forward primer GTPf1 was as shown in SEQ ID NO: 7, and the H2B genotype identification reverse primer GTPr was as shown in SEQ ID NO: 8. PCR was performed using the knocked-in cell DNA sample, the original cell line DNA sample without knocked-in, and a blank control as templates. After agarose gel electrophoresis and exposure, the cell line with the correct knocked-in gene could amplify the band of the corresponding size, while the wild-type cell line without knocked-in and the blank control could not amplify the band.

[0127] (3) Puromycin resistance in knockout cell lines

[0128] The monoclonal cell lines identified above were expanded. When the density reached 70% to 80% confluence, the culture medium was switched to EPSC medium containing 5% FBS and the cells were cultured overnight. On the day of transfection (day 0), the cells were digested with 0.05% trypsin and separated into single cells by pipetting with M10. After centrifugation, 1 to 1.5 × 10 6 100 µL of Opti-MEM (3 µg of pCAG-Cas9-BSD plasmid (see Figure 4 B) and 1.5µg Cre, 1.5µg BSD transient plasmid) were resuspended and electroporated using an Amaxa electroporator (Lonza) with program A-023. After electroporation, the subculture medium was added to blow and aspirate the cells from the electroporation cup and transfer them to the feeder plate in good condition. After the cells were cultured for 24 hours after attachment, the culture medium was aspirated and the target cells were selected by adding EPSCM culture medium containing 10μg / mL BSD. The cells were changed every day, and after 3 days of continuous addition of drugs, they were replaced with normal EPSCM culture medium and continued to be cultured until single cell clones were picked out and passaged and expanded separately. The single clones were transferred into 48 wells at a ratio of 1:3 and cultured using EPSCM culture medium, EPSCM culture medium + 1μg / mL Puromycin, and EPSCM culture medium + 10μg / mL BSD, respectively. After continuous culture for 3 days, the monoclonal cell lines without Puromycin and BSD resistance were selected for expansion and culture for subsequent experiments.

[0129] (4) COL10A1-H2B-eGFP knock-in

[0130] The above-mentioned monoclonal cell line without Puro and BSD resistance was taken, and COL10A1-H2B-eGFP was knocked in according to the method basically the same as step “(1) COL2A1-H2B-mCherry knock-in”, and the monoclonal strain was selected for subculture and amplification. The difference is that the Opti-MEM used contains 3 µg COL10A1-eGFP targeting vector, 1.5 µg pCAG-Cas9-BSD plasmid and 1.5 µg pLenti-U6-gRNA2 COL10A1 targeting gRNA expression vector.

[0131] (5) Cell genotype identification

[0132] The monoclonal cell precipitate from step (4) was collected and genotyped according to the same method as step “(2) Cell genotype identification”. The forward primer GTPf2 for COL10A1 genotype identification was the sequence shown in SEQ ID NO:9. The correctly identified monoclonal cell line was screened and used for later use.

[0133] (6) Puromycin resistance in knockout cell lines

[0134] The monoclonal cell line correctly identified in step (4) was amplified, and the Puromycin resistance in the cell line was knocked out in the same way as in step "(4) Knockout of Puromycin resistance in the cell line" to obtain a pluripotent stem cell line with dual reporter genes, COL2A1, for use in the experiment. mCherry COL10A1 eGFP hEPSC cell lines were expanded and cryopreserved for subsequent experiments.

[0135] 1.2.3 Differentiation of human expanded pluripotent stem cells into hypertrophic chondrocytes

[0136] 1.2.3.1 Induction of cartilage progenitor cell differentiation

[0137] COL2A1 mCherry COL10A1 eGFP hEPSC cell lines were induced to differentiate into chondrogenic progenitor cells in two dimensions (2D). The corresponding differentiation process and strategy are shown in Figure 1 In E. Briefly, the culture plates were pretreated with 1% matrigel and placed in a 37°C cell culture incubator for more than 1 hour. Stem cells were passaged for 2 generations on the pretreated culture plates to remove the feeder cells. When the cell density reached 90%, COL2A1 without feeder was added. mCherry COL10A1 eGFPhEPSCs were digested into appropriate clumps and transferred to matrigel pre-treated culture plates with stem cell culture medium at a ratio of 1:6. After the cells were cultured overnight, the culture medium was aspirated, gently washed with PBS, and replaced with 2D induction culture medium. Its composition is based on 2D basal culture medium. Taking 40mL 2D basal culture medium as an example, the composition includes: 18.5mL IMDM culture medium, 18.5mL Ham's F12 culture medium, 5mg / mL BSA, 1×CD Lipid 400μL, 1×Glutamine Penicillin-Streptomycin 400μL, 1×ITS premix 400μL, 450nMmonothioglycerol.

