CN110628821B

CN110628821B - Cell model and preparation method and application thereof

Info

Publication number: CN110628821B
Application number: CN201810664195.8A
Authority: CN
Inventors: 陈志国; 王淑艳; 徐大为
Original assignee: Xuanwu Hospital
Current assignee: Xuanwu Hospital
Priority date: 2018-06-25
Filing date: 2018-06-25
Publication date: 2022-02-18
Anticipated expiration: 2038-06-25
Also published as: CN110628821A

Abstract

The invention provides a cell model, a preparation method and application thereof, and provides a preparation method for inducing pluripotent stem cells by WS somatic cells, which comprises the following steps: s1, obtaining somatic cells derived from WS patients; s2, transfecting a vector expressing the human telomerase reverse transcriptase and the resistance gene to the obtained somatic cells, culturing, and screening the somatic cells capable of expressing the human telomerase reverse transcriptase; s3, transfecting a vector carrying reprogramming genes for inducing iPSCs into the somatic cells in the S2, and culturing; according to the invention, the vector capable of expressing human telomerase reverse transcriptase hTERT is transferred before the somatic cell of WS is reprogrammed, so that the somatic cell of WS can be reprogrammed into the pluripotent stem cell, the induction efficiency of iPSCs is improved, and the problem that the pluripotent stem cell cannot be successfully induced or the induction efficiency is extremely low by using the somatic cell of a WS patient in the prior art is solved.

Description

Cell model and preparation method and application thereof

Technical Field

The invention belongs to the field of induced pluripotent stem cells in biotechnology, and particularly relates to a cell model and a preparation method and application thereof.

Background

Wolner Syndrome (WS), also called as adult presenility, cataract-scleroderma-presenility Syndrome, is a congenital autosomal recessive hereditary disease, is mostly seen in offspring of blood-related marriage, especially in offspring of brother and sister marriage, the disease gene is positioned at 8p 12-p 11, and the main clinical manifestations include aged face-look, short stature, juvenile poliosis, juvenile cataract, extremity scleroderma-like skin change, osteoporosis, tissue calcification, diabetes and gonadal dysgenesis. WS patients develop a series of obvious features of aging in the body, including: short and small body, face shrinkage, hair whitening and falling off, skin atrophy and sclerosis and pigmentation; heart diseases such as heart failure and myocardial infarction; ③ atherosclerosis and hypertension; insulin-resistant hyperglycemia, hyperlipidemia and hyperuricemia; mental deterioration and brain atrophy; sixthly, the eye part is degenerated early; seventhly, autoimmune diseases such as rheumatoid diseases and the like are easily caused due to abnormal immune system; osteoporosis, joint deformity and restricted movement. It is very noteworthy that WS patients are prone to develop various tumors, especially tumors of mesodermal origin, melanoma and astrocytoma, etc., which is very consistent with the fact that aging and tumor incidence are positively correlated. WS patients have a mean life span of 50 years and mostly die of vascular disease and tumors. WS affects almost all tissues and organs throughout the body, and features that accompany the natural aging process in humans are accelerated in WS individuals. WS is considered to be the most suitable model for studying physiological aging in humans, since it can exhibit more phenotypes and symptoms similar to those of natural aging in humans.

In recent years, the emergence of Induced Pluripotent Stem Cell (iPSC) technology has provided opportunities for the development of models for disease and personalized therapies for family genetic diseases, as well as being well suited for WS research. Induced Pluripotent Stem Cells (iPSCs) are cells having characteristics of embryonic stem cells produced by ectopic expression of 4 pluripotent stem cell-related transcription factors (OCT3/4, SOX2, KLF4, cMYC, called shanzhong factor) in differentiated somatic cells, and show great potential in modeling the disease pathogenesis, screening for new drugs, and developing new therapeutic methods. This is particularly true for genetic diseases, which are reproducible by providing an unprecedented in vitro model, since iPSCs from patient cells share the same genetic background.

However, the study of somatic reprogramming in WS patients has presented more difficulties and challenges than other genetic diseases. The research already shows that the skin fibroblast of WS patients is aged and accompanied with serious chromosome abnormality after in vitro culture, and the reprogramming process can further cause cell events such as active oxygen (ROS) increase, DNA break and cell death (Senescence) to seriously obstruct the reprogramming process of the somatic cells, so that the research on the reprogramming of the somatic cells carrying pathogenic mutant genes of WS patients is difficult and serious, and no successful implementation case is seen at home and abroad.

Disclosure of Invention

Therefore, the technical problem to be solved by the present invention is a cell model, and a preparation method and an application thereof.

To this end, the present invention provides a method for inducing pluripotent stem cells using WS somatic cells, comprising the steps of:

s1, obtaining somatic cells derived from WS patients;

s2, transfecting a vector expressing human telomerase reverse transcriptase (hTERT) and a resistance gene into the somatic cells, culturing, and screening the somatic cells capable of expressing the human telomerase reverse transcriptase;

s3, transfecting a vector carrying reprogramming genes for inducing iPSCs into the somatic cells screened in the S2 step, and culturing the iPSCs.

In the method for inducing pluripotent stem cells by using WS somatic cells, in step S1, the somatic cells derived from WS patient are fibroblasts from skin of WS patient, peripheral blood mononuclear cells from WS patient or bone marrow mesenchymal cells from WS patient.

The method, in step S2, transfects a vector that regulates expression of a foreign gene simultaneously with the transfection of a vector that expresses human telomerase reverse transcriptase.

The method, in step S2, the backbone vector for the regulation foreign gene expression vector is selected from pLVX-Tet-on, Tet-pLKO-neo or pTet-IRES-EGFP.

In the method, in step S2, the backbone vector expressing human telomerase reverse transcriptase is a lentiviral vector. The vector is selected from pLVX-light-Puro, Tet-pLKO-neo or pTet-IRES-EGFP.

The method, the reprogramming genes are at least 4 of OCT4, SOX2, NANOG, LIN28, c-MYC, KLF-4 and SV40 LT.

The reprogramming genes are OCT4, SOX2, c-MYC and KLF-4.

In the method, in step S3, the vector is Sendai virus.

The invention provides application of the induced pluripotent stem cells prepared by the method in the fields of disease pathogenesis, drug screening and treatment method screening of adult premature senility, tumor, atherosclerosis, diabetes or osteoporosis. Because the incidence rates of tumors, atherosclerosis, diabetes and osteoporosis of the population suffering from the premature senility are obviously higher than those of the normal population, and the induced pluripotent stem cells prepared by the method have the multidirectional differentiation potential, the induced pluripotent stem cells have the potential of being applied to the pathogenesis, drug screening and treatment method screening of the diseases in the tumors, atherosclerosis, diabetes and osteoporosis.

The invention provides a cell model, which comprises induced pluripotent stem cells prepared by the method.

The invention provides application of the cell model in the fields of disease pathogenesis research, drug screening and therapeutic method screening of adult premature senility, tumors, atherosclerosis, diabetes or osteoporosis.

The technical scheme of the invention has the following advantages:

1. the invention provides a method for inducing pluripotent stem cells by using WS somatic cells, which comprises the following steps: s1, obtaining somatic cells derived from WS patients; s2, transfecting a vector which simultaneously expresses human telomerase reverse transcriptase and a resistance gene into the somatic cells, culturing, and screening the somatic cells capable of expressing the human telomerase reverse transcriptase; s3, transfecting a vector carrying reprogramming genes for inducing iPSCs into the somatic cells cultured in the S2 step, and culturing the iPSCs; the invention discovers and confirms through a large number of experimental researches that the reason that the somatic cells derived from WS patients cannot be reprogrammed is that telomere elongation and telomerase activation are abnormal in the reprogramming process of the somatic cells of WS patients, and confirms that the problem that the telomere elongation and the telomerase activation are abnormal in the reprogramming process of the somatic cells of WS is solved by transferring a vector capable of expressing hTERT into the somatic cells of WS before the reprogramming of the somatic cells of WS, so that the somatic cells of WS can be reprogrammed into pluripotent stem cells. Therefore, the invention provides the method for inducing the pluripotent stem cells by using the WS somatic cells, which comprises the steps of transfecting a vector which simultaneously expresses human telomerase reverse transcriptase and a resistance gene into the somatic cells of a WS patient, screening out the somatic cells capable of expressing the human telomerase reverse transcriptase, enabling the fibroblast to have basic telomerase activity and telomere length, ensuring the normal reprogramming of the somatic cells, improving the induction efficiency of iPSCs, and solving the problem that the pluripotent stem cells cannot be successfully induced by using the somatic cells of the WS patient in the prior art.

2. The invention provides a method for inducing pluripotent stem cells by WS somatic cells, which comprises the step of S2, wherein a vector for transfecting and expressing human telomerase reverse transcriptase is simultaneously transfected with a vector capable of regulating and controlling the expression of an exogenous gene, the expression of the exogenous gene-hTERT in somatic cells of WS patients, such as fibroblasts, can be regulated and controlled by the vector capable of regulating and controlling the expression of the exogenous gene, the vector capable of regulating and controlling the expression of the exogenous gene is reconstructed by pLVX-Tet-on Advanced, Tet-pLKO-neo or pTet-IRES-EGFP, the pLVX-Tet-on Advanced is a controllable lentiviral vector expression system, the expression of the exogenous gene can be regulated and controlled by increasing and decreasing tetracycline, such as doxycycline hydrochloride, and the expression of the hTERT in the iPS cells can be regulated and controlled.

3. In the method for inducing pluripotent stem cells by using WS somatic cells, provided by the invention, in the step S3, the vector is Sendai virus, and as the exogenous gene introduced by the Sendai virus is not integrated into the genomic DNA of host cells, namely the exogenous gene expression mediated by the Sendai virus does not change the genetic background of the host cells, the Sendai virus self vector and the exogenous gene sequence can be eliminated from the host cells, and the obtained IPSCs cells are induced to have no insertion of the exogenous gene, so that the safety is higher, the infection efficiency of the Sendai virus is higher, the expression level of the target gene is higher, and the induction efficiency of iPSCs can be greatly improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a microscope photograph of WS fibroblasts and WT fibroblasts before and 5 days after transfection of Sendai virus in Experimental example 1 of the present invention;

FIG. 2 shows the results of alkaline phosphatase staining of WS fibroblasts and WT fibroblasts after transfection of Sendai virus at week 3 in Experimental example 1;

FIG. 3 is a graph showing the positive cloning efficiency of alkaline phosphatase staining of WS fibroblasts and WT fibroblasts after transfection of Sendai virus at week 3 in Experimental example 1 of the present invention;

FIG. 4 is a result of analysis of telomerase activity during reprogramming of WS fibroblasts and WT fibroblasts in Experimental example 2 of the present invention;

FIG. 5 is a graph showing the fold-change of telomerase activity during reprogramming of WS fibroblasts and WT fibroblasts in Experimental example 2 of the present invention;

FIG. 6 is a genetic map of plasmid pBABE-puro-hTERT in the present invention;

FIG. 7 is a microscope photograph of WS-hTERT and WT-hTERT before and at 5 days after transfection of Sendai virus in Experimental example 3 of the present invention;

