Method for constructing iPSC-derived IL-10 protein overexpressing MSC cell line

Abstract

The present invention belongs to the field of molecular biology and cell technology, and provides a method for constructing an iPSC-derived IL-10 protein overexpressing MSC cell line. The construction method comprises constructing an exogenous IL-10 protein expression cassette for initiating transcriptional activity by an MSC-specific promoter, and then stably inserting the protein expression cassette into human iPSC cells by CRISPR / Cas 9 technology, and then inducing differentiation of iPSC cells to obtain an MSC cell line overexpressing IL-10 protein. The MSC overexpressing IL-10 protein of the present invention can efficiently regulate the immune environment in the joint, and has a better therapeutic effect than MSCs of natural origin.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and cell technology, and particularly relates to a method for constructing an iPSC-derived IL-10 protein-overexpressing MSC cell line. Background Art

[0002] MSC is an adult stem cell with biological characteristics such as multidirectional differentiation, immunoregulation, anti-fibrosis, anti-inflammation, anti-apoptosis and angiogenesis. It plays an important role in tissue repair, cancer treatment and immune regulation, and is widely used in disease research and regenerative medicine.

[0003] However, the use of MSCs for treatment also has the disadvantages of difficult preparation and unstable effects. For example, the sources of MSCs from umbilical cord tissue and placenta are limited, the storage costs are high, and there are logistical challenges. MSCs derived from bone marrow, fat, and peripheral blood require damaging operations on volunteers, and MSCs derived from natural tissues are limited by their own limited expansion capacity, so they may need to be recruited repeatedly. In this process, different donors, different tissue sources, and differences in cell passaging operations may lead to changes in cell morphology, DNA abnormalities, cell aging, decreased proliferation and differentiation ability, and changes in cell plasticity, thereby affecting the quality and lifespan of MSCs, and thus affecting the repeatability of the experiment, resulting in inconsistent clinical research results.

[0004] To overcome these obstacles, researchers have designed culture systems to produce high-quality MSCs, thereby achieving reproducible results in regenerative medicine. Among these methods, deriving MSCs from iPSCs is a key alternative. iPSC-induced differentiated MSCs have the same in vitro and in vivo multi-lineage differentiation potential and secretory factor function as natural MSCs, and can be prepared on a large scale with more stable and uniform characteristics. In addition, a large number of studies have also confirmed that natural MSCs have some adverse effects during treatment. For example, interleukin-10 (IL-10), one of the most effective anti-inflammatory cytokines in inflammatory responses, is expressed at low levels in natural MSCs, and the therapeutic effect is limited. Therefore, how to improve the therapeutic ability of MSCs is a technical problem that continues to be broken through. Summary of the invention

[0005] In view of the defects of difficult preparation and unstable effect of natural MSCs derived from tissues in treatment, the present invention provides a method for constructing an iPSC-derived IL-10 protein overexpressing MSC cell line.

[0006] In order to achieve the above-mentioned object, one embodiment of the present invention provides a method for constructing an iPSC-derived IL-10 protein-overexpressing MSC cell line, characterized in that it comprises: constructing an exogenous IL-10 protein expression cassette for initiating transcriptional activity by an MSC-specific promoter, stably inserting the protein expression cassette into human iPSC cells by CRISPR / Cas 9 technology, inducing differentiation of iPSC cells, and obtaining an MSC cell line overexpressing IL-10 protein.

[0007] In the method for constructing the iPSC-derived IL-10 protein-overexpressing MSC cell line as described above, the MSC-specific promoter is LY6E, and its sequence is shown in SEQ ID NO:8.

[0008] The method for constructing an iPSC-derived IL-10 protein-overexpressing MSC cell line as described above, wherein the exogenous IL-10 protein expression cassette comprises: a promoter, an IL-10 protein CDS region sequence, and a terminator, the promoter is the MSC-specific promoter LY6E, the terminator is a bGH Poly (A) signal sequence, and the expression cassette sequence is shown in SEQ ID NO: 9.

