CN114350614B - Human induced pluripotent stem cell, and preparation method and application thereof - Google Patents
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/25—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from renal cells, from cells of the urinary tract
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a human induced pluripotent stem cell strain SXMUi001-A-1, a preparation method and application thereof, wherein the human induced pluripotent stem cell strain SXMUi001-A-1 is preserved in China general microbiological culture Collection center (CGMCC) No:23095. the human induced pluripotent stem cell line SXMUi001-a-1 was reprogrammed by extracting urine cells from urine of hemophilia a patients carrying F8 c.2440c > T (p.r814x) nonsense mutation, which is not only similar to embryonic stem cells in cell morphology, gene expression, proliferation and differentiation potential, but also has potential to differentiate toward three germ layers. The human induced pluripotent stem cell strain SXMUi001-A-1 prepared by the invention has important values for molecular mechanism research of nonsense mutation of hemophilia, detection of hemophilia, drug screening, gene therapy and other clinical application researches.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a human induced pluripotent stem cell, a preparation method and application thereof.
Background
Hemophilia is an X chromosome linked recessive hereditary hemorrhagic disease, and is characterized by impaired active thromboplastin production, prolonged clotting time, a tendency to post-traumatic hemorrhage throughout the life, and "spontaneous" bleeding without significant trauma in critically ill patients. Including hemophilia a, a deficiency of the procoagulant component of factor viii (viii: C), also known as AGH deficiency; hemophilia B (hemophilia B), i.e. factor ix deficiency, also known as PTC deficiency, thrombin component deficiency; hemophilia C (hemophilia C), i.e., factor xi (axi) deficiency, also known as PTA deficiency, thromboplastin deficiency.
Among them, hemophilia a is a bleeding disease of recessive inheritance of the X chromosome caused by the deficiency of coagulation factor 8, and clinically manifested as repeated bleeding of skin, joints, muscles, mucous membrane and soft tissues. Molecular defects leading to hemophilia most of the molecular defects are nonsense and missense mutations in the clotting factor gene, in addition to 48% of hemophilia a being intronic, and a few are deletion/insertion mutations, or splicing abnormalities [ bagnals, r.d., n.waseem, p.m. green, and f.giannelli.2002.'Recurrent inversion breaking intron 1of the factor VIII gene is a frequent cause of severe hemophilia A', blood, 99:168-74 ]. Deficiency of the F8 gene results in Hemophilia A (HA) with an incidence of 1/5,000 in the male population [ Yang Renchi. The national expert consensus for hemophilia diagnosis and treatment (2017 edition) [ J ]. J.Chinese hematology, 2017,38 (05): 364-370 ]. Factor VIII Gene (F8) Variant Database (http:// www.factorviii-db. Org /) showed 2015F 8 mutants in 5480 case reports, with 216 nonsense mutations accounting for about 10.7%. There are 32 cases of F8 c.2440C > T reported at present, so that construction of hemophilia A nonsense mutant (c.2440C > T) is of great significance to the study of hemophilia A.
The current treatment method of hemophilia is still mainly infusion coagulation factor replacement therapy, the traditional therapy has huge consumption, great economic burden and living pressure are brought to families of patients [ Chinese guidelines on the treatment of hemophilia (version 2020) ]'.2020.Zhonghua Xue Ye Xue Za Zhi, 41:265-71 ], and coagulation inhibitors can influence curative effects in the treatment process, nonsense mutations are found to be high risk factors for generating the coagulation factor inhibitors [ Gouw, S.C., H.M. van den Berg, J.Oldenburg, J.Astermark, P.G.de Groot, M.Margaglione, A.R.Thompson, W.van Heerde, J.Boekhorst, C.H.Miller, S.le Cessie, and J.G.van der Bom.2012.' F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review and meta-analysis ', blood,119:2922-34 ] ], so that the research on molecular pathogenesis of hemophilia caused by nonsense mutations has important significance and a new treatment scheme. In 2006 Takahashi and Yamanaka [ Yokoyama, y., T.Suzuki, M.Sakata-Yanagimoto, K.Kumano, K.Higashi, T.Takato, M.Kurokawa, S.Ogawa, and s.chiba.2009, "Derivation of functional mature neutrophils from human embryonic stem cells', blood,113:6584-92 ], transferred four transcription factors into specialized adult cells, reprogrammed to induce pluripotent stem cells (ipscs), which had characteristics similar to embryonic stem cells, were able to self-renew and differentiate multipotency, and avoided the ethical and legal problems involved with embryonic stem cells. Hemophilia is taken as a single-gene genetic disease, gene therapy is the only cure means, the clinical test object of the current gene therapy is animals, and animal models can not completely simulate the disease state and the treatment effect of human bodies due to species differences. Dimos et al [ Dimos, J.T., K.T.Rodolfa, K.K.Niakan, L.M.Weisenthal, H.Mitsumoto, W.Chung, G.F.Croft, g.saphier, R.Leibel, R.Goland, H.Wichterle, C.E.Henderson, and k.eggan.2008.'Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons', science,321:1218-21 ] generated iPS cells from Amyotrophic Lateral Sclerosis (ALS) patients. These patient-specific iPS cells have embryonic stem cell characteristics and successfully differentiate directionally into motor neuron cells with genes that lead to ALS. The somatic cell of the patient is utilized to reprogram the specific iPS cell of the disease to be differentiated into the specific cell, the pathological process of the disease can be reproduced, and a reliable basis is provided for the mechanism research and the drug treatment of the disease.