FIG. 8 is a graph showing the positive cloning efficiency of alkaline phosphatase staining of WS-hTERT and WT-hTERT after 3 weeks of Sendai virus transfection in Experimental example 3 of the present invention;

FIG. 9 shows the immunofluorescence staining results of iPSCs obtained after 4 weeks of transfection of Sendai virus by WS-hTERT and WT-hTERT in Experimental example 3 of the present invention;

FIG. 10 shows the RT-PCR detection results of iPSCs obtained after the week 4 of transfection of Sendai virus by WS-hTERT and WT-hTERT in Experimental example 3 of the present invention;

FIG. 11 shows the karyotype detection results of iPSCs obtained after 4 weeks of transfection of Sendai virus by WS-hTERT and WT-hTERT in Experimental example 3 of the present invention;

FIG. 12 shows the karyotype detection result of iPSCs obtained by WT-hTERT at week 4 after transfection of Sendai virus in Experimental example 3 of the present invention;

FIG. 13 shows the result of iPSCs differentiation assay of WS-hTERT obtained after 4 weeks of Sendai virus transfection in Experimental example 3;

FIG. 14 shows the result of iPSCs differentiation assay of WS-hTERT obtained after 4 weeks of Sendai virus transfection in Experimental example 3;

FIG. 15 shows WRN expression immunofluorescence staining results of iPSCs obtained after 4 weeks of transfection of Sendai virus with WS-hTERT and WT-hTERT in Experimental example 3 of the present invention;

FIG. 16 shows the WRN expression immunoblotting results of iPSCs obtained after the week 4 of transfection of Sendai virus by WS-hTERT and WT-hTERT in Experimental example 3 of the present invention;

FIG. 17 shows the results of analysis of telomerase activity during reprogramming of WS-hTERT and WT-hTERT in Experimental example 3 of the present invention;

FIG. 18 is the fold change of telomerase activity during reprogramming of WS-hTERT and WT-hTERT in Experimental example 3 of the present invention;

FIG. 19 is telomere length of WS in cells before and after Sendai virus transfection in Experimental example 3 of the present invention at day 6;

FIG. 20 shows telomere lengths in cells of WT before and after transfection of Sendai virus in Experimental example 3 of the present invention at day 6;

FIG. 21 is telomere length of WS-hTERT in cells before and at day 6 after Sendai virus transfection in Experimental example 3 of the present invention;

FIG. 22 is the telomere length of WT-hTERT in cells on day 6 before and after transfection of Sendai virus in Experimental example 3 of the present invention;

FIG. 23 is a bar graph showing the percentage distribution of telomere length in cells of WS, WT, WS-hTERT and WT-hTERT on day 6 before and after Sendai virus transfection in Experimental example 3 of the present invention;

FIG. 24 is the average telomere length of WS, WT, WS-hTERT and WT-hTERT in cells on day 6 before and after transfection of Sendai virus in Experimental example 3 of the present invention;

FIG. 25 shows the relative telomere lengths of different generations of iPSCs of WT, WS-hTERT and WT-hTERT in Experimental example 3 of the present invention;

FIG. 26 is a map of plasmids pLVX-Tet-on and pLVX-light-hTERT-Puro in Experimental example 4 of the present invention;

FIG. 27 shows hTERT expression at passage 10 of WS-ihTERT iPSCs and WT-ihTERT iPSCs in Experimental example 4 of the present invention;

FIG. 28 is a graph showing the ratio of the total number of BRDU-positive cells obtained by culturing WS-ihTERT iPSCs and WT-ihTERT iPSCs in Experimental example 4 of the present invention in a DOX-containing or DOX-free medium after addition of BRDU;

FIG. 29 is a cell cycle analysis of WS-ihTERT iPSCs and WT-ihTERT iPSCs after synchronization of nocodazole treatment in Experimental example 4 of the present invention;

FIG. 30 shows the percentage of cells in different cycles of WS-ihTERT iPSCs in Experimental example 4 of the present invention after synchronization with nocodazole treatment;

FIG. 31 shows the percentage of cells in different cycles of WT-ihTERT iPSCs in Experimental example 4 of the present invention after synchronization with nocodazole treatment;

FIG. 32 shows the cell ratio of CPT-treated apoptotic iPSCs in Experimental example 5;

FIG. 33 shows the cell ratio of apoptotic iPSCs after treatment with different stressors in Experimental example 5 of the invention;

FIG. 34 is an analysis of the iPSCs of WS-ihTERT-and WT-ihTERT-at different time points in Experimental example 5 of the present invention;

FIG. 35 shows cell cycle analysis of WS-ihTERT-and WT-ihTERT-iPSCs in CPT or in the absence of CPT after synchronization with nocodazole treatment in Experimental example 5 of the invention;

FIG. 36 shows the ratio of the WS-ihTERT-and WT-ihTERT-iSCs cells in S phase in inventive experiment example 5 with or without CPT treatment;

FIG. 37 shows the result of detecting DNA double strand breaks of WS-ihTERT-and WT-ihTERT-in high dose CPT or without CPT treatment in the invention experiment 5;

FIG. 38 is the ratio of gamma H2AX foci in cells after high dose CPT of iPSCs of WS-ihTERT-and WT-ihTERT-in Experimental example 5 of the invention;

FIG. 39 is the Western blotting result of gamma H2AX foci in cells after high dose CPT of iPSCs of WS-ihTERT-and WT-ihTERT-in Experimental example 5;

FIG. 40 is a graph showing the results of Western blotting of gamma H2AX foci in cells following high dose CPT of WS-ihTERT-and WT-ihTERT-iPSCs in FIG. 39;

FIG. 41 is a graph showing the diffusion profiles of RPA32 and Rad51 in cells after CPT treatment of iPSCs of WS-ihTERT-and WT-ihTERT-in Experimental example 5 of the present invention;

FIG. 42 is a graph showing the percentage of cells having the percentage of RPA32+ positive cells in iPSCs of WS-ihTERT-and WT-ihTERT-after treatment with CPT in inventive example 5;

FIG. 43 is a graph of the percentage of cells with Rad51+ positive cells in WS-ihTERT-and WT-ihTERT-iPSCs after treatment with CPT in inventive example 5;

FIG. 44 is the Western blotting result of RPA32 in cells after high dose CPT of WS-ihTERT-and WT-ihTERT-iPSCs in Experimental example 5 of the present invention;

FIG. 45 shows the Western blotting results of Rad51 in cells after high dose CPT of WS-ihTERT-and WT-ihTERT-iPSCs in Experimental example 5;

FIG. 46 is a graph showing the results of immunofluorescent staining of induced pluripotent stem cell iPSCs obtained in example 2 of the present invention;

FIG. 47 is a chart showing the result of karyotype detection of induced pluripotent stem cell iPSCs obtained in example 2 of the present invention;

FIG. 48 is a graph showing the results of the differentiation assay of induced pluripotent stem cell iPSCs obtained in example 2 of the present invention.

Detailed Description

Fibroblast cells (AG03141) and wild-type (WT) fibroblast cells (AG10803) of the skin of Wolner Syndrome (WS) patients, both provided by Coriell biorobot, in the following examples, wherein the nucleotide C at position 2476 of WRN gene of WS fibroblast cell (AG03141) was mutated to T (2476C > T) to generate a stop codon at position 748, i.e., [ Gln748TER (Q748X) ];

the pBABE-hTERT-Puro plasmid capable of expressing human telomerase reverse transcriptase and puromycin resistance genes, the skeleton vector capable of expressing human telomerase reverse transcriptase are lentiviral vectors pLVX-Tight-Puro and the vector for regulating and controlling the expression of exogenous genes reconstructed by pLVX-Tet-on Advanced (map shown in figure 26 right picture) are all provided by Clonetech Laboratories Inc.;

sendai virus carrying OCT4, SOX2, c-MYC, and KLF-4 was supplied by Thermo Fisher Scientific;

transfection reagents were supplied by Thermo Fisher Scientific;

the packaging cell line 293T cells were purchased from the China academy of sciences type culture Collection cell Bank;

packaging plasmids CMV. DELTA.8.74 and pMD.G-2.0 were purchased from Addgene;

immunodeficient SCID mice were purchased from Beijing Wintolite laboratory animal technology, Inc.;

the specific construction method of pLVX-light-hTERT-Puro capable of expressing human telomerase reverse transcriptase and puromycin resistance gene referred to in the following examples is as follows:

1. and (2) amplifying the coding sequence of the hTERT by using a PCR method by taking a plasmid vector pBABE-puro-hTERT containing the hTERT coding sequence as a template, wherein the coding sequence of the hTERT is shown as SEQ ID NO: 1, adding restriction enzyme sites Not I and EcoRI to the 5 'end and the 3' end of the PCR fragment obtained by amplification.

2. And (3) carrying out double digestion on the PCR product obtained in the step (1) and the vector pLVX-light-Puro by using restriction endonucleases Not I and EcoRI respectively, and purifying the digestion product.

3. The PCR product purified in step 2 and the vector were mixed at a molar ratio of 10:1 and ligated using T4 DNA ligase.

4. And (3) taking 5ul of the ligation product obtained in the step (3) to transform competent cells, screening out positive colonies by antibiotics, extracting plasmids to perform enzyme digestion and sequencing identification, wherein a forward primer F used for sequencing is shown as SEQ ID NO: 2, and the reverse primer R is shown as SEQ ID NO: 3, respectively. Selecting a colony amplification extraction plasmid with correct sequencing for the next experiment, wherein the constructed pLVX-light-hTERT-Puro plasmid map is shown in the left picture in the attached figure 26, and the nucleotide sequence of the plasmid map is shown in SEQ ID NO: 4, respectively.