[0009] The method for constructing the iPSC-derived IL-10 protein-overexpressing MSC cell line as described above comprises the following steps:

[0010] Step 1: Construct a sgRNA vector targeting intron 1 of the AAVS1 gene, a safe site in the human genome;

[0011] Step 2: Transfect the sgRNA vector into HEK293T cells, and screen the sgRNA vector with the highest cutting efficiency by T7E1 enzyme cutting detection;

[0012] Step 3, constructing a homologous repair donor template plasmid containing an exogenous IL-10 protein expression cassette;

[0013] Step 4: The sgRNA vector with the highest cutting efficiency and the homologous repair donor template plasmid constructed in step 3 are transfected into human iPSC cells by electrofection, and iPSC cell clones with homozygous knock-in of the exogenous IL-10 protein expression cassette are selected by single clone selection and PCR identification;

[0014] Step 5: Induce the differentiation of the iPSC cell clones homozygous for the exogenous IL-10 protein expression cassette obtained in step 4 to obtain MSCs overexpressing IL-10 protein.

[0015] The present invention has the following advantages:

[0016] 1. The present invention derives MSC cells from iPSCs. The MSCs induced by iPSCs have the same in vitro and in vivo multi-lineage differentiation potential and secretory factor function as natural MSCs, and can be prepared on a large scale with more stable and uniform characteristics.

[0017] 2. The present invention inserts an exogenous IL-10 protein expression cassette into cells, and the obtained IL-10 protein overexpressing MSC cell line can highly express IL-10 and has better immunoregulatory ability.

[0018] 3. The present invention uses the MSC-specific promoter LY6E to initiate the transcription activity of the exogenous gene IL-10. This promoter initiates the transcription activity only after iPSCs differentiate into MSCs. Using this specific promoter, the IL-10 protein can be targeted and the gene can be fixed in the MSC cell stage for expression, thereby avoiding affecting the proliferation and differentiation ability of iPSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the plasmid map of sgRNA No. 1 targeting the AAVS1 site.

[0020] Figure 2 Schematic diagram of the homologous repair donor template plasmid map targeting the AAVS1 site.

[0021] Figure 3 Schematic diagram of PCR sequencing for inserting exogenous human IL-10 protein expression cassette into the AAVS1 site.

[0022] Figure 4 This is a morphological diagram of the positive clone iPSC cell line targeting the AAVS1 site differentiating into MSC.

[0023] Figure 5 This is a diagram of the three-lineage differentiation capabilities of MSC cells.

[0024] Figure 6 This is the relative expression level of IL-10 gene in MSCs.

[0025] Figure 7 This is the WB image of IL-10 protein in MSC cell line.

[0026] Figure 8 The graph shows the proportion of CD73, CD90 and CD105 positive cells in MSC cells carrying different promoter expression cassettes.

[0027] Fig. 9 This is a diagram for evaluating histological indicators of the mouse osteoarthritis model after injection of MSCs overexpressing IL-10. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0029] Example 1: Construction of a sgRNA vector targeting intron 1 of the AAVS1 gene, a safe site in the human genome.

[0030] Using the human genome AAVS1 gene from the NCBI website as a template, the intron between exon 1 and exon 2 was selected, and the DNA sequence located in the middle of the intron was used as the target. http: / / crispor.tefor.net / sgRNA was designed.

[0031] The CACCG sequence was added to the front end of the selected sgRNA sequence as the F primer, and the AAAC sequence was added to the front end of the reverse complementary sequence of the sgRNA as the R primer. A total of three groups of sgRNA were designed, and the relevant primer sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 6. The plasmid for simultaneously expressing Cas 9 protein and sgRNA was obtained from Addgene pX330. The two plasmids of pX330 were cloned by conventional molecular cloning methods. Bbs The sequence between site Ⅰ (site 245 and site 267) was replaced with the corresponding sgRNA sequence, and the sgRNA vector was obtained.

