Method for establishing induced pluripotent stem cell line by using clinical waste nasal polyp tissue and application of induced pluripotent stem cell line

Abstract

The invention belongs to the technical field of biological stem cells, and particularly relates to a method for establishing an induced pluripotent stem cell line by using a clinical waste nasal polyp tissue and application of the induced pluripotent stem cell line. According to the invention, the nasal polyp tissue is cultured into a cell line capable of passage by using a tissue adherence method, and the cell line is identified to have the characteristics of fibroblasts; according to the invention, a nasal polyp-derived cell line is successfully established, and compared with a donor nasal polyp cell line, the invention does not observe that the nasal polyp-derived iPSC has any new mutation in P3, including cancer-related mutation and various mutation types. The P8 mutation number of the iPSC is slightly increased, the mutation numbers of blank mutants and chromosomes 1, 11, 17 and 20 are slightly increased, and the genome stability and safety of further application are not affected. The results show that the stable iPSC system can be constructed by the method, the gene mutation is less, and the early passage of the nasal polyp-derived iPSC can be used for stem cell treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological stem cells, and in particular relates to a method and application of establishing an induced pluripotent stem cell line by using clinically discarded nasal polyp tissue. Background Art

[0002] Nasal polyps are a common noncancerous growth of the nasal mucosa, often associated with chronic sinusitis. Chronic rhinosinusitis with nasal polyps (CRSwNP) usually has symptoms including nasal congestion, nasal discharge, facial discomfort, decreased sense of smell, etc. that last for more than 12 weeks. The incidence rate in adults is about 2.7%, with a higher incidence rate in men, but women diagnosed with CRSwNP experience more severe disease. Compared with CRS, histological analysis of CRSwNP shows matrix swelling and fibrosis, basement membrane thickening, epithelial damage, and reduced blood vessels and glands. The mucosa of the nasal polyp part shows symptoms such as increased goblet cells and basal cell hyperplasia.

[0003] Hematopoietic stem cell transplantation (HSCT) is currently a common cellular immunotherapy that replaces the patient's hematopoietic system by infusing hematopoietic stem cells. This method is often used to treat blood tumors, immune and genetic diseases. There are two main ways of hematopoietic stem cell transplantation, including autologous transplantation and allogeneic transplantation. Among them, allogeneic transplantation is more likely to have complications. Graft-versus-host disease (GVHD) is a complication after allogeneic hematopoietic stem cell transplantation, which significantly increases the morbidity and mortality of patients. About 30-40% of patients will develop this complication after receiving a transplant, which can manifest clinically as acute or chronic.

[0004] Induced pluripotent stem cells (iPSCs) were first reported in 2006. Yamanaka's team screened 24 transcription factors related to stem cells or pluripotency, and successfully reprogrammed mouse embryonic fibroblasts or adult mouse fibroblasts into induced pluripotent stem cells using a group of transcription factors named Yamanaka (OCT4, SOX2, KLF4, C-MYC). In 2007, Thomoson's research group and Yamanaka's research group almost simultaneously reported iPSCs reprogrammed from human somatic cells. Subsequently, Zhao et al. obtained fully developed mice of iPSCs through tetraploid blastocyst complementation experiments, confirming the pluripotency of iPSC development. iPSCs are pluripotent stem cells artificially obtained from non-pluripotent cells by forced induction to express specific genes of pluripotent stem cells. They are similar to pluripotent stem cells in terms of the expression of pluripotency-related genes, DNA methylation patterns, self-renewal ability, embryoid body formation, teratoma formation, and chimera formation. The success of iPSCs is measured by morphological criteria, specific gene expression, and in vitro differentiation capacity. The potential of iPSC technology is not limited to generating cells that can differentiate into any cell type in the body. It includes the ability to model complex human diseases in vitro, screen drugs with greater accuracy, and develop cell therapies that may cure previously untreatable diseases. However, despite its considerable promise, iPSC technology faces challenges that need to be addressed to fully realize its therapeutic potential. These challenges include improving the efficiency and safety of cell reprogramming methods, understanding the mechanisms of pluripotency induction, and developing reliable protocols to differentiate iPSCs into clinically relevant cell types.