Disclosure of Invention
Based on this, it is an object of the present invention to provide a human induced pluripotent stem cell.
The method comprises the following technical scheme:
a human induced pluripotent stem cell strain SXMUi001-A-1, wherein the preservation number of the human induced pluripotent stem cell strain SXMUi001-A-1 is CGMCC No:23095, 10 and 28 days 2021, are deposited with the China general microbiological culture Collection center (China Committee).
It is also an object of the present invention to provide a pharmaceutical composition.
The method comprises the following technical scheme:
a pharmaceutical composition comprises at least one of the above culture or extract of human induced pluripotent stem cell strain SXMUi001-A-1 and human induced pluripotent stem cell strain SXMUi001-A-1 as effective components.
The invention also aims at providing a preparation method of the human induced pluripotent stem cell strain SXMUi001-A-1.
The method comprises the following technical scheme:
collecting somatic cells carrying F8 mutant genes, and performing cell culture;
the method comprises the steps of introducing plasmids containing reprogramming factors into cultured somatic cells, culturing the somatic cells by adopting a reprogramming culture medium, and screening the reprogrammed somatic cells to obtain a human induced pluripotent stem cell strain SXMUi001-A-1, wherein the reprogramming factors are OCT4, SOX2, KLF4 and MYC.
The cell strain SXMUi001-A-1 of the invention is preserved in China general microbiological culture Collection center (CGMCC No) at the day of 10 months and 28 days of 2021: 23095. addresses are Beijing, chaoyang area North Chen Xiyu No. 1, 3, china academy of sciences microbiological institute, post code: 100101.
the inventors of the present invention have found in the study that by extracting urine cells from urine of hemophilia a patients carrying nonsense mutation of F8 c.2440c > T (p.r814x) and successfully reprogramming the somatic cells, a new human induced pluripotent stem cell line SXMUi001-a-1 was obtained, which was found to be similar to embryonic stem cells not only in cell morphology, gene expression, proliferation and differentiation potential but also in potential toward three germ layers. The biological preservation is carried out on the plant in 2021 and 10 months and 28 days (the preservation number is CGMCC No. 23095). The human induced pluripotent stem cell strain SXMUi001-A-1 prepared by the invention has important application value for molecular mechanism research of nonsense mutation of hemophilia, detection of hemophilia, drug screening, gene therapy and other clinical application researches.
Drawings
FIG. 1 is a map of plasmid pCEP4-M2L in example 1.
FIG. 2 is a map of plasmid PEP4-EO2S-ET2K in example 1.
FIG. 3 is a characterization of the human induced pluripotent stem cell SXMUi001-A-1 cell line prepared in example 1, wherein A is the induced reprogramming process of urine cells (1-16 days, D1-D16), D0 is the pre-current state of urine cells when electrotransferred, and stem cell-like clone formation is seen from D1, D7 to D16; p0 is the picked iPS clone, P7 is the 7 th generation iPS, and the scale is 100 μm; b is an alkaline phosphatase experimental detection result of the human induced pluripotent stem cell SXMUi 001-A-1; c is a chromosome karyotype analysis result of the human induced pluripotent stem cells SXMUi 001-A-1; d is Sanger sequencing result of human induced pluripotent stem cells SXMUi 001-A-1; e is an immunofluorescence staining detection result of the induced pluripotent stem cells SXMUi001-A-1 of the human, and the scale is 100 mu m; f is the qPCR analysis and detection result of pluripotency genes NANOG, OCT4 and SOX2 of human induced pluripotent stem cells SXMUi001-A-1, hES (human embryonic stem cells) and UC cells; g is a graph of HE staining slice results in teratoma in vivo differentiation experiments.