[0032] Example 2: Screening out the sgRNA with the highest cutting efficiency.

[0033] The sgRNA vector successfully constructed in Example 1 was transfected into the same amount of HEK293T cells using the Lip 3000 liposome transfection method. The cells were collected 48 hours after transfection and genomic DNA was extracted.

[0034] The target fragment was amplified by PCR, recovered and purified, and the efficiency of NHEJ after target site cleavage was detected by T7E1 enzyme digestion detection. According to the grayscale analysis of the bands after agarose electrophoresis, sgRNA No. 1 with higher cleavage efficiency was selected for subsequent experiments. The corresponding vector sequence is shown in SEQ ID NO: 7, and the plasmid map is shown in Figure 1 shown.

[0035] Example 3: Construction of a homologous repair donor template plasmid containing an exogenous IL-10 protein expression cassette.

[0036] The exogenous IL-10 protein expression cassette mainly includes a promoter, an IL-10 protein CDS region sequence, and a terminator. The promoter is the MSC-specific promoter LY6E, and its sequence is shown in SEQ ID NO: 8. The terminator is the bGH Poly (A) signal sequence, and the expression cassette sequence is shown in SEQ ID NO: 9.

[0037] The IL-10 gene homologous repair template vector selected the pUC57 vector obtained from Addgene as the backbone.

[0038] The left homology arm of AAVS1 was synthesized, 1000 bp; the exogenous IL-10 protein expression cassette was synthesized according to SEQ ID NO: 9; the right homology arm of AAVS1 was synthesized, 500 bp, and the above three sequences were sequentially inserted into the pUC57 vector by conventional molecular cloning methods. EcoR I site to Hind III sites, thus obtaining a donor plasmid carrying left and right homologous arms and a homologous recombination template. The corresponding plasmid sequence is shown in SEQ ID NO: 10, and the plasmid map is shown in Figure 2 shown.

[0039] Example 4: Co-transfection of sgRNA plasmid and donor template plasmid, clone isolation and identification.

[0040] iPSCs were cultured to 90% confluence using standard methods, and the cells were digested and counted. 1 × 10 6 - 1 × 10 7 Centrifuge the cells at low speed, take out the centrifuge tube into the biosafety cabinet, open the tube cover, and discard the supernatant.

[0041] Pipette 0.5 ug - 5 ug of the above-mentioned sgRNA plasmid and homologous donor template plasmid in different proportions, specifically 0.5 ug, 0.6 ug, 0.7 ug, 0.8 ug, 0.9 ug, 1 ug, 1.1 ug, 1.2 ug, 1.3 ug, etc., add to the electrotransfer buffer, mix by pipetting several times, aspirate all the liquid and add to the cell pellet obtained above, resuspend the cells, transfer them to the electrotransfer cup, and then put it into the transfection chamber of the electrotransfer instrument for electrotransfer operation. After the electrotransfer operation is completed, take out the electrotransfer cup and put it into the biosafety cabinet to transfer the cells to the culture dish.

[0042] The culture medium of the cells after electroporation was replaced every day. After two days of culture, the cells were digested, counted, and 5,000 cells were inoculated in a 10-cm culture dish. The culture was continued for 7 days. Single clones were picked in a laminar flow bench and inoculated in 24-well plates. The culture was continued for 7 days to verify the positive clones.

[0043] The obtained iPSC cells were subjected to PCR detection. The genomic DNA of the cells in each well was extracted using a DNA extraction kit, and PCR amplification and sequencing were performed to select cell clones with homozygous insertion sequences. The corresponding primer sequences across homology arms are shown in SEQ ID NO: 11 to SEQ ID NO: 16. The sequencing results of positive clones are shown in Figure 3 As shown, it was shown that the expression cassette sequence of IL-10 was correctly inserted into the target site.

[0044] Example 5: Differentiation of iPSCs into MSCs.