Example 1

This example provides a method for inducing pluripotent stem cells using WS somatic cells, comprising the following steps:

s1, obtaining somatic cells from WS patients: taking fibroblasts (AG03141) of the skin of a patient with Wolner Syndrome (WS), and carrying out in-vitro amplification on the cells, wherein the method comprises the following steps: the fibroblasts were cultured at a density of 2X 10⁵The individual cells were seeded in DMEM medium containing 15% fetal bovine serum at 37 ℃ with 5% CO₂Culturing under 90% humidity, rinsing with PBS without calcium and magnesium ions for 1 time after cultured fibroblasts grow into a single layer, adding PBS digestive juice containing 0.25% pancreatin and 0.02% EDTA for digestion for 2-3min, adding DMEM medium containing 15% fetal calf serum, and blowing to stop digestion at a ratio of 1:2 or 1: 4, at 37 ℃ and 5% CO₂Culturing under the condition of 90% humidity, stopping digestion when cells just become round during the first 3 passages, and collecting the cells without walls to obtain expanded WS fibroblasts;

s2, transfecting a vector for simultaneously expressing human telomerase reverse transcriptase and puromycin resistance genes into the WS fibroblasts in the step S1, culturing, and screening the WS fibroblasts capable of stably expressing the human telomerase reverse transcriptase, wherein the specific steps are as follows:

(1) pBABE-hTERT-Puro virus packaging: recovering 293T cell as packaging cell line, culturing and amplifying in DMEM medium containing 10% fetal calf serum, collecting cells one day before plasmid transfection and counting, and collecting (5-6). times.10⁶293T cells were seeded in 10cm polylysine-coated cell culture dishes at 37 ℃ with 5% CO₂Culturing overnight under the condition of 90% humidity, and performing plasmid transfection when the cells grow to 70-80% confluence; before transfection, 2 polystyrene tubes were prepared and 0.8ml of OPTI-MEM was added to each tube, one of which was 6ug of pBABE-hTERT-Puro plasmid, CMV. DELTA.8.74 ug and pMD.G-2.02 ug, and the other was 32ul of transfection reagent lipofectamine 3000 (supplied by Thermo Fisher Scientific Co., Ltd.), gently mixed and left to stand for 5 minutesAdding the mixed solution in two polystyrene tubes into 293T cell culture medium, culturing overnight, removing supernatant, and replacing with fresh 293T cell culture medium at 37 deg.C and 5% CO₂Culturing for 48h, collecting virus supernatant for infecting WS fibroblasts;

(2) counting, centrifuging and removing supernatant WS fibroblasts collected in step S1, and concentrating cells at a density of 1 × 10⁵Inoculating each cell into one hole of a 6-hole plate, centrifuging for 5 minutes at room temperature and at 200g, and removing supernatant;

(3) adding the virus supernatant collected in step (1) to WS cells at 37 deg.C with 5% CO₂Culturing for 24 hours;

(4) 48h after infection with the virus, 2ml of puromycin-containing medium was placed in each well at 37 ℃ with 5% CO₂Culturing for 7 days under the condition of 90% humidity, screening the WS fibroblasts capable of expressing human telomerase reverse transcriptase, and then carrying out in-vitro amplification culture;

s3, transfecting a vector carrying a reprogramming gene for inducing iPSCs into the fibroblast cells cultured in the S2 step, and culturing the iPSCs, wherein the vector comprises the following specific steps:

(1) WS fibroblasts collected in step S2 were counted, centrifuged, and the supernatant removed, and the cells were cultured at a density of 1X 10⁵Resuspend the cells in 5ml PBS, centrifuge for 5 minutes at room temperature, 200g, remove supernatant;

(2) the cells from step (1) were infected with the Sendai virus carrying OCT4, SOX2, c-MYC and KLF-4 to a viral titer of about 1X 10⁸IU/ml, 5 days after infection, the cells were transferred to trophoblast cells prepared from mouse fibroblasts and cultured in human embryonic stem cell culture media comprising Knock-out DMEM (DMEM medium), 20% KSR, 1% NEAA, 1% GlutaMAX, 0.1mM beta-mercaptoethanol and 10ng/ml bFGF (all provided by Thermo Fisher Scientific, Waltham, MA, USA), and after two weeks of culture, it was seen that iPSCs clones appeared in the culture medium system, i.e., pluripotent stem cells induced by WS somatic cells;

(3) the above cells obtained in step (2) were transferred to Martigel (matrigel) coated 24-well plates, 0.5ml of mTesR medium (supplied by Stemcell, Vancover, BC, Canada) was placed in each well for expansion culture, and after a while, the fused iPSCs were digested with Accutase cell digest for 2min, washed with PBS, and then the cells were scraped from the petri dish using the tip of a 2ml pipette, collected in a centrifuge tube and centrifuged at 1:6, carrying out passage to obtain the product. The induced pluripotent stem cell iPSCs obtained in the above way are preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC 14895.

In order to identify the biological characteristics of iPSCs, the pluripotent markers OCT4, SOX2, TRA-1-81 and SSEA4 were examined by immunocyte tissue staining. injection of iPSCs into the hindlimb muscles of SCID/Beige mice to detect teratomas. RT-PCR was used to detect the expression of the pluripotency genes OCT4, SOX2, NANOG, REX-1, ESG1, and DPPA 4. Karyotyping and performing analysis are performed using conventional methods. Teratoma tissue structures were analyzed using immunohistochemical staining. The above-mentioned identification results are shown in Experimental example 3.

Example 2

s1, obtaining of somatic cells derived from WS patient: taking fibroblasts (AG03141) of the skin of a patient with Wolner Syndrome (WS), and carrying out in-vitro amplification on the cells, wherein the method comprises the following steps: the fibroblasts were cultured at a density of 2X 10⁵The individual cells were seeded in DMEM medium containing 15% fetal bovine serum at 37 ℃ with 5% CO₂Culturing under 90% humidity, rinsing with PBS without calcium and magnesium ions for 1 time after cultured fibroblasts grow into a monolayer, adding PBS digestive juice containing 0.25% pancreatin and 0.02% EDTA for digestion for 2-3min, adding DMEM medium containing 15% fetal calf serum, and blowing to stop digestion at a ratio of 1:2 or 1: 4, at 37 ℃ and 5% CO₂Culturing under 90% humidity condition, terminating digestion when cells just become round in the first 3 passages, and collecting the cells without walls to obtain expanded WS fibroblasts;

s2, transfecting a vector for simultaneously expressing human telomerase reverse transcriptase and puromycin resistance genes and a vector capable of regulating and controlling exogenous gene expression to the WS fibroblasts in the step S1, culturing, and screening the WS fibroblasts capable of stably expressing the human telomerase reverse transcriptase, wherein the specific steps are as follows:

(1) and (3) slow virus packaging: recovering 293T cell as packaging cell line, culturing and amplifying in DMEM medium containing 10% fetal calf serum, collecting cells one day before plasmid transfection and counting, and collecting (5-6). times.10⁶293T cells were seeded in polylysine-coated 10cm cell culture dishes at 37 ℃ with 5% CO₂Culturing overnight under the condition of 90% humidity, and performing plasmid transfection when the cells grow to 70-80% confluence; before transfection, 2 polystyrene tubes were prepared and 0.8ml of OPTI-MEM was added to each of them, one of them was added with 6ug of the vector (or pLVX-light-hTERT-Puro) plasmid for regulating expression of the foreign gene, which was modified from pLVX-Tet-on Advanced, and the packaging plasmids CMV. DELTA.8.74 ug and pMD.G-2.02 ug, the other was added with 32ul of transfection reagent lipofectamine 3000 (provided by Thermo Fisher Scientific Co., Ltd.), the mixture was gently mixed, left to stand for 5 minutes, the mixture of the two polystyrene tubes was added to 293T cell medium, the supernatant was discarded after overnight culture, and the mixture was replaced with 37 ℃ fresh 293T cell medium and 5% CO₂Culturing for 48h, collecting virus supernatant for infecting WS fibroblasts; obtaining two virus supernatants, namely lentivirus pLVX-Tet-on advanced or lentivirus pLVX-light-hTERT-Puro, according to the method;

(3) adding the two virus supernatants prepared in step (1), i.e., lentivirus pLVX-Tet-on advanced and lentivirus pLVX-light-hTERT-Puro, to the WS cells at the same time at 37 ℃ and 5% CO₂Culturing for 24 hours;

(4) 48h after infection with the virus, 2ml of a medium containing puromycin and G418 (Geneticin) was placed in each well at 37 ℃ with 5% CO₂Culturing for 7 days under 90% humidity condition, and screening out the human telomerase reverse transcriptaseWS fibroblasts, then carrying out in-vitro amplification culture, wherein a culture medium for in-vitro amplification contains doxycycline hydrochloride (Dox);

(2) the cells from step (1) were infected with the Sendai virus carrying OCT4, SOX2, c-MYC and KLF-4 to a viral titer of about 1X 10⁸IU/ml, 5 days after infection, the cells were transferred to trophoblast cells prepared from mouse fibroblasts and cultured in human embryonic stem cell culture medium comprising Knock-out DMEM (DMEM medium), 20% KSR, 1% NEAA, 1% GlutaMAX, 0.1mM beta-mercaptoethanol and 10ng/ml bFGF (all provided by Thermo Fisher Scientific, Waltham, MA, USA), and after two weeks of culture, it was seen that iPSCs clones appeared in the medium system, i.e., clones using WS somatic cells

(3) The cells obtained in step (2) above were transferred to Martigel (matrigel) coated 24-well plates, 0.5ml of mTesR medium (supplied by Stemcell, Vancover, BC, Canada) was placed in each well for amplification culture, and after a period of time, the fused iPSCs were digested with Accutase cell digest for 2min, washed with PBS, then the cells were scraped from the culture dish using the tip of a 2ml pipette, collected in a centrifuge tube and centrifuged at 1:6, carrying out passage to obtain the product.

The induced pluripotent stem cell iPSCs obtained in the above way are subjected to pluripotent marker identification, karyotyping and differentiation, the method for pluripotent marker identification refers to the step 1.3 in experimental example 3, the method for karyotyping refers to the step 1.4 in experimental example 3, and the method for differentiation detection refers to the step 1.5 in experimental example 3. The immunofluorescence staining result of the induced pluripotent stem cell iPSCs obtained in the above is shown in figure 46, the karyotype detection result is shown in figure 47, and the differentiation detection result is shown in figure 48. The induced pluripotent stem cell iPSCs obtained in the above way are stored in the common microorganism center of China Committee for culture Collection of microorganisms, the address is No. 3 of No.1 Siro-Shih, North Chen, south China, Beijing, the postal code is 100101, the storage date is 12 months and 12 days in 2017, the storage number is CGMCC No.14895, and the iPSCs are classified and named as human induced pluripotent stem cells.

In the step S2, transfecting two vectors pLVX-Tet-on Advance and pLVX-Tight-hTERT-Puro into the fibroblast, wherein the pLVX-Tet-on Advance is a vector responsible for regulation and expresses tetracycline (tetracycline) regulated transactivator (rtTA-Advanced); and pLVX-light-hTERT-Puro is a reactive vector and is used for expressing a target gene hTERT, and the expression is driven by a modified reaction element promoter of pLVX-Tet-on Advance with tetracycline. In order to realize the expression regulation of the target gene hTERT, pLVX-Tet-on Advance and pLVX-Tight-hTERT-Puro are simultaneously transferred into fibroblasts of a WS patient, rtTA-Advanced expressed by a pLVX-Tet-on Advance vector is combined to a tetracycline reaction element of a promoter region of a reactive vector pLVX-Tight-hTERT-Puro, so that the expression of the exogenous gene hTERT is in a closed state, and when tetracycline is added into a culture solution, the rtTA-Advanced is dissociated from the tetracycline reaction element, and the expression of the exogenous gene hTERT is opened.

Experimental example 1 Induction of fibroblast derived from WS patient by Hill factor

The experimental example aims to investigate whether the fibroblast derived from WS patients can be induced to prepare iPSCs only by using the mountain factors, and comprises the following specific steps:

1. fibroblast cells (AG03141) and wild-type (WT) fibroblast cells (AG10803) from the skin of Wolner Syndrome (WS) were obtained, which were provided by Coriell Bioreponosity, wherein the WRN gene of WS fibroblast cell (AG03141) had a mutation of nucleotide C at position 2476 to T (2476C > T) and a stop codon generated at position 748, i.e., [ Gln748TER (Q748X) ].