[0138] On different days of differentiation induction, different growth factors were added according to the following formula:

[0139] Day 1: 2D basal medium + 20ng / mL bFGF + 10 μM CHIR99021 + 50ng / mL Activin A;

[0140] Day 2: 2D basal medium + 20ng / mL bFGF + 10 μM SB431541 + 3 μM CHIR99021 + 250nM LDN193189;

[0141] Day 3: 2D basal medium + 1μM C59 + 100nM PD173074;

[0142] Day 4-6: 2D basal medium + 100nM SAG + 600nM LDN193189;

[0143] Day 6-12: 2D basal medium + 20ng / mL BMP4.

[0144] The cell medium was changed every day, and the expression of COL2A1-mCherry red fluorescence was observed under a fluorescence microscope. The cells on days 0, 3, 6, 9, and 12 were collected for characterization detection.

[0145] 1.2.3.2 Induction of hypertrophic chondrocyte differentiation

[0146] Obtaining mature chondrocytes in vitro often requires three-dimensional (3D) culture. We combined existing literature and made slight adjustments to make COL2A1 mCherry COL10A1 eGFPhEPSC cells were differentiated into hypertrophic chondrocytes after a 6-day 2D differentiation process and a 42-day 3D differentiation process in vitro. The 2D differentiation process was the same as described in the previous section. On the 6th day of induction, the sclerotome cells were digested and counted, and the 2D culture medium + Y27632 used on days 4 to 6 was used to culture the cells at a rate of 2.5 × 10 5 Each well of the 96-well low-adsorption culture plate was transferred to each cell and centrifuged at 300×g for 5 minutes. After the cells were cultured overnight, they were replaced with 3D induction medium the next day. The composition of the 3D induction medium is based on the 3D basal medium, and different growth factors are added according to the culture time. Taking 50mL of 3D basal medium as an example, the specific components include: 48mL DMEM / F12 medium, 1×ITSpremix 400μL, 0.17mM vitamin C, 0.35mM proline, 0.15% glucose, 1mM Sodium pyruvate, 1×Glutamine Penicillin-Streptomycin 500μL.

[0147] On different days of differentiation induction, different growth factors were added according to the following formula:

[0148] Day 0-6: 3D basal medium + 40ng / mL PDGF-BB + 0.1μM Dexamethasone;

[0149] Day 6-10: 3D basal medium + 40ng / mL PDGF-BB + 0.1 μM Dexamethasone +10ng / mL TGF-β3;

[0150] Day 10-14: 3D basal medium + 0.1 μM Dexamethasone + 10 ng / mL TGF-β3 + 50 ng / mL BMP4;

[0151] Day 14-28: 3D basal medium + 50 ng / mL BMP4 + 10 nM T3;

[0152] Day 28-42 (hypertrophic chondrogenic induction medium): 3D basal medium + 50ng / mL BMP4 + 10nM T3 + 10mM β-glycerophosphate.

[0153] During the cell culture process, the culture medium was replaced every 2-3 days, and the expression of COL2A1-mCherry red fluorescence and COL10A1-eGFP green fluorescence was observed under a fluorescence microscope. The cells were collected on the 14th, 28th, 35th and 42nd days for characterization detection.

[0154] 1.2.3.3 Characterization of differentiated cells

[0155] (1) Flow cytometry (FCM) detection

[0156] The cells in 2D differentiation culture were digested with 0.25% trypsin for two minutes, M10 was added to terminate the digestion and the cells were blown into a single cell suspension. After centrifugation, they were resuspended in PBS, blown, counted, and about 50,000 cells were resuspended in 120 μL of PBS and detected using a multidimensional high-definition flow cytometer (BD LSRFortessa X-20).

