CN101250502A - A preparation method of induced pluripotent stem cells - Google Patents

Abstract

The invention provides a method for preparing induced embryonic stem cells, which comprises following steps: firstly, introducing six transcription factors into adult cells, secondly, culturing the adult cells under the condition for culturing the embryonic stem cells, and enabling the adult cells to form cells with the form of embryonic stem cells. The method of the invention also comprises: cloning the six transcription factors into a carrier and then transforming the six transcription factors into the adult cells. When the method of the invention is adopted to prepare the embryonic stem cells, the efficiency is high, the acute rejection can be avoided, the embryonic stem cells can be differentiated into different tissue cells under special conditions, and the method of the invention has wide applying prospect.

Description

A preparation method of induced pluripotent stem cells

technical field

The invention belongs to the field of biomedicine, and in particular relates to a method for preparing induced pluripotent stem cells (Induced pluripotent stem cells, referred to as iPS cells).

Background technique

Human embryonic stem cells have the potential to differentiate into various types of human cells, and provide the possibility of application for regenerative medicine (Thomson JA., Itskovitz-Eldor J., Shapiro SS., et al.Science.Nov 6 1998; 282( 5391): 1145-1147). However, immune rejection has a great influence on the transplantation therapy of human embryonic stem cells. In order to be able to cultivate patient-specific pluripotent stem cells to eliminate the impact of immune rejection, people have tried many methods, such as nuclear transfer of adult cells (also known as therapeutic cloning), fusion of adult cells and human embryonic stem cells, etc. , but were not successful. The possibility of obtaining patient-specific induced pluripotent stem cells similar to human embryonic stem cells (iPS cells) was established by inducing human embryonic stem cell-like induced pluripotent stem cells (iPS cells) from adult cells by introducing specific transcription factors.

Recently, several laboratories have successfully reprogrammed human differentiated cells into pluripotent stem cells similar to human embryonic stem cells by introducing specific transcription factors into them. Virus combination, a. Oct4, Sox2, C-myc, Klf4 (Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotentstem cells from adult human fibroblasts by defined factors. Cell. Nov 30 2007; 131(5): 861-872); b.Oct4, Sox2, Nanog, Lin28 (Yu J, Vodyanik MA, Smuga-Otto K, et al.Induced pluripotent stem cells derived from human somatic cells.Science.Dec 21 2007; 318(5858): 1917-1920); c.Oct4, Sox2, c-Myc, KLF4, hTert, SV40 large T (Park IH, Zhao R, West JA, et al.Reprogramming of humansomatic cells to pluripotency with defined factors.Nature.Jan 10 2008 ; 451(7175):141-146). Infect human fibroblasts with a combination of the above transcription factors, reprogram human fibroblasts into pluripotent stem cells similar to embryonic stem cells, and prove that these pluripotent stem cells express surface markers of undifferentiated human embryonic stem cells and can differentiate into human The cells of the three germ layers of the embryo. These experimental results make it possible to use human adult cells to generate individual-specific embryonic stem cells, and then differentiate them into the required types of cells, thus providing strong support for transplantation therapy for some diseases, and basically eliminating the risk of immune rejection. The efficiencies reported by the three laboratories were essentially the same. The efficiency is about 10 induced human pluripotent stem cells obtained from 100,000 starting cells, which is relatively low. Low efficiency will seriously affect its application and promotion.

Therefore, there is an urgent need in this field to develop efficient methods for inducing differentiated cells to transform into pluripotent stem cells similar to embryonic stem cells.

Contents of the invention

The purpose of the present invention is to provide a method for preparing induced pluripotent stem cells.

Another object of the present invention is to provide an induced pluripotent stem cell obtained by the preparation method.

Another object of the present invention is to provide a composition comprising induced pluripotent stem cells obtained by the preparation method. The composition can be used for treating related diseases or for transplantation.

The first aspect of the present invention provides a method for preparing induced pluripotent stem cells, characterized in that it comprises the steps of:

(a) Six transcription factors were introduced into adult cells: Oct4, Nanog, Sox2, Lin28, c-myc and Klf4;

(b) culturing the above-mentioned differentiated cells under embryonic stem cell culture conditions to form cells having the morphology of embryonic stem cells.