2. Amplifying WS fibroblasts and WT fibroblasts in step 1 in vitro, counting, centrifuging, removing supernatant, and mixing 2 × 10⁶The cells were resuspended in 5ml PBS, centrifuged for 5 minutes at room temperature, 200g,removing supernatant, and respectively treating at the same density of 2 × 10⁶The cells were plated, observed under a microscope (TS100, Nikon) and photographed, and then infected with Sendai virus carrying mountain midkine (OCT4, SOX2, c-MYC and KLF-4) at a virus titer of 1X 10⁸IU/ml, after the third day of transfection, the morphology of the cells was observed, after the 5 th day of transfection, the number of WS fibroblasts and WT fibroblasts was observed, then the cells were re-cultured on trophoblast cells of mouse embryonic fibroblasts, after the 3 rd week of transfection, suspected iPSCs monoclonals were picked for alkaline phosphatase staining and observed, and the density of 4 dishes in total was 4X 10⁵Isolation of ipscs and re-culture of the cells on trophoblast cells of mouse embryonic fibroblasts was performed on individual WS fibroblasts for 28 days.

The observation results are that WS fibroblasts and WT fibroblasts exhibit an aging phenotype after day 3 of Sendai virus transfection, and the microscope pictures of WS fibroblasts and WT fibroblasts before and after 5 days of Sendai virus transfection are shown in FIG. 1, the left picture is the microscope picture of WS fibroblasts and WT fibroblasts before Sendai virus transfection, the right picture is the microscope picture of WS fibroblasts and WT fibroblasts after 5 days of Sendai virus transfection, and it can be seen from the figure that the number of surviving WS fibroblasts is significantly lower than the number of WT fibroblasts after 5 days of transfection; the results of alkaline phosphatase staining of WS fibroblasts and WT fibroblasts after 3 weeks of sendai virus transfection are shown in fig. 2, the left graph is the result of alkaline phosphatase staining of WS fibroblasts, the right graph is the result of alkaline phosphatase staining of WT fibroblasts, it can be seen from fig. 2 that WS fibroblasts hardly produce positive clones, WT fibroblasts produce positive clones, the positive cloning rate of WS fibroblasts and WT fibroblasts after 3 weeks of sendai virus transfection is shown in fig. 3, the positive cloning rate induced by WT fibroblasts is 0.25%, while the positive cloning rate induced by WS fibroblasts is 0%, the positive cloning rate value indicates the total number of alkaline phosphatase-stained iPSCs clones relative to the total number of cultured cells, N is 3 (this experiment is repeated 3 times), p <0.001(P value <0.05 was considered statistically significant). In conclusion, there was no iPSC cloning in WS fibroblasts, and the results from the above experiments repeated twice were consistent.

From the above, it was concluded that iPSCs could not be successfully prepared by inducing WS patient-derived somatic cells such as fibroblasts using only the midkine.

Experimental example 2 investigation of telomerase Activity and expression during reprogramming of WS fibroblasts

This example examined whether the WS cells did not have reprogramming capability due to hTERT-induced or telomerase-activated defects, and included the following steps:

1. detection of telomerase Activity of WS-and WT-fibroblasts during reprogramming

iPSCs (WS fibroblasts and WT fibroblasts were reprogrammed only by using midkine) were prepared by inducing WS fibroblasts and WT fibroblasts according to the method in experimental example 1, and telomerase activities of WS fibroblasts and WT fibroblasts were detected in cells before (day 0), 6, 14 and 21 days after transfection of sendai virus carrying reprogramming factors (midkine) respectively. Telomerase activity was measured using the Telo TAGGG Telomerase PCR ELISA kit (supplied by Roche Bioscience, Palo Alto, Calif., USA).

2. Detection results of telomerase Activity and expression during reprogramming of WS fibroblasts

Analysis results of telomerase activities of WS fibroblasts and WT fibroblasts during reprogramming as shown in fig. 4, WS fibroblasts (0.0017 telomerase activity at day 0, relative activity, OD/μ g protein) and WT fibroblasts (0.0016 telomerase activity at day 0) were slightly deficient compared to telomerase activity during reprogramming. Fold change in telomerase activity during reprogramming of WS and WT fibroblasts as shown in fig. 5, telomerase activity was increased 84-fold in WT fibroblasts and only 3-fold in WS cells during reprogramming at day 6. From the above, it was found that telomerase activity was low in WS fibroblasts during reprogramming. It follows that WS fibroblasts are not able to reprogram due to defects in hTERT induction or telomerase activation.

Experimental example 3 reprogramming was performed after transferring hTERT into WS and WT fibroblasts before reprogramming

The experimental example investigates whether normal reprogramming can be carried out to prepare iPSCs after hTERT is introduced into WS fibroblasts before reprogramming, and the specific steps are as follows:

1. respectively inducing WS fibroblasts (AG03141) and WT fibroblasts (AG10803) to prepare iPSCs according to the method in example 1, wherein hTERT-expressing plasmids are transferred into WS and WT fibroblasts before reprogramming, the hTERT-expressing plasmids are pBABE-puro-hTERT (the gene map is shown in figure 6), the WS fibroblasts transferred into the hTERT-expressing plasmids are named WS-hTERT, and the WT fibroblasts transferred into the hTERT-expressing plasmids are named WT-hTERT. The following observations and tests were performed during reprogramming and iPSCs culture of WS-hTERT and WT-hTERT:

1.1, observing the survival rate of WS-hTERT and WT-hTERT by observing and photographing through a microscope (TS100, Nikon) 5 days after transfecting Sendai virus carrying the mountain midkine (OCT4, SOX2, c-MYC and KLF-4).

1.2 after 3 weeks of transfection of the Sendai virus, suspected iPSCs monoclonals were picked, stained with alkaline phosphatase, observed, and the positive cloning rate was calculated and compared with that of WS fibroblasts and WT fibroblasts in Experimental example 1 after 5 days of transfection of the virus.

1.3, after 3 weeks of transfecting the Sendai virus, selecting suspected iPSCs monoclonals for culture amplification, after enough cells are obtained (because few cells are in the present stage and the identification of the pluripotent marker is not carried out, in the experiment, a plurality of monoclonals are selected for culture amplification in the stage, after enough cells are obtained, 3 clones are selected for further identification in each group), detecting the pluripotent markers (Oct-4, Sox2, Tra-1-81 and SSEA-4) expressed by iPSCs of WS-hTERT and WT-hTERT by an immunofluorescence detection method, and detecting the pluripotent markers Oct-4, Sox2, NANOG, REX-1, ESG1 and DPPA4 expressed by WS and WT iPSCs by an RT-PCR detection kit (provided by Thermo Fisher Scientific). The immunofluorescence detection method is implemented according to the method disclosed in the literature (Li P, Li M, Tang X, Wang S, Zhang YA, Chen Z. Accelerated generation of oligomeric promoter cells from human induced multiplex cells by expressed expression of Sox10and Olig2.science China Life sciences.2016; 59(11):1131-1138.DOI:10.1007/S11427-016-0165-3), the main antibodies used in the implementation process are shown in Table 1 below,

TABLE 1 Primary antibodies used in immunofluorescence detection methods

Name (R)	Directory	Business company	Dilution of
				anti-OCT4	2750	CST	400
anti-TRA-1-60	MAB4360	EMD Millipore						300
				anti-SSEA4	MAB4304	EMD Millipore			100
anti-SOX2	MAB4343	EMD Millipore						1000
				anti-TRA-1-81	14-8883	eBioscience	200
anti-AFP	14-6583	eBioscience	200
				anti-Tuj-1	MAB1637	EMD Millipor		500
anti-WRN	AB200	Abcam							200
				anti-γH2AX	9718	CST	200
anti-RPA32	2208	CST	400
				anti-Rad51	SC-8349	Santa Cruz	400

1.4, performing karyotype analysis on the WS-hTERT and WT-hTERT fibroblast iPSCs cultured and amplified in the step 1.3, wherein the analysis method comprises the following steps: cells were treated with colchicine (supplied by SIGMA corporation) to arrest in the mitotic phase, fixed with ethanol-glacial acetic acid (Beijing Yili chemical) fixative, and subjected to hypotonic treatment and drop-wise strip analysis.

1.5, digesting the WS-hTERT and WT-hTERT fibroblast iPSCs cultured and amplified in the step 1.3 by using Accutase (provided by Thermo Fisher Scientific company), collecting cells, mixing the cells with 100ul of matrigel (provided by BD bioscience company), injecting the mixture into hindlimb muscles of an immunodeficient SCID mouse, observing regularly every week, taking out tumors after the tumors are formed, fixing the tumors by using 4% paraformaldehyde solution, then slicing paraffin, and analyzing the tissue structure by HE staining.

1.6, taking WS-hTERT and WT-hTERT fibroblast iPSCs cultured and amplified in 1.3, detecting WRN expression in the iPSCs of the WS-hTERT and WT-hTERT fibroblasts, and detecting by adopting an immunofluorescence detection method and immunoblotting detection, wherein the immunofluorescence detection method is according to documents (Li P, Li M, Tang X, Wang S, Zhang YA, Chen Z. Acceleration generation of oligonucleotide primers cells from human induced multiplexed primers cells by using forced expression of X10and Olig2.science Life sciences, 2016; 59; 11: 1131-1138.DOI:10.1007/S11427-016 and 0165-3). Immunoblot analysis (Western blot analysis) methods the main antibodies include anti-WRN (supplied by ab200, Abcam, Cambridge, MA, USA) according to the standard methods available.

1.7 detection of telomerase activity in cells was performed before (day 0), 6, 14 and 21 days after transfection of the Sendai virus, respectively. Telomerase activity was measured using the Telo TAGGG Telomerase PCR ELISA kit (supplied by Roche Bioscience, Palo Alto, Calif., USA).

1.8, detecting whether the expression of the hTERT in the cells can prolong telomeres before (day 0) and 6 days after the transfection of the Sendai virus, and simultaneously detecting the length of the telomeres in the cells before (day 0) and 6 days after the transfection of the Ws cells and the WT cells which are not transferred with the hTERT plasmid. The telomerase Q-FISH method in iPSCs was determined using Q-FISH (quantitative fluorescent in situ hybridization) for telomere lengths before and at day 6 after viral transfection according to the procedures disclosed in the literature (Zhang X, Li B, de Jonge N, Bjorkholm, Xu D. the DNA methylation inhibition primers both kinetic and apoptosis of leukamia cells at which is expressed by telomerase over-expression. oncotarget.2015; 6(7): 4888. 4900. DOI: 10.18632/oncotarget.2917). Briefly, different generations of iPSCs were collected and transferred to frozen glass slides that were subjected to cyclic freeze-thawing with liquid nitrogen, then incubated for 10min in 0.1N (0.1N corresponds to 0.1mol/L) HCL solution, PNA-telomere probes (supplied by anageee inc., Daejeon, Korea) were added to the above incubation solution, and then high resolution images were obtained using successive laser scans at 488nm with Leica Confocal TCS SP5 and collected and analyzed.

1.9, in order to detect whether the telomere continues to extend in the iPSC period, WS-hTERT, WT-hTERT and WT iPSCs (iPSCs in 4 weeks after transfection of the virus) are taken for subculture, the telomere length of different passage numbers (P5, P10, P20, P30 and P40) is determined by using Flow-FISH, and the iPSC passage method is referred to in the literature (Wang S, Differentiation of human induced plotent cells to physical functional purified peptide nerves).