[0005] Fibroblasts are a donor cell for generating iPSCs and an important tool for optimizing reprogramming protocols. After fibroblasts, more and more somatic cells have been successfully reprogrammed. For example, Hanna et al. converted non-terminally differentiated mouse B cells into iPSCs by inducing the expression of four transcription factors (OCT4, SOX2, KLF4, and C-MYC). Loh et al. obtained induced pluripotent stem cells from CD34+ cells in human peripheral blood cells by retroviral transduction of OCT4, SOX2, KLF4, and MYC. Seki et al. generated iPSCs from human terminally differentiated circulating T cells (hTDCTCs) using SeV vectors encoding human OCT3 / 4, SOX2, KLF4, and C-MYC.

[0006] Prior art CN118910194A discloses a collagen biosynthesis method based on induced pluripotent stem cell technology, which specifically includes the following steps: Step 1. Cell reprogramming: extract somatic cells, including fibroblasts and blood cells, from the skin or blood of a donor, and introduce reprogramming factors into somatic cells using a viral vector or a non-viral method, wherein the viral vector is a retrovirus or an adeno-associated virus.

[0007] Prior art CN118995631A discloses a method for constructing a neurodegenerative disease model based on iPSCs. By obtaining skin fibroblasts or blood cells from patients and converting them into iPSCs using reprogramming technology, and then differentiating them into neural progenitor cells and specific types of neural cells, it can accurately simulate the pathological characteristics of neurodegenerative diseases in vitro. At the same time, combined with high-throughput screening technology and CRISPR / Cas9 gene editing technology, it can quickly evaluate the efficacy of candidate drugs and study gene functions, reveal the molecular mechanism of the disease, accelerate the development of new drugs, and provide important tools for the development of personalized treatment strategies.

[0008] Prior art CN113846123A discloses a method for promoting somatic cell reprogramming into induced pluripotent stem cells by low-intensity ultrasound, comprising the following steps: transfecting somatic cells with Oct4, Sox2, Klf4, and c-Myc; 48 hours after transfection, subjecting the cells to ultrasonic irradiation treatment, during which the iPSCs culture medium is replaced; ectopically overexpressing four transcription factors Oct4, Sox2, Klf4, and c-Myc in the cells, culturing until the cell morphology changes and stem cell clones appear, and then replacing the culture medium with stem cell culture medium to obtain induced pluripotent stem cells.

[0009] Prior art CN116064660A discloses a method for preparing sheep induced pluripotent stem cells, which comprises constructing a piggyBac transposon plasmid carrying four factors including sheep-derived transcription factors OCT4, SOX2, KLF4, and c-MYC in series, and two factors including sheep-derived transcription factors NANOG and LIN28 in series, and electroporating and transfecting them into sheep fetal fibroblasts; selecting clones with morphology similar to embryonic stem cells, and then screening cell clones that meet the characteristics of embryonic stem cells for subculture and identification to obtain sheep induced pluripotent stem cells.

[0010] Prior art CN116769836A discloses a method for preparing induced pluripotent stem cells carrying FBN1 gene mutation, comprising: step 1: primary culture of peripheral blood mononuclear cells; step 2: reprogramming of induced pluripotent stem cells; and step 3: identification of stemness of induced pluripotent stem cells.

[0011] Prior art CN107893049A discloses a method for producing iPSCs and target differentiated cells by reprogramming donor cells obtained in a non-invasive manner. Specifically, the donor cells are exfoliated epithelial urine cells. The differentiated cells can be obtained by differentiation of reprogrammed iPSCs or by directly reprogramming urine cells.

[0012] Prior art CN109679922A discloses a method for preparing CADASIL patient-specific vascular smooth muscle cells and endothelial cells, comprising the following steps: a) reprogramming in vitro fibroblasts from CADASIL patients carrying heterozygous mutations in the NOTCH3 gene to obtain an induced pluripotent stem cell line; b) directing differentiation of the induced pluripotent stem cell line into vascular wall cells to obtain a CADASIL disease cell model.

[0013] However, none of the above-mentioned prior arts disclose or involve the use of nasal polyp tissue for stem cell reprogramming. The present invention, on the other hand, removed nasal polyp tissue from a patient who developed GVHD after hematopoietic stem cell transplantation, derived a cell line by in vitro culture, and after optimizing the reprogramming conditions, reprogrammed the cell line into induced pluripotent stem cells for the first time. And the pluripotency experiment verified that the iPSC cell line has typical pluripotent cell characteristics. iPSCs are an important clinical research tool. The present invention retains cells that can be continuously propagated for subsequent research through this means, and hopes that more precious seed cells can be retained through the method to provide an experimental basis for studying related diseases. Summary of the invention

[0014] The present invention first provides a method for establishing an induced pluripotent stem cell line using clinically discarded nasal polyp tissue, comprising the following steps:

[0015] 1) Cultivation of nasal polyp cell lines;

[0016] Optionally, the culturing comprises a tissue adherence method;

[0017] 2) Use optimized electroporation parameters to reprogram somatic cells to obtain induced pluripotent stem cells.