The above-mentioned transcription factors can be transferred into cells in the form of DNA, protein or mRNA.

In a specific embodiment, the above step (a) is to clone the coding sequences of the above six transcription factors into vectors respectively, and then transform the six vectors into adult cells.

The above-mentioned vectors are viral vectors or plasmids. The vector is a lentiviral vector, a retroviral vector or an adenoviral vector, that is, the above-mentioned transcription factors are first packaged into viruses. Preferably, the above-mentioned vector is a lentiviral vector.

Preferably, the above-mentioned adult cells are primate adult cells. The aforementioned primate is human. The above-mentioned adult cells include adult stem cells, adult differentiated cells, or a mixture of adult stem cells and adult differentiated cells. Preferably, the above-mentioned adult cells are human adult cells.

The above-mentioned adult differentiated cells are human differentiated cells.

The above-mentioned human adult cells are selected from the group consisting of epithelial cells, nerve cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes, nucleated erythrocytes, macrophages, monocytes, fibroblasts, myocardial A type of cell.

Preferably, the above-mentioned somatic cells are fibroblasts. The aforementioned fibroblasts are derived from human body.

The above-mentioned embryonic stem cells can express alkaline phosphatase and 2 or more (including 2) antigens among the surface antigens of SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81.

The second aspect of the present invention provides an induced pluripotent stem cell prepared by the above method. The induced pluripotent stem cells can be induced to differentiate into various differentiated cells under corresponding conditions to treat various diseases.

The above-mentioned induced pluripotent stem cells can express alkaline phosphatase and 2 or more antigens among surface antigens such as SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81.

The third aspect of the present invention provides a composition comprising the induced pluripotent stem cells prepared by the above method, and the composition can be used for treating related diseases or transplanting. The above composition includes the induced pluripotent stem cells prepared by the above method and a pharmaceutically acceptable carrier.

The above-mentioned induced pluripotent stem cells have pluripotency and are in an undifferentiated or poorly differentiated state.

The term "transcription factor" in the present invention includes protein, DNA and RNA forms. DNA includes cDNA, genomic DNA and synthetic DNA. Therefore, the induction of adult cells can be achieved by directly introducing the proteins or mRNAs of the six transcription factors, or by cloning the coding sequences of the transcription factors into vectors, and then transforming the six vectors into adult cells.

In order to achieve the purpose of the present invention, the inventors referred to some experimental methods of the pioneers. Since four factors were used in their experiments, two of them overlapped, so a total of six factors were used. (Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. Nov30 2007; 131(5): 861-872; Yu J, Vodyanik MA, Smuga-Otto K , et al. Induced pluripotent stem celllines derived from human somatic cells. Science. Dec 21 2007; 318(5858): 1917-1920.). The inventors synthesized the transcription factors they used in the experiment, and transferred six transcription factors Oct4, Nanog, Sox2, Lin28, c-myc and Klf4 into the differentiated cells, because of the efficiency of Yamnaka laboratory and Thomson laboratory Basically the same, so we only use the four transcription factors in Thomson's laboratory to transfer the differentiated cells at the same time as a control to induce the production of iPS cells. The experiment was successful, and the efficiency of transferring 6 transcription factors to induce iPS was 10.4 times that of only transferring 4 transcription factors.

The invention has the advantages of high efficiency, and the obtained induced pluripotent stem cells (like embryonic stem cells) have differentiation potential, which provides more convenience for the treatment of related diseases and the generation of patient-specific cell types in transplantation therapy.

Human embryonic stem cells can specifically express alkaline phosphatase and SSEA-3, SSEA-4, Tra-1-60, Tra-1-81 these surface antigens; express alkaline phosphatase and SSEA-3, SSEA-4, These surface antigens Tra-1-60, Tra-1-81 are the criteria for judging that the cells are similar to undifferentiated human embryonic stem cells. The criterion for judging the pluripotency of a cell is: when the cell differentiates into an embryoid body (EB), it can form the cells of the three germ layers of the human embryo and express the genes unique to the three germ layers.