Detailed Description
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or" unless the context clearly dictates otherwise.
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The abbreviations and terms involved in the present invention are defined as follows:
"induced pluripotent stem cells" (Induced pluripotent stem cells, iPSCs) refer to pluripotent stem cells that have been reprogrammed by introduction of specific transcription factors into terminally differentiated somatic cells. After the differentiated cells are reversed under specific conditions, they return to a totipotent state, either to form embryonic stem cell lines, or to develop further into new individuals, i.e., cell reprogramming (Cell reprogramming). Differentiation is the result of selective expression of genes, without altering genetic material, while reprogramming is in a sense that it is a reversal of differentiation. Unlike classical embryonic stem cell technology and somatic cell nuclear transfer technology, iPSCs technology does not use embryonic or egg cells and therefore has no ethical problems.
The present invention will be described in further detail with reference to specific examples.
Some embodiments of the invention provide a human induced pluripotent stem cell strain SXMUi001-A-1 with a preservation number of CGMCC No:23095, which was deposited at the China general microbiological culture Collection center, with the China Committee for culture Collection of microorganisms, at 10 and 28 of 2021.
In some embodiments, the human induced pluripotent stem cell strain SXMUi001-a-1 classification is named: human induced pluripotent stem cells. In some of these embodiments, the human induced pluripotent stem cell strain SXMUi001-a-1 described above has one or more of the following properties: pluripotency; pluripotency; the ability to form teratomas; normal diploid karyotype; has the capacity of unlimited proliferation; telomeres shorter than human embryonic stem cells; the ability to proliferate under atmospheric oxygen conditions (e.g., greater than 5% oxygen to about 21% oxygen) to have an undifferentiated phenotype; colonies proliferate. In some of these embodiments, the induced pluripotent stem cell strain SXMUi001-a-1 is capable of expressing embryonic stem cell transcription factor OCT4, and/or specific surface markers SSEA4, TRA-1-60, TRA-1-81.
In some embodiments, the method for preparing the human induced pluripotent stem cell strain SXMUi001-A-1 comprises the following steps: the preparation method of the human induced pluripotent stem cell strain SXMUi001-A-1 comprises the following steps:
the F8 gene is subjected to c.2440C & gtT nonsense mutation, and reprogramming factors are introduced into somatic cells of a hemophilia A patient; the reprogramming factors include OCT4, SOX2, KLF4, MYC
And (5) performing cell reprogramming and screening to obtain the cell.
In some of these embodiments, the somatic cells include, but are not limited to, urine, blood, adipose tissue, amniotic fluid, placental chorion, amniotic membrane, umbilical cord blood, periosteal cells at various sites, or skin fibroblasts.
In some embodiments, the somatic cells are urine cells, avoiding pain from skin sampling and injury to the patient.
In some of these embodiments, the reprogramming factor MYC described above is preferably c-Myc.
Some embodiments of the present invention provide a pharmaceutical composition, the active ingredient of which comprises any one of the above-mentioned human induced pluripotent stem cell strain SXMUi001-a-1, a culture and/or extract of the human induced pluripotent stem cell strain SXMUi001-a-1.
In some embodiments, the active ingredient in the pharmaceutical composition comprises a culture of the human induced pluripotent stem cell strain SXMUi001-a-1, further comprising: a purified population of human induced pluripotent stem cells; cells (e.g., purified populations of differentiated cells) that induce differentiation of pluripotent stem cells from humans. These differentiated stem cells include, but are not limited to: pancreatic beta cells, neural stem cells, cortical neurons, dopaminergic neurons, oligodendrocyte (oligodendrocyte) or oligodendrocyte progenitor cells, liver cells, hepatic stem cells, cardiac muscle cells, or renal epithelial cells.