[0018] In certain embodiments, the nasal polyp cell line is cultured using NP medium.

[0019] In certain embodiments, the nasal polyp cell line is isolated from clinically discarded nasal polyp tissue.

[0020] In certain embodiments, the optimized electroporation parameters are 150 V and 10 ms-50 ms.

[0021] In certain embodiments, the optimized electroporation parameters are 150 V and 20 ms.

[0022] In certain embodiments, the induced pluripotent stem cells do not comprise new cancer-associated mutations compared to the parental cell line.

[0023] In certain embodiments, the induced pluripotent stem cell does not comprise a BCOR mutation.

[0024] The present invention also provides applications of the above method, wherein the application is a) preparing a stem cell treatment reagent, medicine or composition;

[0025] b) Drug screening.

[0026] The present invention also provides induced pluripotent stem cells prepared by the above method.

[0027] Finally, the present invention provides the application of the above-mentioned induced pluripotent stem cells, wherein the application is a) preparing a stem cell therapeutic agent, a medicine or a composition;

[0028] b) Drug screening.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] (1) Currently, there are not many reports on cell lines derived from nasal polyp tissue, and the culture conditions for stable cell lines are not clear. There are even fewer reports on reprogramming derived cell lines into induced pluripotent stem cell lines. The present invention uses the tissue adhesion method to culture nasal polyp tissue into a cell line that can be propagated. The cell line has been identified to have the characteristics of fibroblasts. And the iPSCs reprogrammed using this cell line have the characteristics of an embryonic stem cell model. And using the second-generation sequencing methods RNA-seq and WES, the test results found that the expression gene pattern of iPSCs derived from nasal polyp cell lines is similar to that of embryonic stem cells, and using WES detection, it was found that compared with the parental cell line, the reprogramming process did not introduce new cancer-related mutations, confirming that the reprogramming process of the present invention is safe.

[0031] (2) The present invention did not observe any BCOR mutations in all cell lines, which indicates that early-passage nasal polyp-derived iPSCs may still have prospects for clinical treatment. The nasal polyp cell line was derived from a patient who developed GVHD symptoms after hematopoietic stem cell transplantation. The present invention passed the primary cells to passage 6 and reprogrammed them. We mapped the nasal polyp cells and nasal polyp-derived iPSCs to the ClinVar database and classified the gene mutations through the InterVar database. We found a total of 2 cancer-related mutations, TSC2 and RNF43, among 771 cancer-related genes. The nasal polyp cell line and nasal polyp-derived iPSCs had exactly the same cancer-related mutations in passages 3 and 8, which indicates that the reprogramming and in vitro culture process did not introduce new cancer-related mutations, and nasal polyp-derived iPSCs are safe for cell therapy.

[0032] (3) The present invention also compared the correlation between the total mutations of the nasal polyp cell line and the iPSC derived from nasal polyps (P3 and P8). There are 301 (74.9%) mutant genes in nasal polyps and iPSC derived from nasal polyps (P3, P8). The iPSC-P3 derived from nasal polyps and nasal polyps share exactly the same mutant genes, while the iPSC-P8 derived from nasal polyps has 58 more mutant genes, indicating that the gene mutation is increased during the in vitro culture process rather than during the reprogramming process. At the same time, we compared the mutation types of the parental nasal polyp cell line and the iPSC derived from nasal polyps (P3, P8). The images show that SNVs account for the vast majority of gene mutations, and the number of SNVs decreases at passage 8. The mutation type marked as "blank" accounts for the vast majority of gene mutations during in vitro passage, which indicates that iPSC-P8 derived from nasal polyps is safe in stem cell applications. Further, we explored the proportion of single substitution types, and the images showed that in all cell lines, G>A, C>T, T>C, and A>G accounted for the majority of SNV mutations.