Therefore, in the experiments of the present invention, the inventors transferred six transcription factors Oct4, Nanog, Sox2, Lin28, c-myc and Klf4 into differentiated human cells, so that the differentiated cells regained the morphology of embryonic stem cells , their alkaline phosphatase activity, and surface antigens such as SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81 were detected, and the results of the detection also fully showed that the clones obtained by the inventors similar to human embryonic stem cells. When we formed the obtained cells into embryoid bodies, the specific genes of the three germ layers were all expressed, indicating that the cells had the pluripotency to form the three germ layers of human embryos.

Embryonic stem cells are totipotent and can differentiate into any type of cell in the body. Under specific induction conditions, embryonic stem cells can differentiate into a certain line, type or type of cell. The embryonic stem cells obtained in the present invention also have the above characteristics. At present, embryonic stem cells have been successfully differentiated into various cells, such as hematopoietic cells (Ronald, et al. PNAS, 1995, 92: 7530-7534), nerve cells (J Dinsmore, et al. Theriogenology, 1998, 49 : 145-151), muscle cells (Benjamin E, et al, Nature Biotechnology, 2000, 18 April: 399-406), fat cells (Dani C Smith, et al.J cell Sci., 1997, Jun, 110: 1279 -1285), endothelial cells (Dniel Vittet, et al. Blood, 1996, 88 (9): 3424-3442) and the like. Therefore, obtaining the patient's adult cells and inducing them to transform into embryonic stem cells or embryonic stem cells by infecting vectors carrying six factors can induce them to differentiate into the cells required by the patient in vitro, because the obtained cells and tissues are similar to The patient's genes are completely identical, which is an autologous transplant in the cell or tissue transplantation treatment and will not be rejected by the patient's immune system.

The "embryonic stem cells", "pluripotent stem cells similar to embryonic stem cells", "iPS cells", "induced pluripotent stem cells" and "iPS-S" in the present invention can be used interchangeably, and refer to those induced by adult cells. , cells with embryonic stem cell morphology and differentiation potential. The embryonic stem cells can express alkaline phosphatase and more than two antigens among surface antigens such as SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81.

The embryonic stem cell condition of the present invention can adopt commercially available embryonic stem cell medium culture or other culture conditions that can keep embryonic stem cell undifferentiated state (Thomson JA., Itskovitz-Eldor J., Shapiro SS., et al.Science.Nov 6 1998; 282(5391): 1145-1147; Xu, C., et al. Nat Biotechnol. 2001, 19(10): 971-4.). The preferred culture condition is to culture adult cells for 5-15 days with the standard culture medium of embryonic stem cells, and then change to the conditional culture medium of fibroblasts (according to literature Xu, C., et al.Nat Biotechnol.2001, 19( 10): 971-4. Production) of the above cells to cells forming embryonic stem cells. Those skilled in the art can use different cell culture media and adjust the number of days of cell culture according to the specific conditions of cell culture.

The adult cells of the present invention include adult stem cells, adult differentiated cells or a mixture of adult stem cells and adult differentiated cells.

The somatic cells in the present invention are preferably fibroblasts. Human adult cells can be obtained by known techniques. Adult cells that can be used in the present invention include, but are not limited to, epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes, nucleated red blood cells, macrophages, monocytes, Fibroblasts, cardiomyocytes and other muscle cells, etc.

The adult cells of the present invention can be derived from different species, including but not limited to human, gorilla, monkey, cow, horse, sheep, pig, dog, cat, guinea pig, rat, mouse, goldfish, Xenopus and Striped fish etc. The adult cells of the present invention can be isolated from the above species or purchased from relevant cell banks. Human adult cells are preferred.