In some embodiments, the active ingredient in the pharmaceutical composition comprises an extract of the human induced pluripotent stem cell strain SXMUi001-a-1, further comprising, but not limited to: nucleic acids, amino acids, small molecule compounds, antibodies, polypeptides, organelles, and the like.
Some embodiments of the present invention provide the use of the human induced pluripotent stem cell strain SXMUi001-a-1 described above and/or the pharmaceutical composition described above for constructing a hemophilia cell model.
Some embodiments of the invention provide the use of the human induced pluripotent stem cell strain SXMUi001-a-1 described above for the preparation and/or screening of a hemophilia treatment drug.
In some of these embodiments, the human induced pluripotent stem cell strain SXMUi001-a-1 has low immunogenicity. Therefore, it can become a novel source of transplant donor with low immune rejection reaction, and can be applied to hemophilia transplant treatment.
In some embodiments, the hemophilia is a bleeding disorder with inherited coagulation dysfunction, comprising: hemophilia a, hemophilia B; hemophilia C. In one embodiment, hemophilia a.
Some embodiments of the present invention provide for the use of the human induced pluripotent stem cell strain SXMUi001-a-1 described above for the preparation of a reagent for detecting, classifying or predicting, monitoring therapy, prognosis or otherwise evaluating hemophilia.
Some embodiments of the present invention provide a preparation method of the human induced pluripotent stem cell strain SXMUi001-A-1, comprising the following steps:
collecting somatic cells carrying F8 mutant genes, and performing cell culture;
the plasmids containing reprogramming factors OCT4, SOX2, KLF4 and MYC are introduced into somatic cells after culture, and the somatic cells are cultured by adopting a reprogramming culture medium, and the reprogrammed cells are screened to obtain the human induced pluripotent stem cell strain SXMUi001-A-1.
Preferably, plasmids containing reprogramming factors OCT4, SOX2, KLF4 and c-Myc are introduced into somatic cells after culture, and the somatic cells are cultured by adopting a reprogramming culture medium, and the reprogrammed cells are screened to obtain the human induced pluripotent stem cell strain SXMUi001-A-1.
In some embodiments, the somatic cells carrying the F8 mutant gene are urine cells, particularly urine cells in which the F8 gene is subjected to nonsense mutation of c.2440C > T.
In some embodiments, the above-described introduction is by electroporation transfection of plasmids PEP4-EO2S-ET2K and pCEP4-M2L by a nuclear transfer apparatus, comprising: the procedure was selected by passing 6. Mu.g of plasmid PEP4-EO2S-ET2K 6. Mu.g and 4. Mu.g of pCEP4-M2L containing reprogramming factors OCT4, SOX2, KLF4, c-Myc through an electrotransducer LONZA Nucleofecto 2b Device: t-020, 1X 10 by electroporation transfection 6 In human urine cells.
Example 1
The inventors have previously screened for the type of gene mutation in hemophilia patients in Shanxi area, wherein the nonsense mutation F8 c.2440C > T was detected in a hemophilia A patient in a second hospital of Shanxi medical university, confirmed (http:// www.factorix.org /) that the mutation was nonsense.
1. Preparation of human induced pluripotent stem cells
(1) Collecting urine cells:
urine shed cells were isolated from a sample of hemophilia patient urine and subjected to proliferation culture [ Zhou, t., C.Benda, S.Dunzinger, Y.Huang, J.C.Ho, J.Yang, y. Wang, Y.Zhang, Q.Zhuang, Y.Li, X.Bao, H.F.Tse, J.Grillari, R.Grillari-Voglauer, d.pei, and m.a. esteban.2012.'Generation of human induced pluripotent stem cells from urine samples', nat Protoc,7:2080-9 ].
Midrange urine was collected, human Urine Cells (HUCs) were isolated by centrifugation at 400g for 10min at room temperature, cell pellet was washed twice with DPBS (Gibco), and resuspended in 1ml of urine cell primary medium containing DMEM/F12 1:1 mixture, REGM SingleQuot growth factor additive, 10% FBS, 100×Penicillin-Streptomycin, 1ml urine cell primary culture medium, 37℃and 5% CO 2 Culturing in an incubator, which is day 1of urine cell culture. 1ml of culture medium is added every day for 24-72 hours, most of the culture medium in the well plate is removed on the 4 th day, 1ml of RE/MC proliferation culture medium is added, 1ml of culture medium is removed every day, 1ml of culture medium is added, renal epithelial cell adhesion occurs during the period, and after the cell density reaches 80% -90%, the cells are transferred to a new six-well plate for continuous culture.