SciRep.

2015Mar 18; 5:9232.). The Flow-FISH method for determination of telomerase Q-FISH in iPSCs was carried out according to the method disclosed in the literature (Roos G, Hultdin M. Flow cytometry of molecular length. cytometry.2001; 45(1):79-80.), the Flow cytometer used in the experiments was supplied by BD Bioscience, machine model Calibur II, Flow Jo. software for analysis.

2. Observation and test results

2.1 cell viability before and 5 days after transfection of WS-hTERT and WT-hTERT

The survival rate of cells before and after transfection of WS-hTERT and WT-hTERT at day 5 is shown in FIG. 7, the left graph shows the survival rate before transfection of Sendai virus, and the right graph shows the survival rate at day 5 after transfection of Sendai virus, and it can be seen that WS-hTERT fibroblasts show better survival rate at day 5 after transfection with the expression of hTERT.

2.2 Positive cloning efficiency of alkaline phosphatase staining at week 3 after transfection of WS-hTERT and WT-hTERT

The positive cloning rate of alkaline phosphatase staining at 3 weeks after WS-hTERT and WT-hTERT transfection is shown in fig. 8, and although the positive cloning rate of WS-hTERT was lower than WT-hTERT, it can also be seen therefrom that the alkaline phosphatase staining positive iPSC clones of WS-hTERT cells were higher than those of WS fibroblasts, and that of WT-hTERT cells were significantly higher than those of WT fibroblasts, the positive cloning rate values indicating that the alkaline phosphatase staining positive iPSCs clones were equal to 3 (experimental replicates 3) relative to the total number of cultured cells, and P <0.001(P value <0.05 is considered to be statistically significant).

2.3 pluripotent markers for iPSCs expression of WS-hTERT and WT-hTERT

The pluripotent markers Oct-4, Sox2, Tra-1-81 and SSEA-4 expressed by iPSCs of WS-hTERT and WT-hTERT are detected by an immunofluorescence detection method, and the result is shown in figure 9, which shows that the pluripotent markers Oct-4, Sox2, Tra-1-81 and SSEA-4 are expressed in iPSCs of WS-hTERT. The results of detecting pluripotent markers Oct-4, Sox2, NANOG, REX-1, ESG1 and DPPA4 expressed by iPSCs of WS-hTERT and WT-hTERT by RT-PCR are shown in FIG. 10, and show that iPSCs of WS-hTERT and WT-hTERT can express pluripotent markers Oct-4, Sox2, NANOG, REX-1, ESG1 and DPPA 4.

2.4 karyotype of iPSCs for WS-hTERT and WT-hTERT

The iPSC line karyotype detection results of WS-hTERT and WT-hTERT fibroblasts are shown in FIG. 11-FIG. 12, FIG. 11 is the iPSC line karyotype detection result of WS-hTERT, FIG. 12 is the iPSC line karyotype detection result of WT-hTERT, and the comparison shows that the iPSC lines of WS-hTERT and WT-hTERT fibroblasts all show normal karyotypes.

2.5 differentiation of iPSCs for WS-hTERT and WT-hTERT

The differentiation results of iPSCs of WS-hTERT and WT-hTERT are shown in FIGS. 13-14, and it is known that iPSCs of WS-hTERT and WT-hTERT fibroblasts can form teratomas and can differentiate into 3 kinds of germ layer cells including endoderm (AFP positive), mesoderm (alpha-SMA positive) and ectoderm (Tuj-1 positive) cells when injected into immunodeficient SCID mice.

2.6 WRN expression of iPSCs for WS-hTERT and WT-hTERT

WRN expression immunofluorescence staining results of iPSCs obtained after transfection of Sendai virus by WS-hTERT and WT-hTERT for week 4 are shown in FIG. 15, and WRN expression immunoblotting results of iPSCs obtained after transfection of Sendai virus by WS-hTERT and WT-hTERT for week 4 are shown in FIG. 16.

2.7 detection of telomerase Activity at different time points during reprogramming of WS-hTERT and WT-hTERT

The results of analysis of telomerase activities at different time points during reprogramming of WS-hTERT and WT-hTERT are shown in FIG. 17 (the histograms of both WS-hTERT and WT-hTERT in FIG. 17 are from left to right for

days

0, 6, 14 and 21), and the results of fold change of telomerase activities at different time points during reprogramming of WS-hTERT and WT-hTERT are shown in FIG. 18 (the histograms of both WS-hTERT and WT-hTERT in FIG. 18 are from left to right for

days

0, 6, 14 and 21), and it is known from FIGS. 17-18 that the telomerase activities in both WS-hTERT and WT-hTERT fibroblasts are maintained at high levels during reprogramming.

2.8 telomere length in cells of WS-hTERT and WT-hTERT before and on day 6 after Sendai virus transfection

The telomere lengths of WS, WT, WS-hTERT and WT-hTERT in the cells before and 6 days after Sendai virus transfection are shown in FIGS. 19, 20, 21 and 22 (the abscissa of FIGS. 19 to 22 is relative fluorescence), respectively, the left graph in the above 4 figures is the telomere length of the cells before Sendai virus transfection, the right graph is the telomere length of the cells 6 days after Sendai virus transfection, as can be seen from FIGS. 19 to 20, on day 0 (before infection), 0.25% of WS fibroblasts showed telomere lengths longer than 1000 units (the unit is relative fluorescence intensity of the abscissa, the relative fluorescence intensity is used as an index of relative telomere length in this test), 2.88% of the WS fibroblasts showed telomere lengths of 500-1000 units, and less than that of WT fibroblasts (the length of 0.38% of WT >1000 units; the WT length of 4.51% of WT 500-1000 units M). Stable expression of hTERT can increase the proportion of fibroblasts with longer telomeres, such as in WS-hTERT cells (0.98%, > 1000; 7.94%, 500-. The average telomere length was also increased in total cells due to hTERT expression, as shown in figure 24, WS, from 79-92.4; WS-hTERT, from 103.2-161.9; WT, 768 "-116.6; WT-hTERT from 123.5-179.

The proportion of cells with longer telomeres increased during 6 days of reprogramming as shown in FIGS. 19-22, however, the proportion of longer telomeres in WS cells (2.45%, > 1000; 4.92%, 500-.

2.9 relative telomere lengths of different generations of WS-hTERT, WT-hTERT and WT iPSCs

The relative telomere lengths of different generations of WS-hTERT, WT-hTERT and iPSCs of WT are shown in FIG. 25 (Fib in FIG. 25 indicates fibroblasts, the bar graphs are WS-hTERT, WT-hTERT and WT from left to right, and the P5 and P40 generations have no relative telomere length of WT), and the value of the relative telomere length represents the fluorescence intensity of a telomere DNA specific probe, 4X 10 for each analysis⁴The number of the iPSCs is one,repeating for 4 times, it can be seen that the relative length of telomeres of iPSCs was extended to a similar level at the 5 th generation of iPSCs and remained stable up to the 40 th generation despite the difference in telomere length of fibroblast cell lines.

3. The results show that the activity of telomerase can enable WS fibroblasts to carry out normal reprogramming, namely, after an hTERT expression plasmid is introduced into the WS fibroblasts before reprogramming, iPSCs can be prepared by normal reprogramming. The results also show that iPSCs (WS-hTERT iPSCs) prepared by normal reprogramming after introducing the hTERT expression plasmid into the WS fibroblasts before reprogramming have the multipotentiality characteristic, and the expression multipotential markers and the differentiation capacity are equivalent to those of WT-iPSCs or WT-hTERT iPSCs (iPSCs prepared by normal reprogramming after introducing the hTERT expression plasmid into the WT fibroblasts before reprogramming).

Experimental example 4 overexpression of hTERT is a necessary condition for the smooth progress of cell cycle of WS-derived iPSCs

This experimental example investigated whether exogenous hTERT expression is a necessary condition for cell cycle progression of iPSCs derived from WS, since exogenous hTERT expression may mask defects caused by WRN mutations in WS fibroblasts, the following experiments were performed for this purpose:

1.1 this example used a doxycycline hydrochloride (Dox) -inducible system (supplied by Clontech) to generate iPSCs. And (3) taking fibroblasts (AG03141) and Wild Type (WT) fibroblasts (AG10803) of the skin of a Wolner Syndrome (WS) patient to prepare WS-ihTERT iPSCs and WT-ihTERT iPSCs respectively according to the method in the example 2. The WS-ihTERT iPSCs and WT-ihTERT iPSCs obtained above were subjected to amplification culture according to the amplification culture procedure in example 2, and the amplification culture was performed in a ratio of 1:6, and determining Telomerase activities expressed by WS-ihTERT iPSCs and WT-ihTERT iPSCs when cultured to generation 10 (P10), wherein DOX is not added to the culture medium when generation 10 is cultured, and the Telomerase activities are determined using Telo TAGGG Telomerase PCR ELISA kit (supplied by Roche Bioscience, Palo Alto, Calif., USA).

1.2, in order to further detect the proliferation capacity of WS-ihTERT iPSCs and WT-ihTERT iPSCs, the WS-ihTERT iPSCs, WT-iTERT iPSCs and WT iPSCs (prepared in experimental example 1) obtained in 1.1 are subjected to subculture for about 10 th cells, BRDU (bromodeoxyuridine) is released for 2 or 4 hours in a pulsed manner, the WS-ihTERT iPSCs and WT-ihTERT iPSCs are cultured in a medium containing DOX and a medium not containing DOX respectively, the cells which are BRDU-positive are counted, and the proportion of the BRDU-positive cells in the total number of the cells is calculated.

1.3 WS-ihTERT iPSCs and WT-ihTERT iPSCs (the iPSCs mentioned above are cells around 10 th band after passage) obtained in 1.1 were cultured at the same density on the first day, and cultured to day 3, the cells were treated by 200ng/ml nocodazole (provided by Sigma-Aldrich, St. Louis, MO, USA) for 16h, so that the cells were kept synchronized at the G2/M stage, and then the cells were cultured in iPSCs medium for 5 hours (at this culture stage, iPSCs were started to enter S-phase), the cells were collected at 2 hour intervals and fixed with 70% ethanol, and in order to induce DNA replication stress at S-phase, the cells were treated by 250nM CPT (Sigma-Aldrich), then stained with ammonium iodide (PI) and analyzed by flow cytometry (BD).

2. Observation and test results

2.1 expression of hTERT in WS-ihTERT iPSCs and WT-ihTERT iPSCs at passage 10

The expression of hTERT in WS-ihTERT iPSCs and WT-ihTERT iPSCs at the 10 th generation is shown in FIG. 27 (in FIG. 27, the + and the-respectively indicate DOX + (in the culture medium containing DOX), DOX- (in the culture medium without DOX), and the ordinate indicates hTERT expression), and it can be seen that when the cells are cultured in the culture medium without DOX at the 10 th generation, the expression of hTERT in WS-ihTERT iPSCs and WT-iTERT iPSCs is remarkably reduced, and WS-iTERT iPSCs express lower endogenous hTERT mRNA and are lower than WT-iTERT iPSCs 1/3. During the 10 th generation culture, WS-ihTERT iPSCs were observed to exhibit lower anchorage rates and longer proliferative replication times than WT-ihTERT iPSCs. WT-ihTERT iPSCs are usually passaged at a 1:6 ratio and then become fused within 4-5 days, as opposed to WS-ihTERT iPSCs which must be subcultured at a 1:3-4 ratio and doubled in average 7 days.