[0033] (4) Since chromosome 12 is rich in cell cycle-related genes and carries the pluripotency-related gene NANOG, trisomy 12 may confer selective proliferation and reprogramming advantages to pluripotent stem cells. Gain of 12p is a characteristic of testicular germ cell tumors. Other types of chromosomal aneuploidy frequently observed in human pluripotent stem cells are trisomy 8 and 20q. Other typical chromosomal aneuploidies in human ESCs exist in chromosomes 1, 17, 20, and X. Given that trisomy 1, 12, 17, and 20 account for the majority of aneuploidy in iPSCs, we compared the mutation burden of chromosome 1, chromosome 8, chromosome 11, chromosome 12, chromosome 17, chromosome 20, and sex chromosomes. The results showed that the number of POLYP-P8 mutations increased slightly on chromosomes 1, 17, and 20, and increased dramatically on chromosome 11.

[0034] (5) The present invention successfully established a nasal polyp-derived cell line, and compared with the donor nasal polyp cell line, the present invention did not observe any new mutations in the nasal polyp-derived iPSC at P3, including cancer-related mutations and various mutation types. The number of mutations in the iPSC increased slightly at P8, among which the number of "blank" mutations and chromosomes 1, 11, 17, and 20 mutations increased slightly, which did not affect the genomic stability and safety of further applications. These results show that this method can construct a stable iPSC line with fewer gene mutations, and the early passage of nasal polyp-derived iPSC can be used for stem cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 , Culture results of nasal polyp tissue-derived cell lines. Morphology of nasal polyp tissue-derived cell lines at different generations under bright field.

[0036] Figure 2 , The optimization results of the reprogramming conditions of nasal polyp cell lines. The electroporation efficiency was the highest under the conditions of 150V and 20ms.

[0037] Figure 3 , results of the reprogramming process of nasal polyp cell lines.

[0038] Figure 4 , Identification results of iPSCs derived from nasal polyp cell lines; a. Morphology of embryonic stem cells and iPSCs derived from nasal polyps. b. AP staining of embryonic stem cells and iPSCs derived from nasal polyps; c. Identification of pluripotency markers (NANOG, OCT4, and SOX2) in embryonic stem cells and iPSCs derived from nasal polyps; d, e Immunofluorescence identification of EBs of embryonic stem cells and iPSCs derived from nasal polyps and markers of the three germ layers of EB formation (SOX17, BRACHYURY, and PAX6).

[0039] Figure 5 , the transcriptome expression of iPSC derived from nasal polyp cell line (iPSC-POLYP) and the identification results of related gene mutations; a. Differences in transcriptome levels between nasal polyp-derived cell lines, iPSC derived from nasal polyp tissues and embryonic stem cells; c. The number of cancer-related gene mutations in nasal polyp tissue-derived cell lines and iPSC derived from nasal polyp tissues; c. Venn diagram of mutated genes in iPSC-POLYP-P3, P8 and parental cells; d. Types of gene mutations in iPSC-POLYP-P3, P8 and parental cells. E. Comparison of single base mutations between parental cell lines and iPSC-POLYP (P3, P8); f. Number of major chromosome mutations in iPSC-POLYP and parental cells. DETAILED DESCRIPTION

[0040] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0041] Example 1

[0042] (1) Culture of nasal polyp cell lines:

[0043] Under sterile conditions, nasal polyp tissue was cut into small pieces of 1-2 mm in size to promote cell migration and proliferation from tissue structures. These tissues were then evenly spread on the surface of a 100 mm culture dish. After about 20 minutes, the tissue fragments were allowed to slightly adhere to the bottom of the culture dish, and NP medium was carefully added to the culture dish. NP medium is a growth medium (100×) containing 90% DMEM / F-12, 10% fetal bovine serum, and 1% penicillin-streptomycin, which is specifically designed to promote the growth of nasal polyp cells. The medium was carefully added drop by drop around the tissue fragments to prevent displacement, and then gently swirled in the culture dish to achieve uniform distribution. The culture dish was then placed in an incubator at 37°C and 5% CO2. The medium was changed every two days to support normal cell growth and remove waste, while minimizing interference with adherent tissue pieces and new cell growth. Once a large number of cells proliferated significantly, usually forming a layer of cells around the tissue pieces, the cells were ready for subculture. First, the cells were washed twice with DPBS, then digested with 0.25% Trypsin-EDTA solution, and then an equal volume of NP medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 min to remove the trypsin, and then resuspended in new NP medium and transferred to a new T25 culture flask. The cell generation was recorded as P1. The morphology of nasal polyp tissue-derived cell lines at different generations was identified under bright field, and the results are shown in Figure 1. Figure 1 shown.