(2) Reprogramming:
taking HUCs 1×10 with good culture condition 6 After resuspension of the cells with the electrotransfer reagent according to Amaxa Basic Nucleofector Kit for primary mammalian Epithelial cells kit instructions, 6ug PEP4-EO2S-ET2K and 4ug pCEP4-M2L (synthesized according to a profile, wherein the profile of pCEP4-M2L (Plasmid: # 2096) is shown in FIG. 1 and the profile of PEP4-EO2S-ET2K (Plasmid: # 20927) is shown in FIG. 2) were added, mixed and transferred to an electrotransfer cup, and an electrotransfer instrument (LONZA Nucleofector 2b Device) was turned on for selection procedure: t-020, putting the electric rotating cup into an electric rotating instrument, starting a program and completing electric rotation. After the electrotransformation, 1ml RE/MC proliferation culture medium is added, the mixture is uniformly mixed and transferred to a culture plate on which Matrigel (BD, america) is paved in advance, and then a proper amount of RE/MC proliferation culture medium is added, the temperature is 37 ℃, and the CO is 5 percent 2 Culturing in an incubator. The next Day after electrotransformation, the mTeSR1 medium, labeled Day 1 (D1), was changed, followed by passaging every 3-4 days with 0.25% trypsin (Sigma) solution. As shown in FIG. 3A, which is an induced reprogramming process of urine cells (1-16 days, D1-D16), D0 is the pre-electrotransformation state of urine cells, D16 visible stem cell (hESC) like formation after electrotransformation, human Embryonic Stem Cell (HESC) colonies were picked up after day 18 and expanded in mTESR1 mediumIn the enrichment culture, P0 is the picked iPS clone, P7 is the 7 th generation iPS, and the typical hESC morphology is presented, and the cell line is named as F8-hiPSC (namely SXMUi 001-A-1). The microbial strain is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (China) with the preservation number of CGMCC No:23095. addresses are Beijing, chaoyang area North Chen Xiyu No. 1, 3, china academy of sciences microbiological institute, post code: 100101. the cell line is subjected to amplification culture by using mTESR1 culture medium, and cells obtained by culture are collected by conventional cells and used for subsequent identification and research.
2. Identification of human induced pluripotent stem cells
(1) Alkaline phosphatase expression identification:
since the activity of alkaline phosphatase in embryonic stem cells is relatively high, the activity of alkaline phosphatase can be relatively known from the staining result, and whether the experimental cells have pluripotency can be demonstrated from the side. Clone cells expanded on the above cell line F8-hiPSC were stained with alkaline phosphatase, medium was removed from the well plate, and TBST was washed 3 times for 5min each. Adding alkaline phosphatase chromogenic buffer solution, standing for 5min, sucking off the buffer solution, adding AP chromogenic solution, incubating at room temperature in dark place for 5-10min, removing the staining solution, washing with distilled water for 1-2 times to terminate the reaction, and observing under a mirror. The results of the detection are shown in FIG. 3B, and the cytoplasm contains a dark blue positive reaction product. The F8-iPSC expresses AP as high as ES cells, and the primary identification cell line F8-hiPSC has multipotency.
(2) Karyotype analysis:
the induced pluripotent stem cells undergo severe changes in the induction process, so that the problem of cell division is likely to cause abnormality of cell karyotypes, and the abnormality of cell karyotypes is also likely to be caused, so that karyotype detection is required to be carried out regularly in the cell subculture of the iPS so as to ensure the normal of the cells.
The cell line F8-hiPSC generation 5 was subjected to karyotyping, and the P5-generation iPS was collected for detection, and when the cells reached the logarithmic phase, colcemid was added to a solution having a final concentration of 20. Mu.g/ml for 2 hours. The supernatant was removed and the pellet was resuspended in 8ml of 0.075M KCl and incubated at 37℃for 20 minutes. Cells were fixed with fresh Carnoy fixative (3:1 ratio of methanol to glacial acetic acid). 20 metaphase cells were analyzed on an Olympus BX51 microscope using Ikaros (MetaStstems, germany) with a 450-500 band resolution.