2.2 proliferative Capacity of WS-ihTERT iPSCs and WT-iTERT iPSCs

The proliferation potency of WS-ihTERT iPSCs and WT-ihTERT iPSCs is shown in FIG. 28 (in FIG. 28, the abscissa of DOX + indicates the culture in the medium containing DOX, and the ordinate indicates the proportion of BRDU-positive cells in the medium without DOX), and the proportion of WT-ihTERT iPSCs is equivalent to that of WT-iPSCs (derived from fibroblasts that do not express abnormal hTERT) and BRDU-positive cells obtained from Dox-treated WS-ihTERT iPSCs regardless of the addition of DOX. However, WS-ihTERT iPSCs lacking Dox obtained significantly lower proportion of BRDU-positive cells than the other groups.

2.3 cell cycle progression of WS-ihTERT iPSCs and WT-ihTERT iPSCs

The cell cycle progression analysis of WS-ihTERT iPSCs and WT-ihTERT iPSCs is shown in FIGS. 29-31, when the cells are transformed into normal iPSC culture medium after 5 hours, the proportion (23 +/-6%) of the cells in the G2/M phase in the WT-ihTERT iPSCs group is higher than that (17 +/-7%) in the WS-ihTERT iPSCs group, and at 7 hours, 62 +/-5% of the WT-ihTERT iPSCs enter the S phase, however, only 40 +/-4% of the WS-ihTERT iPSCs enter the S phase, and therefore, the WS-ihTERT iPSCs enter the S phase at a slower speed.

3. The results show that when the exogenous hTERT expression is closed, the cell passage cycle of the WS group iPSCs is obviously prolonged, and the cell proliferation speed is reduced. Further experiments found that Werner' S group iPSCs (WS-ihTERT) cells entered S phase significantly later than wild type (WT-ihTERT) cells when the cell cycle was synchronized by Nocodozal after G2/M phase. From the above experimental results, it can be concluded that there is cell cycle abnormality in WS-iPSCs, and after synchronization of G2/M phase, the proportion of cells entering S phase at the same time point is significantly lower than that of WT-iPSCs, suggesting that the cell proliferation of WS-iPSCs has correlation with the expression of exogenous hTERT.

Experimental example 5 sensitivity of WS iPSCs to DNA damaging Agents

The experimental example investigates the response of WS-iPSCs and WT-iPSCs to different stressors. 4 stressors are adopted to be applied to WS-iPSCs and WT-iPSCs cells. The 4 stressors are Camptothecin (CPT), bleomycin, a protein transport inhibitor (Brefeldin a) and hydrogen peroxide, respectively. Camptothecin (CPT), a cytotoxic quinoline alkaloid, inhibits DNA topoisomerase I. CPT induces replication arrest and disrupts DNA in a concentration-dependent manner. Low doses (25-100nM) of CPT induce replication fork slowing and reversion such that double-stranded DNA breaks do not reach detectable levels, and higher concentrations (> 100nM) of CPT induce DNA fragmentation (Ray Chaudhuri A, Hashimoto Y, Herrador R, et al.Topoisomere I position damages in PARP-mediated replication for k replication. Nat Structure biol. 2012; 19(4):417-423.DOI: 10.1038/nsmb.2258; Berti M, Ray Chaudhuri A, Thangvel S, et al.Human RECQ1 proteins restart of replication for replicated by DNA ligation I position interruption. Nat. Biol.2013; 20-3. I: 347/10.1038). Bleomycin, another anticancer drug, can induce DNA strand breaks. Protein transport inhibitors (Brefeldin a) inhibit protein transport from the endoplasmic reticulum into the golgi apparatus while exhibiting cytotoxicity to certain cancer cell lines. Hydrogen peroxide is a reactive oxygen species that can readily participate in reactions and disrupt important cellular processes or components, such as DNA synthesis and mitochondrial function.

Different iPSCs groups are set, and WS-ihTERT +, WS-ihTERT-, WT-ihTERT +, WT-ihTERT-, WT (fibroblast AG10803, used as a control group) are sequentially arranged, the WS-ihTERT +, WS-ihTERT-, WT-ihTERT +, WT-ihTERT-is prepared and obtained by taking the WS-ihTERT + and WT-ihTERT + as experimental groups and from experimental example 4, the WS-ihTERT + and WT-ihTERT + are treated by DOX, and the WS-ihTERT-and WT-ihTERT-are not treated by DOX. The proportion of the different iPSCs groups subjected to individual stressors and apoptosis (annexin V +) cells was determined by flow cytometry. The 4 groups of cells were cultured in a feeder-free system, human embryonic stem cell medium mTeSR.

1.1, the 4 groups of cells are respectively treated by the 4 stressors: when the CPT treatment is used, the following procedure is followed: CPT treatment at 250nM (dosing treatment when iPSCs grow to 70-80% confluence, adding CPT at 250nM to the experimental group culture medium and PBS to the control group), and 5% CO at 37 deg.C₂Culturing for 1 hour; when treated with bleomycin, according toThe method comprises the following steps: adding medicine when iPSCs grow to 70-80% confluence, adding 50 μ M bleomycin into experimental group culture medium, adding PBS into control group, and adding 5% CO at 37 deg.C₂Culturing for 1 hour; when Brefeldin a treatment is used, the following procedure is followed: 50 μ M Brefeldin A treatment (dosing when iPSCs grow to 70-80% confluency, 50 μ M Brefeldin A in the experimental group medium and PBS in the control group) at 37 deg.C with 5% CO₂Culturing for 5 min; when using H₂O₂During treatment, the following method is adopted: 500 μ M H₂O₂Treating (adding medicine when iPSCs grow to 70-80% confluence, adding 500 μ M H into experimental group culture medium₂O₂PBS was added to the control group) at 37 ℃ in 5% CO₂And culturing for 5 min. Meanwhile, a normal saline group is set, the cells of the normal saline group are not treated by the 4 stress sources, and the division process of the cells reaches the standard form of apoptosis. Cells from each of the above groups were stained with fluorescein isothiocyanate (Annexin V-FITC) and PI according to Annexin V-FITC/PI Apoptosis Detection Kit.) the labeled cells were analyzed using flow cytometry (BD, Callibur II).

1.2 WS-ihTERT-and WT-ihTERT-iPSCs were cultured at the same density on day one and up to day 3, and the cells were treated by 200ng/ml nocodazole (Nocardiazole) (supplied by Sigma-Aldrich, St. Louis, MO, USA) (by adding 200ng/ml nocodazole to the culture medium when iPSCs were grown to 70-80% confluency) at 37 ℃ with 5% CO₂And culturing for 16h so that the cells are synchronized in the G2/M phase, then transferring the cells to iPSCs culture medium for 5 hours (in this culture phase, iPSCs begin to enter S-phase), collecting the cells every 2 hours and fixing with 70% ethanol, treating a part of the cells with 250nM CPT (Sigma-Aldrich) in order to induce DNA replication stress in S-phase, and removing nocodazole after 13 hours, i.e., replacing normal culture medium, without adding nocodazole. Staining with ammonium iodide (PI) and analysis with flow cytometer ((BD, calilibur II)).

1.3 high doses of CPT in the S phase of WS iPSCs lead to DNA double strand breaks. For detection of DNA double strand breaks, cells were stained for γ H2AX foci (DNA double strand break molecular marker). Method following the implementation in step 1.2, after culturing iPSCs of WS-ihTERT-and WT-ihTERT-in iPSCs medium for 5 hours (iPSCs begin to enter S-phase at this stage of culture), a portion of the cells were treated with 1. mu.M CPT (Sigma-Aldrich) for 1 hour, and a portion were not treated with CPT, and then γ H2AX foci (molecular marker for double strand break DNA) of the cells were stained with ammonium iodide (PI), and observed and photographed under a microscope while γ H2AX foci were detected by the blotting method, and RPA32 and RAD51 in the cells were detected by the immunostaining method and Western blotting method.

2 the results are as follows

2.1 test results of the 4 stressor treatments

The cell ratios of apoptotic iPSCs treated with CPT are shown in fig. 32 (the horizontal coordinates in fig. 32 represent Saline (salt) and CPT-treated groups, respectively, and the histograms in the two groups represent WS ihert +, WS ihert-, WT ihert +, WT-ihert-, WT, and WT, respectively, in order from left to right, respectively, and the cell ratios of apoptotic iPSCs treated with different stressors are shown in fig. 33 (the horizontal coordinates in fig. 33 represent Saline (salt), CPT-treated, Bleomycin (Bleomycin) treated, Brefeldin a treated, and H₂O₂The treatment group, the bar graphs in the 5 groups respectively show WS ihTERT +, WS ihTERT-, WT ihTERT +, WS-ihTERT-, WT from left to right in sequence, only CPT shows different effects on WS iPSCs and WT iPSCs in the 4 stressors, and after CPT treatment, 42.9 +/-3.7% of WS-ihTERT + (containing Dox) and 32.9 +/-4.8% of WS-ihTERT- (not containing Dox) iPSCs show positive for annexin, which is obviously higher than that of other groups (14.7 +/-2.5% of WT-ihTERT +, 18.0 +/-2.1% of WT-ihTERT-, and 10.2 +/-0.7% of WT.

The data above indicate that iPSCs with WRN loss of function are more sensitive to CPT-induced apoptosis.

2.2 cell cycle analysis of iPSCs for WS-ihTERT-and WT-ihTERT-

Cell cycle results of WS-ihTERT-and WT-ihTERT-iPSCs with or without CPT treatment are shown in FIGS. 34-35, FIG. 34 is an analysis of the cell cycle of WS-ihTERT-and WT-ihTERT-iPSCs at different time points, FIG. 35 is an analysis of the cell cycle of WS-ihTERT-and WT-ihTERT-iPSCs with or without CPT after synchronization with nocodazole treatment, FIG. 35 is a cell cycle analysis of nocodal + without CPT treatment, CPT-without CPT treatment, FIG. 35(a) is a cell cycle analysis of WS-iTERT-iSCs without CPT treatment after synchronization with nocodazole treatment, FIG. 35(b) is a cell cycle analysis of WS-iSCT-after synchronization with nocodazole treatment, and FIG. 35 is a cell cycle analysis of CPT-iTERT-treated iSCs with CPT after synchronization with nocodazole treatment, and FIG. 35(b) is a cell cycle analysis of WS-iTERT-iSTES with CPT-and WT-iTERT-with or WT-iSCs without CPT treatment Cell cycle analysis under treatment, FIG. 35(d) is cell cycle analysis under CPT treatment of WT-ihTERT-iPSCs after synchronization of Nokodazole treatment, and the abscissa in FIG. 35 is the number of cells and the ordinate is the fluorescence intensity. The ratio of cells of WS-ihTERT-and WT-ihTERT-treated iPSCs with or without CPT in S phase is shown in FIG. 36 (in FIG. 36, WS represents WS-ihTERT without CPT treatment, WS-CPT represents WS-ihTERT with CPT treatment, the same WT represents WT-ihTERT without CPT treatment, WT-CPT represents WT-ihTERT with CPT treatment), and after 13 hours nocodal was removed, more than 60% of the iPSCs with CPT-treated WS-ihTERT were in S phase, which was much higher than the other 3 groups, thus suggesting that CPT may be the S phase capture of iPSCs in WS-ihTERT-.