[0044] (2) Somatic cell reprogramming:

[0045] First, the present invention optimized the electroporation conditions of the fibroblast-like nasal polyp cell line. Considering that the size and morphology of the nasal polyp cell line are similar to those of mesenchymal stem cells, the electroporation conditions were optimized based on the successful mesenchymal stem cell reprogramming work in the laboratory. The present invention used the BEX electroporator and adjusted the current from 10ms to 50ms with a gradient of 10ms. The results are shown in Figure 2. Figure 2 As shown, nasal polyp cells maintain optimal viability within 10-20 ms, and the number of dead cells and cell fragments increases with the increase of current. The electroporation efficiency reaches the maximum value under the conditions of 150V and 30ms. However, considering the viability and electroporation efficiency after electroporation, the present invention selects 150V and 20ms as the reprogramming conditions.

[0046] One day before the transfection experiment, plate the fibroblastoid nasal polyp cells in a 6-well plate so that the cell confluency is approximately 50-80% on the day of transfection. Allow a vial of FuGENE 6 transfection reagent to reach room temperature before use. Mix by inversion or rotation. Add 6 μL of FuGENE 6 transfection reagent to 93 μL of Opti-MEM medium as medium. Mix and incubate for 5 minutes. Add 1 μL of pCMV-EGFP plasmid or 1 μL of pEF1α-EGFP plasmid to FuGENE 6 to achieve the appropriate ratio of reagent to DNA. Mix and incubate for 15 minutes. Add 100 μL of FuGENE 6 transfection reagent / DNA mixture to each well of transfected cells and mix gently. Incubate for 24-48 hours to determine transfection efficiency. Change the medium every other day until colonies appear.

[0047] The present invention successfully generated iPSCs from P6 nasal polyps. Reprogrammed cells emerged from the intercellular spaces, with dense cell morphology and uniform distribution. Figure 3 As shown, over time, cell clones became larger and larger, and iPSCs were subcultured on the 26th day of iPSCs clone formation.

[0048] (3) RT-qPCR:

[0049] Discard the iPSCs culture medium and rinse once with DPBS. 2 Add 1ml TransZol Up to the growth area, place horizontally for a while to allow the lysate to be evenly distributed on the cell surface to lyse the cells, and then use a pipette to blow the cells to make them fall off. Transfer the cell lysate into a centrifuge tube, add 200μL RNA extractant, and use a pipette to repeatedly blow and aspirate until there is no obvious precipitation in the lysate, and vortex and shake for 5min at room temperature. Then centrifuge at 10,000×g and 4℃ for 15 minutes, transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of 100% ethanol, and gently invert to mix. Add the resulting solution and precipitate to the centrifuge column, centrifuge at 12,000×g for 30s, and discard the centrifugation product. Repeat this step once. Add 500μL WB9, centrifuge at 12,000×g for 30s, and discard the centrifugation product. Repeat this step once. Centrifuge at 12,000×g for 2 minutes to completely remove residual ethanol, place the centrifuge column in an RNase-free tube, add 50-200μL RNase-free water, let stand at room temperature for 1 minute, centrifuge at 12,000×g for 1 minute to elute RNA. RNA is stored at -80℃.

[0050] RNA was reverse transcribed into first-strand cDNA using PerfectStart Uni RT-qPCR Kit (TransGen Biotech). The reaction system was prepared according to the kit instructions. Gently mix, incubate at 50°C for 5 minutes, and heat at 85°C for 2 minutes to inactivate TransScript Uni RT / RI and gDNA Remover.

[0051] Quantitative reverse transcription PCR (RT-qPCR) analysis was performed on Step One 7500 (Applied Biosystems) to quantify the mRNA expression of target genes in iPSCs and the copy number changes in hiPSC lines. The genomic DNA of iPSCs was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0. Briefly, 100 ng of cDNA or gDNA was added to the reaction system. The reaction mixture was incubated at 95°C for 5 min, followed by incubation at 95°C for 15 s, at 60°C for 15 s, and at 72°C for 15 s for a total of 40 cycles. The data were analyzed using Applied Biosystems Step One Software v2.2. GAPDH was amplified as an internal reference to control the amount of cDNA or gDNA in each reaction. The size of the fluorescence threshold cycle (Ct value) can reflect the expression of mRNA. The larger the Ct value, the less mRNA expression.