The vectors of the present invention include viral vectors and plasmids. At present, plasmids have a very mature commercial supply. For example, pBR322 is 4.3kb in length and contains ampicillin and tetracycline resistance genes (Ampr and Tetr). There are restriction endonuclease sites in the middle of the ampicillin and tetracycline resistance genes, which are convenient for the insertion and screening of foreign genes. The pUC series plasmids, 2.6kb in length, are composed of the ampicillin resistance gene of pBR322, the replication initiation site and the lac Z gene fragment of Escherichia coli. The lac Z gene fragment includes the regulatory sequence of the 8-galactosidase gene and the enzyme The coding sequence for the first 146 amino acids. A multi-cloning site is added to the lac Z gene for the insertion of foreign genes for color screening. The difference between different members of the pUC series is that the nucleotide sequence of the multiple cloning site is different for different restriction endonuclease cutting and insertion of foreign genes. In addition, there is the pSP series. Generally, plasmids can only accommodate foreign DNA fragments less than 10kb, and are mainly used as subcloning vectors. The viral vectors include lentiviral vectors, retroviral vectors and adenoviruses. These vectors can be purchased from companies such as Invitrogen or Clontech.

The inserted transcription factor sequence of the present invention is the sequence of the open reading frame of Oct4, Nanog, Sox2, Lin28, c-myc and Klf4.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Description of drawings

Figure 1. Counting results of alkaline phosphatase-positive clones after transfer of specific factors.

The abscissa is 0.1×10 ⁶ initial human foreskin fibroblasts were used respectively with GFP lentivirus, lentivirus combination containing four factors Oct4, Nanog, Sox2 and Lin28 and six factors Oct4, Nanog, Sox2, Lin28, 17 days after c-Myc and Klf4 lentivirus combination infection, the counting results of alkaline phosphatase-positive clones.

The ordinate is the count of alkaline phosphatase-positive clones of embryonic stem cells.

Figure 2. Morphology of three types of human cells

From left to right, human embryonic stem cells (HuES17), human foreskin fibroblasts, and human iPS cells transformed with six factors (iPS-S).

Fig. 3. Results of determination of alkaline phosphatase activity in three kinds of human cells.

The description is the same as that in Figure 2.

Fig. 4. Measurement results of three kinds of human cell-specific surface antigens.

From left to right: human embryonic stem cells (HuES17), human foreskin fibroblasts (Foreskin fibroblast), human iPS cells transformed with six transcription factors (iPS-S); from top to bottom: SSEA3, SSEA4, Tra -Measurement results of 1-60 and Tra-1-81.

Figure 5. iPS-S cells have the pluripotency to differentiate into three embryonic germ layers.

RT-PCR was used for detection.

HuES17: human ES cells; FSF: human foreskin fibroblasts; iPS-S: human iPS cells transfected with six transcription factors; HuES17/EB: embryoid bodies derived from human embryonic stem cells; iPS-S/EB: human iPS Cell-derived embryoid bodies.

The ordinate is the change multiple, and there is a cutoff between 10 times and 60 times the ordinate in Figure 5B, so the fourth column also has a cutoff.

Figure 6. The GFP lentiviral vector constructed by the inventor

The exogenous gene is enhanced green fluorescent protein (EGFP).

Figure 7. Oct4 lentiviral vector.

Insert Oct4 lentiviral vector; for other lentiviral vectors, just replace Oct4 with Nanog, Sox2, Klf4, C-myc or Lin28, so other vector diagrams are omitted.

Detailed ways

Preparation of Example 1 Class Embryonic Stem Cells

Coding regions of specific genes (Oct4, Nanog, Sox2, Lin28, c-myc, and Klf4) were amplified by polymerase chain reaction (PCR) from total human cDNA, and ligated into lentiviral vectors (construct As shown in Figure 7, Invitrogen Company), so that it can be brought into cells by the produced lentivirus for expression. Oct-4 (also known as Oct-3, Pou5Fl), GenBank accession number is NM_002701; Nanog, GenBank accession number is NP_079141; Sox2, GenBank accession number is NP_003097; Lin28, GenBank accession number is NP_078950; is NP_002458; Klf4, GenBank accession number is NP_004226. Amplification primers are listed in Table 1:

Primer name Primer sequence lin28 5' primer SEQ ID NO: 1 atgggctccgtgtccaaccag lin28 3' Primer SEQ ID NO: 2 tcaattctgtgcctccgggag C-myc 5' primer SEQ ID NO: 3 atgcccctcaacgttagcttca C-myc 3' Primer SEQ ID NO: 4 ttacgcacaagagttccgtagc klf4 5' primer SEQ ID NO: 5 atgaggcagccacctggcgag klf4 3' primer SEQ ID NO: 6 ttaaaaatgcctcttcatgtg