2.3 DNA double strand break detection after high dose CPT treatment of iPSCs of WS-ihTERT-and WT-ihTERT-

The results of the double strand break detection of WS-ihTERT-and WT-ihTERT-iPSCs in high dose CPT or without CPT are shown in FIG. 37 (the left panel in FIG. 37 is the result of the double strand break detection of the CPT-untreated WS-and WT-ihTERT-iPSCs, and the right panel is the result of the double strand break detection of 1 μ M CPT-treated WS-and WT-ihTERT-iPSCs), and the ratio of gamma H2AX foci in the cells of the CPT-and WT-ihTERT-iPSCs after high dose CPT is shown in FIG. 38, which shows that the CPT-treated iWS-has a higher ratio of gamma H2AX foci relative to the CPT-treated WT-iTERT-iSCs. The results, which also yielded approximations by using Western blotting assays, are shown in FIGS. 39 and 40. Taken together, γ H2AX expression was higher in WS cells than in WT.

This example examined the binding of proteins by the recombination mechanism of RPA32 and Rad51, two single stranded DNAs (ssDNA), in DSBs (double strand breaks in DNA) and arrested branches. The distribution patterns of RPA32 and Rad51 in cells after CPT treatment of WS-ihTERT-and WT-ihTERT-iPSCs are shown in FIG. 41 (WS in FIG. 41 represents WS-ihTERT-, WT represents WT-ihTERT-); in contrast, RPA32 and Rad51 in iPSCs of WS-ihTERT after CPT treatment were phosphorylated in the nuclear DNA lesion area and immunofluorescent staining was shown as a highly exposed focal spot (Distingtive focus) in the nucleus, as shown in FIGS. 42 and 43. The RPA32 antibody could exhibit total endogenous levels of RPA32, which is not unlike WS and WS-ihhtert-iPSCs. However, the reason why western blot showed two distinct RPA32 bands in WS-ihTERT-iPSCs but not two distinct RPA32 bands in WT-ihTERT iPSCs after CPT treatment (as shown in FIG. 44) is not clear. Previous reports showed that cleavage of Rad51(DNA double strand break repair protein) into 31-kDa proteins occurred in apoptotic cells, with two Rad51 bands detected in WS-ihTERT's iPSCs, but not WT-ihTERT's iPSCs, as shown in FIG. 45.

From the above results, it is known that the sensitivity of iPSCs to DNA damaging drug CPT is increased and DNA repair ability is impaired due to WRN mutation. The induced pluripotent stem cells prepared by the method can be used for drug screening.

It should be understood that the above-described embodiments are merely examples for clarity of description and are not intended to limit the scope of the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This list is neither intended to be exhaustive nor exhaustive. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

<110> Xuanwu hospital of capital medical university

<120> cell model, preparation method and application thereof

<130> HA201501044

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3399

<212> DNA

<213> Artificial Synthesis (hTERT)

<400> 1

atgccgcgcg ctccccgctg ccgagccgtg cgctccctgc tgcgcagcca ctaccgcgag 60

gtgctgccgc tggccacgtt cgtgcggcgc ctggggcccc agggctggcg gctggtgcag 120

cgcggggacc cggcggcttt ccgcgcgctg gtggcccagt gcctggtgtg cgtgccctgg 180

gacgcacggc cgccccccgc cgccccctcc ttccgccagg tgtcctgcct gaaggagctg 240

gtggcccgag tgctgcagag gctgtgcgag cgcggcgcga agaacgtgct ggccttcggc 300

ttcgcgctgc tggacggggc ccgcgggggc ccccccgagg ccttcaccac cagcgtgcgc 360

agctacctgc ccaacacggt gaccgacgca ctgcggggga gcggggcgtg ggggctgctg 420

ctgcgccgcg tgggcgacga cgtgctggtt cacctgctgg cacgctgcgc gctctttgtg 480

ctggtggctc ccagctgcgc ctaccaggtg tgcgggccgc cgctgtacca gctcggcgct 540

gccactcagg cccggccccc gccacacgct agtggacccc gaaggcgtct gggatgcgaa 600

cgggcctgga accatagcgt cagggaggcc ggggtccccc tgggcctgcc agccccgggt 660

gcgaggaggc gcgggggcag tgccagccga agtctgccgt tgcccaagag gcccaggcgt 720

ggcgctgccc ctgagccgga gcggacgccc gttgggcagg ggtcctgggc ccacccgggc 780

aggacgcgtg gaccgagtga ccgtggtttc tgtgtggtgt cacctgccag acccgccgaa 840

gaagccacct ctttggaggg tgcgctctct ggcacgcgcc actcccaccc atccgtgggc 900

cgccagcacc acgcgggccc cccatccaca tcgcggccac cacgtccctg ggacacgcct 960

tgtcccccgg tgtacgccga gaccaagcac ttcctctact cctcaggcga caaggagcag 1020

ctgcggccct ccttcctact cagctctctg aggcccagcc tgactggcgc tcggaggctc 1080

gtggagacca tctttctggg ttccaggccc tggatgccag ggactccccg caggttgccc 1140

cgcctgcccc agcgctactg gcaaatgcgg cccctgtttc tggagctgct tgggaaccac 1200

gcgcagtgcc cctacggggt gctcctcaag acgcactgcc cgctgcgagc tgcggtcacc 1260

ccagcagccg gtgtctgtgc ccgggagaag ccccagggct ctgtggcggc ccccgaggag 1320

gaggacacag acccccgtcg cctggtgcag ctgctccgcc agcacagcag cccctggcag 1380

gtgtacggct tcgtgcgggc ctgcctgcgc cggctggtgc ccccaggcct ctggggctcc 1440

aggcacaacg aacgccgctt cctcaggaac accaagaagt tcatctccct ggggaagcat 1500

gccaagctct cgctgcagga gctgacgtgg aagatgagcg tgcgggactg cgcttggctg 1560

cgcaggagcc caggggttgg ctgtgttccg gccgcagagc accgtctgcg tgaggagatc 1620

ctggccaagt tcctgcactg gctgatgagt gtgtacgtcg tcgagctgct caggtctttc 1680

ttttatgtca cggagaccac gtttcaaaag aacaggctct ttttctaccg gaagagtgtc 1740

tggagcaagt tgcaaagcat tggaatcaga cagcacttga agagggtgca gctgcgggag 1800

ctgtcggaag cagaggtcag gcagcatcgg gaagccaggc ccgccctgct gacgtccaga 1860

ctccgcttca tccccaagcc tgacgggctg cggccgattg tgaacatgga ctacgtcgtg 1920

ggagccagaa cgttccgcag agaaaagagg gccgagcgtc tcacctcgag ggtgaaggca 1980

ctgttcagcg tgctcaacta cgagcgggcg cggcgccccg gcctcctggg cgcctctgtg 2040

ctgggcctgg acgatatcca cagggcctgg cgcaccttcg tgctgcgtgt gcgggcccag 2100

gacccgccgc ctgagctgta ctttgtcaag gtggatgtga cgggcgcgta cgacaccatc 2160

ccccaggaca ggctcacgga ggtcatcgcc agcatcatca aaccccagaa cacgtactgc 2220

gtgcgtcggt atgccgtggt ccagaaggcc gcccatgggc acgtccgcaa ggccttcaag 2280

agccacgtct ctaccttgac agacctccag ccgtacatgc gacagttcgt ggctcacctg 2340

caggagacca gcccgctgag ggatgccgtc gtcatcgagc agagctcctc cctgaatgag 2400

gccagcagtg gcctcttcga cgtcttccta cgcttcatgt gccaccacgc cgtgcgcatc 2460

aggggcaagt cctacgtcca gtgccagggg atcccgcagg gctccatcct ctccacgctg 2520

ctctgcagcc tgtgctacgg cgacatggag aacaagctgt ttgcggggat tcggcgggac 2580

gggctgctcc tgcgtttggt ggatgatttc ttgttggtga cacctcacct cacccacgcg 2640

aaaaccttcc tcaggaccct ggtccgaggt gtccctgagt atggctgcgt ggtgaacttg 2700

cggaagacag tggtgaactt ccctgtagaa gacgaggccc tgggtggcac ggcttttgtt 2760

cagatgccgg cccacggcct attcccctgg tgcggcctgc tgctggatac ccggaccctg 2820

gaggtgcaga gcgactactc cagctatgcc cggacctcca tcagagccag tctcaccttc 2880

aaccgcggct tcaaggctgg gaggaacatg cgtcgcaaac tctttggggt cttgcggctg 2940

aagtgtcaca gcctgtttct ggatttgcag gtgaacagcc tccagacggt gtgcaccaac 3000

atctacaaga tcctcctgct gcaggcgtac aggtttcacg catgtgtgct gcagctccca 3060

tttcatcagc aagtttggaa gaaccccaca tttttcctgc gcgtcatctc tgacacggcc 3120

tccctctgct actccatcct gaaagccaag aacgcaggga tgtcgctggg ggccaagggc 3180

gccgccggcc ctctgccctc cgaggccgtg cagtggctgt gccaccaagc attcctgctc 3240

aagctgactc gacaccgtgt cacctacgtg ccactcctgg ggtcactcag gacagcccag 3300

acgcagctga gtcggaagct cccggggacg acgctgactg ccctggaggc cgcagccaac 3360

ccggcactgc cctcagactt caagaccatc ctggactga 3399

<210> 2

<211> 23

<212> DNA

<213> Artificial Synthesis (F)

<400> 2

gcatacagct ccatccgcac atg 23

<210> 3

<211> 23

<212> DNA

<213> Artificial Synthesis (R)

<400> 3

cgacccgtga accgcgatgt gtt 23

<210> 4

<211> 11165

<212> DNA

<213> plasmid pLVX-light-hTERT-Puro (pLVX-light-hTERT-Puro)