[0052] (4) Immunofluorescence:

[0053] To detect pluripotency and differentiation markers, cells grown in 12-well plates were fixed with 4% paraformaldehyde for 20 minutes. They were permeabilized with 0.1% Triton X-100 in PBS for 20 minutes and blocked with 5% BSA at 37°C for 30 minutes. Subsequently, the cells were stained with primary antibodies at appropriate dilution ratios at 4°C. The next day, the cells were washed 3 times with PBS for 3 minutes each time and incubated with secondary antibodies labeled with fluorescent dyes in 5% BSA at room temperature for 1 hour. The cells were then washed 3 times with PBS for 5 minutes each time and the cell nuclei were stained with DAPI solution. The primary antibodies for detecting pluripotency markers were anti-OCT4 antibody, anti-SOX2 antibody, anti-NANOG antibody, and anti-SSEA4 antibody. The secondary antibodies were Goat anti-Mouse Alexa Fluor 488 and Goat anti-Rabbit Alexa Fluor 568, respectively. Images were captured and quantified using the LAS X imaging system, and the results are shown in Figure 4 As shown, the cell line can normally express the marker genes of pluripotent stem cells and has a certain differentiation potential.

[0054] (5) Embryoid Body (EB) differentiation:

[0055] iPSCs were digested with 0.5mM EDTA and resuspended in EB medium. 7000 cells were transferred to the wells of an ultra-lowattachment U-bottom 96-well plate. Centrifuge at 300×g for 3 minutes to promote cell aggregation, and then carefully transfer the plate to a 37°C, 5% CO2 incubator to allow EB formation. After 7 days of culture, EBs were seeded on Vitronectin-coated plates for 14 days for further differentiation. The culture medium was changed every two days. Immunofluorescence staining was used to detect three germ layer markers. The primary antibody for detecting endoderm markers was anti-SOX17, the primary antibody for detecting mesoderm markers was anti-BRACHYURY, and the primary antibody for detecting ectoderm markers was anti-PAX6. The secondary antibodies were Goat anti-Rabbit AlexaFluor 488 and Goat anti-Mouse Alexa Fluor 568, respectively.

[0056] (6) RNA-seq:

[0057] RNA extraction kits were used to extract RNA from undifferentiated hESC / hiPSC cells and early tissue cell lines after 15 passages. The quality and quantity of total input mRNA were determined using the Agilent RNA 6000Nano kit on the Agilent Bioanalyzer 2100. According to the instructions, 1 μg of total RNA was extracted from each sample as input, and the library was prepared using the Illumina TruSeq RNA Sample Preparation Kit. The size, distribution, and quantity of the library were determined on the BioAnalyzer 2100 using the Agilent High Sensitivity DNA Kit. The libraries were then pooled together at equimolar concentrations and sequenced on the Illumina HiSeq2000. All hESC and hiPSC samples for RNA-Seq analysis were prepared by the same person on the same day and then sequenced simultaneously in the same run. Bowtie 0.12.7 software was used to map RNA-seq reads to the human transcriptome sequence library obtained from the ENSEMBL (GRCh38) reference chromosome, allowing up to 2 mismatches, and then merging the same data. The transcriptome includes protein-coding genes and non-coding genes such as lincRNAs. EMSAR was used to quantify the expression levels in TPM (Transcripts Per Million) and infer the read counts of individual genes. Differentially expressed genes were identified using edgeR 3.4.2 and calculated using DESeq 1.8.3.

[0058] (7) Whole exome sequencing

[0059] The present invention performed whole exome sequencing (WES) (100-fold coverage) on parental nasal polyp cells (P6) and iPSC-POLYP (P3, P8) to determine the somatic genomic mutation load. WES refers to the use of sequence capture technology to capture and enrich DNA in the exon region of the entire genome, and perform high-throughput sequencing, which can directly discover genetic variations associated with protein functional variations. Although exons only account for 1% of the genome, the protein coding region of the human genome contains about 85% of pathogenic mutations. When performing data analysis on sequencing sites, the present invention only retains exons and splicing sites, as well as sites with a population frequency below 0.001 (databases include ESP6500, 1000Genomes, gnomAD, and ExAC). At the same time, the present invention filters out the points marked as benign or likely benign (databases include ClinVar and InterVar) and the points with a quality value of HARD_FILTER, deletes all points with a sequencing depth of less than 10 times, and all sites with mutation reads greater than 3 in the sample. The results are as follows Figure 5 As shown, 2 cancer-related mutations, TSC2 and RNF43, were found in 771 cancer-related genes. The nasal polyp cell line and nasal polyp-derived iPSCs had exactly the same cancer-related mutations at passage 3 and passage 8, indicating that the reprogramming and in vitro culture process did not introduce new cancer-related mutations, and nasal polyp-derived iPSCs were safe for cell therapy.