[0157] The 3D differentiated cartilage microspheres were collected into 15mL centrifuge tubes. Each group of 10 microspheres was added with 3mL digestion solution (DMEM+10% FBS+1% P / S+2mg / mL collagenase II). The centrifuge tubes were wrapped with sealing film and placed in a 37℃ shaker at 120rpm / min for 2-4h. After the microspheres were completely digested, they were centrifuged, counted, and 50,000 cells were resuspended in 120μL PBS for flow analysis.

[0158] (2) Real-time fluorescence quantitative polymerase chain reaction (qPCR) detection

[0159] Digest the differentiated cells cultured in 2D and the cartilage microspheres cultured in 3D according to the above steps, and collect the cell pellets. Note that to ensure the quality of RNA extraction, the number of cells in each sample should be greater than 300,000. Wash the cell pellet with 1 mL of PBS, centrifuge at 12,000 rpm for 2 minutes, and discard the supernatant. Add 800 μL of Trizol and blow the cells with an enzyme-free pipette tip to completely dissolve them until no clumps of cells can be seen with the naked eye. Let it stand at room temperature for 10 minutes, add 160 μL of chloroform, cover the lid, shake the turbine vigorously for 15 seconds, and let it stand at room temperature for 5 minutes. After standing, use a 4°C low-temperature centrifuge and centrifuge at 12,000 rpm for 15 minutes. Carefully aspirate the upper aqueous phase and transfer it to a new 1.5 mL enzyme-free centrifuge tube. Add an equal volume of -20°C pre-cooled isopropanol and let it stand at room temperature for 20 minutes. After standing, centrifuge at 12,000 rpm at 4°C for 10 minutes. Discard the supernatant, add 1mL -20℃ precooled 75% anhydrous ethanol, centrifuge at 12000rpm, 4℃ for 5 minutes. Discard the supernatant, let it dry at room temperature for 3-5 minutes, add 40μL ddH2O, let it stand on ice for 15 minutes until the RNA is dissolved, and measure the RNA concentration with an IMPLEN ultra-micro spectrophotometer. Use RNase-free EP tubes and pipette tips throughout the process.

[0160] The HiScript Q RT SuperMix for qPCR (Vazyme, R123-01) premix kit was used to perform a two-step reverse transcription reaction to obtain cDNA. Finally, qPCR was performed using cDNA as a template and qPCR Master Mix (Q331-02) premix using QuantStudio 5 (Thermo Fisher). Gene expression was analyzed using the ΔΔCT method.

[0161] (3) Western blot detection

[0162] Collect 2D differentiated cells on day 9, discard the culture supernatant, add 4℃ pre-cooled PBS to wash twice, add protein lysis buffer, and stand on ice for lysis for 15 minutes; scrape the cells from the bottom of the culture plate with a cell scraper, transfer to a centrifuge tube, and use an ultrasonic disruptor to ultrasonically disrupt. Centrifuge at 4℃, 12000rpm for 10 minutes, and collect the supernatant. Measure the volume of the collected protein supernatant with a pipette, add 1× Loading buffer, and heat it in a 100℃ metal bath for 10~15 minutes. Prepare separation gel, add 1mL of anhydrous ethanol to flatten the liquid surface, stand at room temperature for more than 1 hour, and prepare concentrated gel after the separation gel is completely solidified. Clamp the prepared gel into the electrophoresis clamp, add the newly prepared electrophoresis solution into the tank, pull out the comb, and add 10μg of protein according to the standard curve equation. Add protein markers on both sides of the sample. Connect the positive and negative poles of the power supply correctly, run the concentrated gel at 60V constant voltage for 30 minutes, and run the separation gel at 90V constant voltage for 90 minutes. Precool the transfer solution in advance, cut a 0.45μm PVDF membrane of the same size as the separation gel, and transfer the membrane at a constant flow of 250mA for 60 minutes. Block with skim milk and block at room temperature for 1 hour. Wash with PBST 3 times, 10 minutes each time. Dilute the primary antibody according to the antibody instructions and incubate overnight at 4℃. After washing, select the secondary antibody of the corresponding species and incubate at room temperature for 1 hour. Use ECL chemiluminescent solution to develop and expose.