[0045] When the iPSC clones that have positive gene insertion and passed the test are cultured to a confluence of 70% - 80%, the differentiation induction experiment is performed. The maintenance medium is aspirated, and the residual medium is washed away with PBS solution. Accutase or TrypLE digestion solution is added, and the cells are placed in an incubator at 37°C, 5% CO2 concentration, and saturated humidity for 3 - 8 minutes. Gently shake to detach the cells from the bottom of the culture plate.

[0046] Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Then remove the supernatant and add 1 mL of fresh neural crest cell induction medium to resuspend the cells. Gently pipette 1-2 times. Count the cells and inoculate them in a Fibronectin / iMatrix-coated plate at a density of 0.1-1. 10 4 cells / cm 2 Then continue to culture for 10 days, replace fresh medium every day, and use 0.2 - 0.4 mL / cm 2 .

[0047] Replace the cells with fresh MSC differentiation medium and continue to culture them in an incubator at 37°C, 5% CO2 concentration, and saturated humidity. Replace the medium with fresh medium every day. The amount of medium used is 0.2 - 0.4 mL / cm 2 After 6 days of continuous culture, the cells were observed and photographed. Figure 4 As shown, it has the morphology of MSC cells.

[0048] The MSCs were induced to differentiate into osteoblasts, chondrocytes and adipocytes using bone, cartilage and adipocyte differentiation medium. Figure 5 As shown, the positive clones were capable of tri-lineage differentiation.

[0049] qPCR assay was performed on cells with MSC cell morphology and tri-lineage differentiation ability. Total RNA was extracted from some cells. After reverse transcription to obtain cDNA, qPCR assay was performed on the target gene expression. The quantitative primers were shown in SEQ ID NO: 17 to SEQ ID NO: 20. The quantitative results were shown in Figure 6 As shown, it shows that the target RNA transcription level is high.

[0050] The cells with higher qPCR results were further verified by WB method to detect whether the protein expression of the target gene IL-10 was increased. The specific results are as follows Figure 7 As shown, the expression of IL-10 protein was significantly increased compared with that of wild-type MSCs.

[0051] Example 6: Effects of exogenous expression of specific promoters and constitutive promoters on the differentiation ability of iPSCs into MSCs.

[0052] A similar method as in Example 3 was used to construct a homologous repair donor template plasmid containing an exogenous IL-10 protein expression cassette, with the only difference being that the constitutive expression promoter CAG was used to replace the MSC-specific promoter LY6E, thereby obtaining an exogenous expression cassette driven by a constitutive promoter.

[0053] Then, the iPSC cells were treated using the same plasmid transfection, clone picking, positive cell identification, MSC induction differentiation method as in Examples 4 and 5 to obtain exogenous expression MSC cells carrying a constitutive promoter.

[0054] After the induction differentiation experiment, MSC cells carrying the LY6E-IL-10 expression cassette and MSC cells carrying the CAG-IL-10 expression cassette were collected, and the two groups of MSC cells were treated with MSC surface markers CD73, CD90 and CD105 flow cytometry antibodies. Then, the proportion of CD73, CD90 and CD105 fluorescence positive cells in the two groups of cells was analyzed by flow cytometry. The results are shown in Figure 8 As shown, the proportion of cells with positive fluorescence of CD73, CD90 and CD105 in MSC cells carrying CAG-IL-10 expression cassette was lower than that in MSC cells carrying LY6E-IL-10 expression cassette, indicating that among the cells expressing exogenous genes using constitutive expression promoter, there are still some cells that fail to successfully differentiate into MSCs, further confirming that the MSC-specific promoter used in the present invention does not interfere with the differentiation ability of iPSCs.

[0055] Example 7: The therapeutic effect of IL-10 overexpressing MSC cell line on osteoarthritis was verified by model mice.

[0056] According to the methods of Examples 1 to 3 above, sgRNA targeting the mouse AAVS1 site and the corresponding homologous repair template plasmid for inserting the exogenous mouse IL-10 gene were designed respectively.