As shown in FIG. 3C, the karyotype analysis is performed on the 5 th generation iPS through G band analysis, so that diploid 46, XY are shown, the cell chromosome is normal, and the cell line F8-hiPSC prepared by the method is safer and has better genome stability.
(3) Sanger sequencing:
the passaged cells of the above cell line F8-hiPSC were collected, and the genomic DNA of the cell line F8-hiPSC was extracted by TIANamp Genomic DNA Kit (day root) and the sequenced fragments were amplified by PCR. Sanger sequencing was performed by biological engineering (Shanghai) Inc.
The sequencing results are shown in FIG. 3D, and the blue position is a mutation site, which shows that Sanger sequencing confirms that the C.2440 of the target site (F8 gene) has C > T nonsense mutation.
(4) STR analysis:
cross-contamination can cause a series of serious problems and consequences during cell culture, and is therefore particularly important for the identification of cell lines. At present, STR genotyping is one of the most effective and accurate methods for carrying out cell cross contamination and property identification, after genomic DNA extraction of cells of the induced cell line F8-hiPSC, STR analysis is carried out by entrusted biological engineering (Shanghai) Inc., cell line comparison is carried out by using DSMZ tools (which contain cell line STR data from ATCC, DSMZ, JCRB, CLASTR and RIKEN databases), a cell line is matched in a cell bank CLASTR, as shown in the following table 1-1, DNA genotyping detection is further carried out by adopting a typing detection scheme shown in the following table 1-2 for the matched cell line NKB-1 and the cell line, and detection results are shown in the following tables 1-3.
TABLE 1-1
| Customer sample numbering | Company numbering | Multiple alleles | Matched cell lines | Cell bank | EV value of |
| HIPS-F8 | 20210325-001 | Without any means for | NKB-1 | CLASTR | 75.86％ |
TABLE 1-2
| Scheme 1 | Scheme 2 | Scheme 3 | Scheme 4 | |
| 1 | AMEL | VWA | D19S433 | D5S818 |
| 2 | D3S1358 | D6S1043 | TH01 | D1S1656 |
| 3 | D13S317 | D2S1338 | D12S391 | D8S1179 |
| 4 | D7S820 | D21S11 | D16S539 | TPOX |
| 5 | D18S51 | FGA | CSF1PO | |
| 6 | PENTAD | PENTAE |
Tables 1 to 3
Wherein the detection sites comprise: the total of 21 of D5S818, D13S317, D7S820, D16S539, VWA, TH01, AMEL, TPOX, CSF PO, D3S1358, D18S51, PENTAD, D6S1043, D2S1338, D21S11, D19S433, D12S391, FGA, D1S1656, D8S1179 and PENTAE was found no matching cell line in the cell line search upon detection of the cell DNA typing of this strain. No multiallelic gene was found in this cell line in this test (multiallelic gene refers to the phenomenon of three or more alleles). The examined cell line F8-hiPSC was also shown to be uncontaminated by other cell lines.
(5) Immunofluorescent staining:
immunofluorescent staining was performed at room temperature and cell line F8-hiPSC cells were fixed in 4% paraformaldehyde for 15 min. After 5 minutes of permeation with 0.5% Triton X-100 in PBS, the cells were blocked with 0.5% Triton X-100 and 10% goat serum for 30 minutes. Next, the cells were incubated overnight at 4℃with primary antibodies diluted in 10% goat serum (OCT 4 antibody: BD Pharmingen,560307, oct3/4Alexa 647 100Tst; SSEA antibody: SSEA4 santa SC-21704; TRA-1-60:BD Pharmingen,560850,Hu TRA-1-60Ag Alexa 647TRA-1-60 100Tst; TRA-1-81:BD Pharmingen,560793, hu TRA-1-81Alexa 647TRA-1-81 100 Tst), respectively, and then incubated at room temperature for 1 hour with the corresponding secondary antibodies diluted in 10% goat serum. Nuclei were counterstained with DAPI for 5min. Images were acquired using a confocal system (Zeiss LSM800, germany).
The immunofluorescence staining results are shown in fig. 3E, and the detection results show that the cell line F8-iPSC can express the embryonic stem cell transcription factor OCT4 and specific surface markers SSEA4, TRA-1-60 and TRA-1-81, so that the pluripotency characteristics of the cell line are further confirmed.