[0163] (4) Immunofluorescence detection

[0164] The 2D differentiation-induced cells were seeded on cell slides and induced to differentiate according to the normal process until day 9. The cell slides were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes before immunofluorescence staining.

[0165] The 3D differentiated cartilage microspheres were fixed with 4% paraformaldehyde overnight, and dehydrated with 30% sucrose solution for 48 hours the next day until the microspheres sank to the bottom of the tube. OTC was used for embedding and frozen sections (8 μm). The sections were subjected to subsequent immunofluorescence staining steps.

[0166] After the cell slides / frozen sections were hydrated with PBS, they were treated with IF Buffer (PBS + 0.5% BSA + 0.1% Triton) at room temperature for 30 minutes, and the primary antibody was diluted 1:200 according to the antibody and diluent, and incubated at 4°C overnight. The next day, the slides were washed three times with IF Buffer, each for 10 minutes. The secondary antibody was diluted 1:500 according to the target protein antibody and primary antibody diluent, incubated at room temperature in the dark for 1 hour, and IF Buffer was added to wash three times, each for 10 minutes, and the slides were sealed with a sealing agent containing DAPI, and imaged using a laser confocal microscope (FV3000, Olympus). Specific antibody information is shown in Table 4.

[0167] (5) Safranin O staining

[0168] After the frozen sections of cartilage microspheres were hydrated with PBS, they were added to 0.5% Safranin O (Saf-O) working solution for staining for 3 minutes, quickly immersed in deionized water for 1 second, and the excess stain was removed. They were quickly differentiated in 3% acetic acid solution for 3-5 times, and the color was observed under the microscope. After drying, the sections were sealed with neutral resin. Images were taken under a bright field microscope (Olympus).

[0169] 1.2.4 Statistical analysis

[0170] qPCR data were expressed as mean and standard deviation. The data between two groups were compared by t-test. One-way analysis of variance was used for comparison among multiple groups. p < 0.05 was considered statistically significant (not statistically significant, ns; p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001). GraphPad Prism 8 software was used for statistical analysis.

[0171] 1.3 Results

[0172] 1.3.1 COL2A1 mCherry COL10A1 eGFP Genotyping of hEPSC lines

[0173] In order to observe the chondrogenesis and hypertrophy process in real time in vitro, we constructed a hEPSC dual fluorescence reporter gene cell line using CRISPR / Cas9 technology to express mCherry and eGFP from the COL2A1 and COL10A1 loci, respectively. Figure 1A and B are schematic diagrams of introducing mCherry and eGFP into the COL2A1 (COL2) and COL10A1 (COL10) gene loci, respectively. GTPf and GTPr are primers designed on the homology arm and H2B, respectively, for identifying wild-type and knock-in cell lines. Specific primer information is shown in Table 1. The mCherry and eGFP elements are located downstream of the stop codons of COL2A1 and COL10A1, respectively, and translation is mediated by T2A and internal ribosome entry site (IRES), respectively. The cells underwent four rounds of gene editing. The first two rounds of editing were to obtain COL2A1 that removed puro resistance. mCherry hEPSC cell line, after a second round of gene knock-in, genotype identification primers were designed on the homology arm side and H2B original, and finally the COL2A1 with correct genotype identification was successfully obtained. mCherry COL10A1 eGFP hEPSC cell lines, identification results as Figure 1 C and D, Figure 1 COL2A1 GTPf1 and GTPr primers were used to identify COL2A1 mCherry hEPSCs were genotyped. DNA samples were derived from wild-type and gene-edited cells, and two correct knock-in clones were identified (red arrows). These two clones were selected for the next step of knockout puromycin resistance and COL10A1-eGFP insertion experiments; Figure 1 COL10A1 GTPf2 and GTPr primers were used to identify COL10A1 mCherry hEPSCs were genotyped and DNA samples were obtained from control cell lines and gene-edited cells.