Table 1

clone formation

1. Purpose: To reprogram differentiated cells to form embryonic stem cell-like clones by introducing specific transcription factors

2. Method

1. Infect 0.5× ¹⁰⁶ neonatal foreskin fibroblasts (purchased from ATCC, catalog number is CRL-2097) with the constructed lentivirus, and the experiment is divided into 3 groups:

a. Blank control group: lentivirus carrying green fluorescent protein (GFP);

b. Experimental group 1: a lentivirus combination containing 4 factors, Oct4, Nanog, Sox2 and Lin28;

c. Experimental group 2: a lentivirus combination containing 6 factors, Oct4Nanog, Sox2, Lin28, c-Myc and Klf4,

2. After 24 hours, these cells were trypsinized and transferred to culture flasks lined with mouse embryonic fibroblasts, initially cultured with standard human embryonic stem cell culture medium, and replaced with conditioned medium after 10 days. According to the literature (Thomson JA., Itskovitz-Eldor J., Shapiro SS., et al. Science. Nov 6 1998; 282 (5391): 1145-1147 and Xu, C., et al. Nat Biotechnol. 2001, 19(10): 971-4.) Preparation. The culture medium of human embryonic stem cells is composed of: 80% DMEM/F12 culture medium (purchased from Invitrogen, catalog number: 11330057); 20% serum substitute (purchased from Invitrogen, catalog number: 10828028); 1mM L-glucose 0.1 mM β-mercaptoethanol; 1% non-essential amino acids (purchased from Invitrogen, catalog number: 11140050). The human embryonic stem cell line HuES17 comes from Harvard University; general cell culture products are purchased from Invitrogen, and the conditioned medium (Conditioned media, abbreviated as CM) is according to the literature (Xu, C., et al.Nat Biotechnol.2001, 19 (10 ): 971-4.) Preparation.

3. Results

There was no colony formation in the blank control group, but colony formation could be seen after 12 days in the experiment of transferring four factors. In the experiment of transferring 6 factors, colony formation could be seen after 7 days. 4 factors induced 0.1×10 ⁶ initial human foreskin fibroblasts to form 16±3 iPS cell colonies, and 6 factors induced 0.1×10 ⁶ initial human foreskin fibroblasts to form 166±6 iPS cell colonies ( see picture 1). The morphology of iPS cell colonies was indistinguishable from that of normal human embryonic stem cells (hESCs) HuES17 in our laboratory, and completely different from that of human foreskin fibroblasts (see Figure 2).

3. Conclusion

Introducing specific factors can indeed reprogram differentiated cells to form embryonic stem cell-like clones. Six factors are 10.4 times more efficient than four factors.

The mensuration of embodiment 2 alkaline phosphatase

1. Purpose: To identify whether the formed clone has the characteristics of embryonic stem cells by detecting the activity of alkaline phosphatase, and compare the results of the two experimental groups

2. Method

For the experimental group transferred with 4 factors in Example 1, clones were picked on the 26th day; while for the experimental group transferred with 6 factors, clones were picked on the 17th day, which is called iPS-S. The selected clones were cultured on mouse embryonic fibroblast trophoblasts using standard human embryonic stem cell culture media and methods (Thomson JA, Itskovitz-Eldor J, ShapiroSS, et al. Embryonic stem cell lines derived from human blastocysts.Science.Nov 61998; 282(5391):1145-1147) In order to quantify the efficiency of reprogramming, we selected a bottle of cells in each experiment and fixed them on day 17, and detected their alkaline phosphatase expression.

3. Results

In the experimental group transferred with 4 factors, 16±3 clones were positive for alkaline phosphatase out of 0.1×10 ⁶ initial fibroblasts; in the experimental group transferred with 6 factors, 0.1×10 ⁶ 166 ± 6 clones were positive for alkaline phosphatase in each starting fibroblast; no clones were formed in cells infected only with GFP lentivirus (see Figure 1).