<400> 4

tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60

cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120

tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180

ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240

agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300

agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360

ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420

cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480

gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540

tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600

agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660

cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720

caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780

aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840

aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900

caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960

gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020

cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080

ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140

aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200

tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260

aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320

gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380

cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440

gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500

ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560

actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620

gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680

aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740

ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800

tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860

actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920

cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980

cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040

gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100

aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160

acagcagaga tccagtttat cgatgaggcc ctttcgtctt cactcgagtt tactccctat 2220

cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac gatgtcgagt 2280

ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa 2340

cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt atccctatca 2400

gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgaggt 2460

aggcgtgtac ggtgggaggc ctatataagc agagctcgtt tagtgaaccg tcagatcgcc 2520

tggagaagga tccgcggccg catgccgcgc gctccccgct gccgagccgt gcgctccctg 2580

ctgcgcagcc actaccgcga ggtgctgccg ctggccacgt tcgtgcggcg cctggggccc 2640

cagggctggc ggctggtgca gcgcggggac ccggcggctt tccgcgcgct ggtggcccag 2700

tgcctggtgt gcgtgccctg ggacgcacgg ccgccccccg ccgccccctc cttccgccag 2760

gtgtcctgcc tgaaggagct ggtggcccga gtgctgcaga ggctgtgcga gcgcggcgcg 2820

aagaacgtgc tggccttcgg cttcgcgctg ctggacgggg cccgcggggg cccccccgag 2880

gccttcacca ccagcgtgcg cagctacctg cccaacacgg tgaccgacgc actgcggggg 2940

agcggggcgt gggggctgct gctgcgccgc gtgggcgacg acgtgctggt tcacctgctg 3000

gcacgctgcg cgctctttgt gctggtggct cccagctgcg cctaccaggt gtgcgggccg 3060

ccgctgtacc agctcggcgc tgccactcag gcccggcccc cgccacacgc tagtggaccc 3120

cgaaggcgtc tgggatgcga acgggcctgg aaccatagcg tcagggaggc cggggtcccc 3180

ctgggcctgc cagccccggg tgcgaggagg cgcgggggca gtgccagccg aagtctgccg 3240

ttgcccaaga ggcccaggcg tggcgctgcc cctgagccgg agcggacgcc cgttgggcag 3300

gggtcctggg cccacccggg caggacgcgt ggaccgagtg accgtggttt ctgtgtggtg 3360

tcacctgcca gacccgccga agaagccacc tctttggagg gtgcgctctc tggcacgcgc 3420

cactcccacc catccgtggg ccgccagcac cacgcgggcc ccccatccac atcgcggcca 3480

ccacgtccct gggacacgcc ttgtcccccg gtgtacgccg agaccaagca cttcctctac 3540

tcctcaggcg acaaggagca gctgcggccc tccttcctac tcagctctct gaggcccagc 3600

ctgactggcg ctcggaggct cgtggagacc atctttctgg gttccaggcc ctggatgcca 3660

gggactcccc gcaggttgcc ccgcctgccc cagcgctact ggcaaatgcg gcccctgttt 3720

ctggagctgc ttgggaacca cgcgcagtgc ccctacgggg tgctcctcaa gacgcactgc 3780

ccgctgcgag ctgcggtcac cccagcagcc ggtgtctgtg cccgggagaa gccccagggc 3840

tctgtggcgg cccccgagga ggaggacaca gacccccgtc gcctggtgca gctgctccgc 3900

cagcacagca gcccctggca ggtgtacggc ttcgtgcggg cctgcctgcg ccggctggtg 3960

cccccaggcc tctggggctc caggcacaac gaacgccgct tcctcaggaa caccaagaag 4020

ttcatctccc tggggaagca tgccaagctc tcgctgcagg agctgacgtg gaagatgagc 4080

gtgcgggact gcgcttggct gcgcaggagc ccaggggttg gctgtgttcc ggccgcagag 4140

caccgtctgc gtgaggagat cctggccaag ttcctgcact ggctgatgag tgtgtacgtc 4200

gtcgagctgc tcaggtcttt cttttatgtc acggagacca cgtttcaaaa gaacaggctc 4260

tttttctacc ggaagagtgt ctggagcaag ttgcaaagca ttggaatcag acagcacttg 4320

aagagggtgc agctgcggga gctgtcggaa gcagaggtca ggcagcatcg ggaagccagg 4380

cccgccctgc tgacgtccag actccgcttc atccccaagc ctgacgggct gcggccgatt 4440

gtgaacatgg actacgtcgt gggagccaga acgttccgca gagaaaagag ggccgagcgt 4500

ctcacctcga gggtgaaggc actgttcagc gtgctcaact acgagcgggc gcggcgcccc 4560

ggcctcctgg gcgcctctgt gctgggcctg gacgatatcc acagggcctg gcgcaccttc 4620

gtgctgcgtg tgcgggccca ggacccgccg cctgagctgt actttgtcaa ggtggatgtg 4680

acgggcgcgt acgacaccat cccccaggac aggctcacgg aggtcatcgc cagcatcatc 4740

aaaccccaga acacgtactg cgtgcgtcgg tatgccgtgg tccagaaggc cgcccatggg 4800

cacgtccgca aggccttcaa gagccacgtc tctaccttga cagacctcca gccgtacatg 4860

cgacagttcg tggctcacct gcaggagacc agcccgctga gggatgccgt cgtcatcgag 4920

cagagctcct ccctgaatga ggccagcagt ggcctcttcg acgtcttcct acgcttcatg 4980

tgccaccacg ccgtgcgcat caggggcaag tcctacgtcc agtgccaggg gatcccgcag 5040

ggctccatcc tctccacgct gctctgcagc ctgtgctacg gcgacatgga gaacaagctg 5100

tttgcgggga ttcggcggga cgggctgctc ctgcgtttgg tggatgattt cttgttggtg 5160

acacctcacc tcacccacgc gaaaaccttc ctcaggaccc tggtccgagg tgtccctgag 5220

tatggctgcg tggtgaactt gcggaagaca gtggtgaact tccctgtaga agacgaggcc 5280

ctgggtggca cggcttttgt tcagatgccg gcccacggcc tattcccctg gtgcggcctg 5340

ctgctggata cccggaccct ggaggtgcag agcgactact ccagctatgc ccggacctcc 5400

atcagagcca gtctcacctt caaccgcggc ttcaaggctg ggaggaacat gcgtcgcaaa 5460

ctctttgggg tcttgcggct gaagtgtcac agcctgtttc tggatttgca ggtgaacagc 5520

ctccagacgg tgtgcaccaa catctacaag atcctcctgc tgcaggcgta caggtttcac 5580

gcatgtgtgc tgcagctccc atttcatcag caagtttgga agaaccccac atttttcctg 5640

cgcgtcatct ctgacacggc ctccctctgc tactccatcc tgaaagccaa gaacgcaggg 5700

atgtcgctgg gggccaaggg cgccgccggc cctctgccct ccgaggccgt gcagtggctg 5760

tgccaccaag cattcctgct caagctgact cgacaccgtg tcacctacgt gccactcctg 5820

gggtcactca ggacagccca gacgcagctg agtcggaagc tcccggggac gacgctgact 5880

gccctggagg ccgcagccaa cccggcactg ccctcagact tcaagaccat cctggactga 5940

gaattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca 6000

gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc 6060

accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct 6120

cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg 6180

gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg 6240

gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg 6300

ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc 6360

cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg 6420

ttctcctctt cctcatctcc gggcctttcg acctgcagcc caagcttacc atgaccgagt 6480

acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg 6540

ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg 6600

agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg 6660

tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag 6720

cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg 6780

ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt 6840

tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg 6900

tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg 6960

cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg 7020

tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgggcgcgtc 7080

tggaacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 7140

gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 7200

tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 7260

gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 7320

cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 7380

ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 7440

cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg 7500

ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 7560

gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 7620

cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga 7680

attaattctg cagtcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag 7740

ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag 7800

tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact 7860

ttttaaaaga aaagagggga ctggaagggc taattcactc ccaacgaaga caagatatcc 7920

ttgatctgtg gatctaccac acacaaggct acttccctga ttagcagaac tacacaccag 7980

ggccaggggt cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc 8040

cagataaggt agaagaggcc aataaaggag agaacaccag cttgttacac cctgtgagcc 8100

tgcatgggat ggatgacccg gagagagaag tgttagagtg gaggtttgac agccgcctag 8160

catttcatca cgtggcccga gagctgcatc cggagtactt caagaactgc tgatatcgag 8220

cttgctacaa gggactttcc gctggggact ttccagggag gcgtggcctg ggcgggactg 8280

gggagtggcg agccctcaga tcctgcatat aagcagctgc tttttgcctg tactgggtct 8340

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 8400

aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 8460

tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt 8520

agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt 8580

gagaggcctt gacattgcta gcgtttaccg tcgacctcta gctagagctt ggcgtaatca 8640

tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 8700

gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 8760

gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 8820

atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 8880

actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 8940

gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 9000

cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 9060

ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 9120

ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 9180

ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 9240

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 9300

cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 9360

aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 9420

gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 9480

agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 9540

ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 9600

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 9660

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 9720

aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 9780

tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 9840

atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 9900

cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 9960

gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 10020

gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 10080

tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 10140

tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 10200

tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 10260

aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 10320

atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 10380

tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 10440

catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 10500

aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 10560

tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 10620

gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 10680

tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 10740

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 10800

gacggatcgg gagatcaact tgtttattgc agcttataat ggttacaaat aaagcaatag 10860

catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 10920

actcatcaat gtatcttatc atgtctggat caactggata actcaagcta accaaaatca 10980

tcccaaactt cccaccccat accctattac cactgccaat tacctgtggt ttcatttact 11040

ctaaacctgt gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg 11100

tactacaaac ttagtagttt ttaaagaaat tgtatttgtt aaatatgtac tacaaactta 11160

gtagt 11165

Claims

1. A method of inducing pluripotent stem cells using somatic cells from a patient with Woner Syndrome (WS), comprising the steps of:

s1, obtaining somatic cells derived from WS patients; the obtained WS somatic cells are fibroblast AG 03141;

s2, transfecting a vector which simultaneously expresses human telomerase reverse transcriptase and a resistance gene into the somatic cells, culturing, and screening the somatic cells capable of expressing the human telomerase reverse transcriptase; the described skeleton carrier for expressing human telomerase reverse transcriptase is lentivirus carrier; the lentiviral vector is pLVX-light-Puro;

s3, transfecting a vector carrying reprogramming genes for inducing iPSCs into the somatic cells screened in the S2, and culturing the iPSCs; the reprogramming genes are OCT4, SOX2, c-MYC and KLF-4; the vector is Sendai virus.

2. The method according to claim 1, wherein the vector for regulating the expression of the foreign gene is transfected at the same time as the vector for expressing human telomerase reverse transcriptase is transfected at step S2.

3. The method according to claim 2, wherein the backbone vector for the regulatory foreign gene expression vector is pLVX-Tet-on Advanced in step S2.

4. The induced pluripotent stem cell prepared by the method of any one of claims 1 to 3, which is named as human induced pluripotent stem cell and is deposited in China general microbiological culture Collection center with the deposit number of CGMCC No. 14895.

5. Use of the induced pluripotent stem cell prepared by the method according to any one of claims 1 to 3 or the induced pluripotent stem cell according to claim 4 in the research of disease pathogenesis or drug screening of early adult aging, tumor, atherosclerosis, diabetes or osteoporosis.

6. A cell model comprising the induced pluripotent stem cell produced by the method of any one of claims 1 to 3 or the induced pluripotent stem cell of claim 4.

7. The use of the cell model of claim 6 in the field of disease pathogenesis research or drug screening of early adult aging, tumors, atherosclerosis, diabetes or osteoporosis.