[0174] 1.3.2 COL2A1 mCherry COL10A1 eGFP Functional validation of hEPSC cell lines

[0175] The dual fluorescent reporter cell lines were used for 12 days of 2D chondrogenic progenitor induction. Figure 1 Figure F is the statistical result of identifying COL2A1-mCherry+ cells using flow cytometry at different differentiation time points. Fluorescence observation and flow cytometry detection found that COL2A1-mCherry fluorescence began to appear on the 6th day of induction and reached the highest level on D9 (9th day). This change trend is consistent with previous reports. Figure 1 Middle G is the statistical analysis of the percentage of COL2-mCherry+ cells at different time points, and the values ​​are shown as mean ± SEM (n = 3 independent experiments), indicating that the mRNA expression level of COL2A1 is also consistent with the change trend of FCM data. Figure 1Middle H is the relative mRNA expression of COL2A1, SOX9, FOXC2 and SOX6 detected by qPCR from differentiation day 0 to day 12. Values ​​are shown as mean ± SEM (n = 3 independent experiments), (blue line: WT hEPSC, red line: COL2A1 mCherry / COL10A1 eGFP hEPSC). During the induction of chondrogenic progenitor cells, there was no significant difference in the mRNA expression levels of COL2A1 and chondrogenic progenitor cell markers such as SOX9, SOX6, and FOXC2 between the dual fluorescent cell line and the WT cell line, indicating that the gene-edited cell line did not affect the normal differentiation of cells. Figure 1 Middle I is a representative immunofluorescence image of cells on day 9 of differentiation, in which COL2A1-H2B-mCherry and SOX9 are co-expressed. The scale bar is 100 μm. The experiment was repeated three times independently with similar results. Immunofluorescence detection demonstrated that SOX9 and mCherry were co-expressed in differentiated cells on day 9. Figure 1 The J in the middle is from COL2A1 mCherry / COL10A1 eGFP Representative immunoblotting results of COL2A1 and mCherry proteins in hEPSC differentiation day 0 and day 9 cells, indicating that at the protein level, the expression of mCherry is consistent with the expression of COL2A1. These results confirm that gene editing cell lines do not affect the normal differentiation of cells and that mCherry fluorescence can correctly reflect the expression of COL2A1. Figure 1 D0, D3, D6, D9, and D12 refer to COL2A1 on day 0, day 3, day 6, day 9, and day 12 of 2D differentiation, respectively. mCherry COL10A1 eGFP hEPSC cells.

[0176] In order to induce chondrocyte hypertrophy in vitro, we combined the previously reported induction protocol, digested the 6-day-cultured chondrocytes, and continued to culture them under 3D conditions for 42 days to allow the chondrocytes to continue to mature and further induce hypertrophy. During the 3D culture process, the cells aggregated into microspheres and gradually increased in size over time. Figure 2 In center A, the entire microsphere showed strong red fluorescence from the 28th to the 42nd day of culture. From the 35th day, GFP fluorescence began to appear. On the 42nd day, the edge of the microsphere emitted obvious green fluorescence. Figure 2The first row in center A is a schematic diagram of the directed differentiation of hEPSCs into hypertrophic cartilage organoids. The schematic diagram shows the addition of different small molecules or growth factors during 2D and 3D differentiation. The middle row is a microscope image of representative time points of differentiation. The bottom row is a representative fluorescence (red and green) image of cartilage microspheres during hypertrophic maturation. The scale bar is 200μm. Flow cytometry further demonstrated this trend, see Figure 2 In B and C, Figure 2 Middle B is a representative picture of flow cytometry results of COL2A1 and COL10A1 positive cells in cells at different stages of differentiation, including osteogenic nodes, day 28, day 35, and day 42. After 28 days of 3D culture, the FCM image showed two separated subpopulations (COL2+COL10-, COL2+COL10+); Figure 2 Middle C shows the percentage of COL2+COL10- and COL2+COL10+ cells in cells at different stages of differentiation, including osteoblasts, day 28, day 35, and day 42. Values ​​are shown as mean ± SEM (n = 3 independent experiments). No COL10A1+ cells were detected on day 28, while COL10A1+ cells on days 35 and 42 were approximately 6% and 21%, respectively. This suggests that the stage from day 28 to day 42 is the hypertrophic maturation stage.