(6) Real-time fluorescent quantitative PCR
Collecting the F8-iPSC cells and hES (human embryonic stem cells from Guangzhou biomedical science and Chinese academy of sciences of China)The health institute gives away) and UC cells (urine cells of the above patients), and the total RNA (F8-iPSC, hES, UC) of each group was extracted with EZbioscience kit, and genomic DNA was removed usingIII RT SuperMix for qPCR (Vazyme) reverse transcribes mRNA into cDNA. The cDNA was quantitatively measured on CFX Connect (BioRad) using 2X ChamQ Universal SYBR qPCR Master Mix (Vazyme) to determine the relative expression levels of the pluripotency genes Nanog, oct4, sox2 (reference gene GAPDH), and the primer sequences of the specific genes Nanog, OCT4, and SOX2 are shown in tables 1 to 4 below.
Tables 1 to 4
The real-time fluorescence quantitative PCR result is shown in FIG. 3F, the expression of F8-iPSC pluripotency marker genes Nanog, oct4 and Sox2 is obviously increased compared with UC, and the expression level of the pluripotency genes is similar to that of hES.
(7) Teratoma in vivo differentiation experiment
Collecting the F8-iPSC cells of the cell line of normal subculture about 2×10 6 The ability of F8-iPSC (SXMUi 001-A-1) to differentiate into three germ layers in vivo was further demonstrated by resuspending the cell pellet with DMEM/F12 basal medium, adding appropriate amount of diluted Matrigel, injecting the cloned cell suspension subcutaneously or intramuscularly into SCID mice, removing teratomas after 4 weeks, and after HE staining of tissue sections, the apparent three germ layer structure (endoderm, mesoderm, ectoderm, respectively, as shown in FIG. 3G) was seen.
As is clear from the above experiments, the invention makes it possible to treat patient-specific somatic cell-derived stem cells (e.g., correction of hemophilia by iPS gene editing, etc.) by extracting urine cells from urine of hemophilia A patients carrying F8 c.2440C > T (p.R814X) nonsense mutation and reprogramming the somatic cells to obtain a cell line F8-hiPSC, which is a human-induced pluripotent stem cell (SXMUi 001-A-1, accession number CGMCC No. 23095) that can be induced to differentiate into different cells and tissues in vitro, as embryonic stem cells. Unlike traditional stem cell therapy, the iPSCs transplantation therapy avoids the limitations of ethics, immune rejection and the like of the traditional stem cell therapy.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
SEQUENCE LISTING
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<212> DNA
<213> Artificial Sequence
<400> 5
aaggtcccgg tcaagaaaca g 21
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 6
cttctgcgtc acaccattgc 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
gtggacctga cctgccgtct 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 8
ggaggagtgg gtgtcgctgt 20
Claims (4)
1. The human induced pluripotent stem cell strain SXMUi001-A-1 is characterized in that the preservation number of the human induced pluripotent stem cell strain SXMUi001-A-1 is CGMCC No:23095, deposited at the national center for culture collection at 10 and 28 of 2021, is derived from somatic cells of hemophilia a patients, whose F8 gene has undergone a nonsense mutation of c.2440c > T, which are urine cells.
2. The human induced pluripotent stem cell strain SXMUi001-a-1 according to claim 1, wherein the human induced pluripotent stem cell strain SXMUi001-a-1 is capable of expressing embryonic stem cell transcription factor OCT4, and/or specific surface markers SSEA4, TRA-1-60, TRA-1-81.
3. A pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises any one of the human induced pluripotent stem cell strain SXMUi001-a-1 and the culture of the human induced pluripotent stem cell strain SXMUi001-a-1 according to any one of claims 1 to 2.
4. Use of the human induced pluripotent stem cell strain SXMUi001-a-1 according to any one of claims 1-2 for preparing a hemophilia cell model.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108064280A (en) * | 2015-01-06 | 2018-05-22 | 延世大学校产学协力团 | It utilizes using Coagulation factor VIII gene as the haemophilia A therapeutic composition of the endonuclease of target |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108064280A (en) * | 2015-01-06 | 2018-05-22 | 延世大学校产学协力团 | It utilizes using Coagulation factor VIII gene as the haemophilia A therapeutic composition of the endonuclease of target |
Non-Patent Citations (1)
| Title |
|---|
| ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs;Zhiqing Hu，et al;《molecular therapy》;第17卷;第199页左栏第2段至右栏第1段、第205页材料与方法 * |
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