[0057] Mouse MSC cells were cultured and then transfected with plasmids. The overall mRNA expression level was detected to confirm that the exogenous IL-10 gene was successfully transferred into mouse MSCs.

[0058] The mouse model of destabilized medial meniscus (DMM) was established by surgical operation. This is a mouse model commonly used in osteoarthritis research. Phosphate buffered saline (PBS), wild-type MSCs, and MSCs overexpressing IL-10 were injected into the joint cavity twice a week for 8 consecutive weeks. Fig. 9 As shown in the results, through histological analysis of the OARSI score, chondrocyte number, proteoglycan loss, and cartilage thickness of osteoarthritis, we confirmed that the group injected with MSCs overexpressing IL-10 protein could significantly protect the structure of articular cartilage and more effectively inhibit the development of osteoarthritis.

[0059] As can be seen above, the present invention is based on engineering means such as iPSC technology, CRISPR / Cas 9 gene editing technology, and iPSC induced differentiation technology to develop suitable engineered MSCs, and provide suitable cells for human disease research and regenerative medicine. First, the immune regulation function of MSCs is transformed. Considering that iPSCs have better amplification ability than MSCs, and in order to ensure the stability between cell batches, it is a better choice to put the gene modification stage in the iPSC cell period. However, most of the existing mammalian promoters are constitutive expression promoters. After being transferred into cells, they will maintain a certain degree of expression and will not be affected by the cell type. Premature gene expression may affect the proliferation and differentiation ability of iPSCs. Therefore, the present invention uses the MSC-specific promoter LY6E to start the transcription activity of exogenous genes. The promoter only starts transcription activity after iPSCs differentiate into MSCs. Using this specific promoter, exogenous genes can be targeted and located, and genes can be fixed in a certain tissue or cell for expression, which reduces the loss of energy and material in the body and the impact on the body's metabolic activities, while also being able to achieve the effect of directional improvement of a certain cell characteristic.

[0060] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for constructing an iPSC-derived IL-10 protein-overexpressing MSC cell line, characterized in that: The method comprises: constructing an exogenous IL-10 protein expression cassette that uses an MSC-specific promoter to initiate transcriptional activity; stably inserting the protein expression cassette into human iPSC cells through CRISPR / Cas 9 technology; inducing differentiation of iPSC cells to obtain an MSC cell line that overexpresses IL-10 protein; the MSC-specific promoter is LY6E, and its sequence is shown in SEQ ID NO:

8.

2. The method for constructing an iPSC-derived IL-10 protein-overexpressing MSC cell line according to claim 1, characterized in that: The exogenous IL-10 protein expression cassette includes: a promoter, an IL-10 protein CDS region sequence, and a terminator. The promoter is the MSC-specific promoter LY6E, and the terminator is the bGH Poly (A) signal sequence. The expression cassette sequence is shown in SEQ ID NO:

9.

3. The method for constructing an iPSC-derived IL-10 protein-overexpressing MSC cell line according to claim 1, characterized in that: The following steps are involved: Step 1: Construct a sgRNA vector targeting intron 1 of the AAVS1 gene, a safe site in the human genome; Step 2: Transfect the sgRNA vector into HEK293T cells, and screen the sgRNA vector with the highest cutting efficiency by T7E1 enzyme cutting detection; Step 3, constructing a homologous repair donor template plasmid containing an exogenous IL-10 protein expression cassette; Step 4: The sgRNA vector with the highest cutting efficiency and the homologous repair donor template plasmid constructed in step 3 are transfected into human iPSC cells by electrofection, and iPSC cell clones with homozygous knock-in of the exogenous IL-10 protein expression cassette are selected by single clone selection and PCR identification; Step 5: Induce the differentiation of the iPSC cell clones homozygous for the exogenous IL-10 protein expression cassette obtained in step 4 to obtain MSCs overexpressing IL-10 protein.

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