[0177] Subsequently, we further analyzed the day 42 microspheres using immunofluorescence. Figure 2 Middle D is a representative immunofluorescence image of frozen sections of hypertrophic cartilage organoids on day 42. The left image shows that COL2A1-H2B-mCherry (red) is expressed throughout the organoid, COL10A1-H2B-eGFP (green) is mainly expressed at the edge of the organoid, the middle image shows that SOX9 (red) is mainly expressed inside the organoid, and the right image shows that ACAN (purple) is highly expressed in the center of the organoid. The scale bar is 200μm. Figure 2 E is a schematic diagram of the distribution of cell types in organoids. Figure 2 In center D, we found that the microspheres had a distinct hierarchical regional structure, with COL10A1+ cells distributed in the outermost layer of the microspheres, defined as the hypertrophic layer. COL2A1 expression was highest in the middle layer, while aggrecan (ACAN) and SOX9 were highly expressed in the deep layer. This hierarchical regional structure resembles the unique and highly ordered structure with characteristic zoning (superficial, middle, and deep layers) observed in normal articular cartilage. COL10A1-eGFP expression was highly concentrated in the outermost layer of the microspheres, suggesting that direct contact with triiodothyronine (T3) and sodium β-glycerophosphate in the hypertrophy-inducing medium may accelerate hypertrophy. Figure 2D0, D7, D14, D21, D28, D35 and D42 refer to COL2A1 on differentiation day 0, 3D differentiation day 7, 14, 21, 28, 35 and 42, respectively. mCherry COL10A1 eGFP hEPSC cells.

[0178] The mRNA levels of chondrocyte markers were detected from day 0 to day 42 of microsphere culture. Figure 3 Middle A is the relative expression of mRNA of chondrocyte (upper row) and mast cell (lower row) markers in cells at different stages of differentiation, including osteoblast, day 28, day 35, and day 42, by qPCR. Data are expressed as mean ± SD (n = 3 independent experiments), *p < 0.05, **p < 0.01, ***p < 0.001, and it was found that COL2A1 and SOX6 peaked on day 28 and then declined, ACAN accumulated over time, and hypertrophy-related markers COL10A1, RUNX2, IHH, COL1A1, and MMP13 mainly appeared on day 28 and increased significantly on day 42. Figure 3 Middle B shows cartilage microspheres at day 28 and day 42 stained with safranin O. The scale bar is 200 μm. The figure shows representative images from six independent repeated experiments. Both the microspheres at day 28 and day 42 showed abundant proteoglycan staining, but the periphery of the microspheres at day 42 showed mast cell morphology. Figure 3 Middle C is a representative immunofluorescence image of frozen sections of cartilage microspheres on day 28 and day 42, with a scale bar of 200 μm. COL2A1-H2B-mCherry and COL10A1-H2B-eGFP were co-expressed with SOX9, ACAN, IHH, RUNX2, and MMP13, and the expressions of IHH, RUNX2, and MMP13 were all higher than those of microspheres on day 28. Figure 3 hEPSC represents gene-edited but undifferentiated cells, Sclerotome represents osteogenic nodule, D14, D21, and D28 refer to the 14th, 28th, and 42nd days of 3D differentiation, respectively. mCherry COL10A1 eGFP hEPSC cells.

[0179] The above experiment used the CRISPR-Cas9 system to construct COL2A1 mCherry COL10A1 eGFPhEPSC cell lines, combined with existing chondrocyte differentiation protocols, successfully induced hypertrophic cartilage organoids with sustained expression of COL2A1 and expression of COL10A1 during the hypertrophy induction stage. In addition to stable and reproducible reporter gene expression, the organoids also showed structural hierarchies and cell characteristics similar to those in human articular cartilage. Mast cells mainly appeared on the outermost side of the microspheres, suggesting that direct contact with the induction fluid may promote the occurrence of cell hypertrophy to a certain extent. This phenomenon is similar to the structure of articular cartilage, where hypertrophic and calcified chondrocytes are close to the subchondral bone (rich in blood and nutrient supply), while hyaline chondrocytes are close to the joint cavity (no blood supply and insufficient nutrient supply). These results show that the hypertrophic cartilage organoids we constructed in vitro reproduce the characteristics of human cells to a certain extent, and the expression of two reporter genes can accurately study the process of cartilage formation and hypertrophy.