[0060] In addition, the present invention also compares the correlation between the total mutations of the nasal polyp cell line and the iPSC derived from nasal polyps (P3 and P8). Nasal polyps and iPSC derived from nasal polyps (P3, P8) have a total of 301 (74.9%) mutant genes. The iPSC-P3 derived from nasal polyps and nasal polyps share exactly the same mutant genes, while the iPSC-P8 derived from nasal polyps has 58 more mutant genes, indicating that the gene mutation is increased during in vitro culture rather than during reprogramming. At the same time, the present invention compares the mutation types of the parental nasal polyp cell line and the iPSC derived from nasal polyps (P3, P8). The images show that SNVs account for the vast majority of gene mutations, and the number of SNVs decreases at passage 8. The mutation type marked as "blank" accounts for the vast majority of gene mutations during in vitro passage, indicating that iPSC-P8 derived from nasal polyps is safe in stem cell applications. Furthermore, the present invention explored the proportion of single substitution types, and the images showed that in all cell lines, G>A, C>T, T>C and A>G accounted for the majority of SNV mutations.

[0061] Since chromosome 12 is rich in cell cycle-related genes and carries the pluripotency-related gene NANOG, trisomy 12 may confer selective proliferation and reprogramming advantages to pluripotent stem cells. Gain of 12p is a characteristic of testicular germ cell tumors. Other types of chromosomal aneuploidy frequently observed in human pluripotent stem cells are trisomy 8 and 20q. Other typical chromosomal aneuploidies in human ESCs are present in chromosomes 1, 17, 20, and X. Given that trisomy 1, 12, 17, and 20 account for the majority of aneuploidy in iPSCs, we compared the mutation burden of chromosome 1, chromosome 8, chromosome 11, chromosome 12, chromosome 17, chromosome 20, and sex chromosomes. The results showed that the number of POLYP-P8 mutations increased slightly on chromosomes 1, 17, and 20, and increased dramatically on chromosome 11.

[0062] In summary, the present invention found that compared with the donor cell line, the cell line has obvious transcriptome differences, and the expression of pluripotency genes is completely different from that of the donor cells, which proves the stability of the reprogramming method. When WES analysis was performed again, it was found that compared with the donor cells, the reprogramming process did not introduce new cancer-related gene mutations, confirming the advantages of the reprogramming method of the present invention.

[0063] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for establishing an induced pluripotent stem cell line using clinically discarded nasal polyp tissue, characterized in that: The steps include: 1) Cultivation of nasal polyp cell lines; Optionally, the culturing comprises a tissue adherence method; 2) Use optimized electroporation parameters to reprogram somatic cells to obtain induced pluripotent stem cells.

2. The method according to claim 1, characterized in that: The nasal polyp cell line was cultured using NP medium.

3. The method according to claim 1, characterized in that: The nasal polyp cell line is isolated from clinically discarded nasal polyp tissues.

4. The method according to claim 1, characterized in that: The optimized electroporation parameters are 150V and 10ms-50ms.

5. The method according to claim 4, characterized in that: The optimized electroporation parameters are 150V and 20ms.

6. The method according to claim 1, characterized in that: The induced pluripotent stem cells do not contain new cancer-associated mutations compared to the parental cell line.

7. The method according to claim 1, characterized in that: The induced pluripotent stem cells do not contain a BCOR mutation.

8. An application of the method according to claims 1-7, characterized in that: The application is a) preparing stem cell treatment reagents, drugs or compositions; b) Drug screening.

9. Induced pluripotent stem cells prepared by the method of claims 1 to 7.

10. The use of the induced pluripotent stem cells according to claim 9, characterized in that: The application is a) preparing stem cell treatment reagents, drugs or compositions; b) Drug screening.

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