4. Conclusion

Most of the formed clones have the characteristics of embryonic stem cells and express alkaline phosphatase (see Figure 3). Both H17 and iPS-S express alkaline phosphatase.

The determination of embodiment 3 specific surface antigen

1. Purpose: To further identify whether the obtained clone has the characteristics of embryonic stem cells by detecting whether it has the specific surface antigen of embryonic stem cells.

2. Method

Using literature (Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. Nov 6 1998; 282(5391): 1145-1147 and Xiao L, Yuan X, SharkisSJ. Activin A Maintains self-renewal and regulates fibroblast growth factor, Wnt, and bonemorphogenic protein pathways in human embryonic stem cells.Stem Cells.Jun2006; 24(6):1476-1486), respectively detect the embodiment by immunofluorescence 1 The expression of SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81, which are specific surface antigens of embryonic stem cells, in the obtained clones.

3. Results

The iPS cells produced in this experiment were positive for SSEA-3, SSEA-4, Tra-1-60 and Tra-a-81 antigens, while foreskin fibroblasts did not (see Figure 4).

4. Conclusion

It is further proved that the iPS cells generated in this experiment have the characteristics of human embryonic stem cells, that is, express specific markers of embryonic stem cells.

Expression identification of three germ layer marker genes in the embryoid body of embodiment 4

1. Purpose: To verify that the obtained clones have multi-tissue differentiation ability by the expression of three germ layer marker genes in embryoid bodies.

2. Method

In order to verify the pluripotency of iPS-S cells, we naturally differentiated them to form embryoid bodies (EBs) and detected the expression of different marker genes of the three germ layers by real-time quantitative PCR.

3. Results

We found that iPS-S cells were able to differentiate to form all three germ layers in the embryoid body because it expressed the alpha-feto protein of the endoderm, the Bra gene of the mesoderm and the Sox1 gene of the ectoderm (Fig. 5).

4. Conclusion

It is further proved that most of the clones formed have the characteristics of embryonic stem cells and have the potential to differentiate into different tissue cells.

In summary, our study shows that the transfer of 6 specific factors can form human iPS cells more efficiently and faster than the transfer of 4 specific factors. The iPS-S cells transformed with six specific factors can express some markers of undifferentiated human embryonic stem cells, and have the potential to differentiate into three different germ layers.

Sequence Listing

Claims (12)

1. A method for preparing induced pluripotent stem cells, comprising the steps of: (a) Six transcription factors were introduced into adult cells: Oct4, Nanog, Sox2, Lin28, c-myc and Klf4; (b) cultivating the above-mentioned adult cells under embryonic stem cell culture conditions to form cells having the morphology of embryonic stem cells. 2. The preparation method according to claim 1, characterized in that, the transcription factor is in the form of protein or mRNA or DNA. 3. the described preparation method of claim 1 is characterized in that, described step (a) is that Oct4, Nanog, Sox2, Lin28, the coding sequence of these 6 kinds of transcription factors of c-myc and Klf4 are respectively cloned on the carrier, The six vectors were then transformed into adult cells. 4. The preparation method according to claim 3, characterized in that, the carrier is a viral vector or a plasmid. 5. The preparation method according to claim 3 or 4, characterized in that, the vector is a lentiviral vector, a retroviral vector or an adenoviral vector. 6. The preparation method according to claim 4, characterized in that, the carrier is a lentiviral vector. 7. The preparation method according to claim 1, wherein the adult cells are from primates. 8. The preparation method according to claim 1 or 6, characterized in that the adult cells are human adult cells. 9. The preparation method according to claim 8, wherein said human adult cells are selected from epithelial cells, nerve cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes, One of nuclear erythrocytes, macrophages, monocytes, fibroblasts, and cardiomyocytes. 10. The preparation method according to claim 8, characterized in that the somatic cells are fibroblasts. 11. An induced pluripotent stem cell prepared by the preparation method according to claim 1. 12. A composition comprising induced pluripotent stem cells prepared by the preparation method according to any one of claims 1-4, 6-7, 9-10.

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