[0180] The hypertrophic cartilage organoids constructed in the above experiment have three-dimensional cellular structural characteristics and gene expression profiles similar to human cartilage, which can well reflect the structural hierarchy and physiological characteristics of human cartilage, and can stably expand in vitro and maintain genetic stability. It provides an excellent personalized model for exploring the development and disease mechanisms of cartilage, and also has great application prospects in drug screening, organ regeneration and other fields.

[0181] In summary, the cell line with dual reporter genes in the present application can be used to construct human hypertrophic cartilage organoids in vitro, has stable and reproducible dual fluorescent reporter gene expression, can track cartilage differentiation and hypertrophy in real time in vitro, and is similar to the composition and hierarchy of natural chondrocytes, and is expected to be applied to disease modeling, drug screening, tissue transplantation and other fields.

[0182] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0183] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. A method for constructing a cell line of induced pluripotent stem cells with dual reporter genes, characterized in that: The induced pluripotent stem cell with dual reporter genes comprises a first reporter gene and an endogenous COL2A1 gene in the COL2A1 locus of its genome, and comprises a second reporter gene and an endogenous COL10A1 gene in the COL10A1 locus, wherein the first reporter gene and the second reporter gene respectively encode proteins capable of generating different reporter signals, and the induced pluripotent stem cell with dual reporter genes has the ability to differentiate into chondrocytes and further induce the generation of hypertrophic cartilage organoids; The method for constructing a cell line of induced pluripotent stem cells having dual reporter genes comprises integrating the first reporter gene and the second reporter gene into the COL2A1 locus and the COL10A1 locus of the induced pluripotent stem cells to be genetically modified, respectively, comprising: (1) introducing a first donor DNA molecule into the induced pluripotent stem cell to be genetically modified, wherein the first donor DNA molecule comprises a first exogenous sequence flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm provide homologous recombination between the first donor DNA molecule and the COL2A1 locus of the induced pluripotent stem cell to be genetically modified, and the first exogenous sequence comprises a first reporter gene; providing a Cas9 protein and a first gRNA targeting the COL2A1 gene in the induced pluripotent stem cell to be genetically modified; (2) introducing a second donor DNA molecule into the induced pluripotent stem cell to be genetically modified, wherein the second donor DNA molecule comprises a second exogenous sequence flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm provide homologous recombination between the second donor DNA molecule and the COL10A1 locus of the induced pluripotent stem cell to be genetically modified, and the second exogenous sequence comprises a second reporter gene; providing a Cas9 protein and a second gRNA targeting the COL10A1 gene in the induced pluripotent stem cell to be genetically modified; wherein steps (1) to (2) are performed in any order; The sequence of the first gRNA is 5'-TTGTGTTGTTTCTGGGTTCAGG-3'; The sequence of the second gRNA is 5'-TTTCTAGTTAGCAATCTTAAGG-3'; The first reporter gene can be co-expressed with the endogenous COL2A1 gene, and the second reporter gene can be co-expressed with the endogenous COL10A1 gene; The 5' homology arm and the 3' homology arm described in step (1) have homology with the upstream sequence of the stop codon and the downstream sequence of the stop codon of the COL2A1 locus, respectively; The 5' homology arm and the 3' homology arm described in step (2) have homology to the upstream sequence of the stop codon and the downstream sequence of the stop codon of the COL10A1 locus, respectively.

2. The method for constructing a cell line of induced pluripotent stem cells having dual reporter genes according to claim 1, characterized in that: The first reporter gene is located downstream of the stop codon of the COL2A1 gene; The second reporter gene is located downstream of the stop codon of the COL10A1 gene.

3. The method for constructing a cell line of induced pluripotent stem cells having dual reporter genes according to claim 1, characterized in that: The first reporter gene encodes a first fluorescent protein, and the second reporter gene encodes a second fluorescent protein; The colors of the fluorescence emission lights of the first fluorescent protein and the second fluorescent protein are different.

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