Application of MSI1 C-terminal protein short peptide in inducing and maintaining the naive state of human embryonic stem cells

Abstract

The present invention relates to the field of biotechnology, and specifically relates to the use of a C-terminal peptide of the MSI1 protein in inducing and maintaining the naive state of human embryonic stem cells. The present invention provides the use of a C-terminal peptide of the MSI1 protein in preparing a kit for inducing and maintaining the naive state of embryonic stem cells. The present invention discovers for the first time that a C-terminal peptide of the MSI1 protein can improve the conversion efficiency of human embryonic stem cells. Results demonstrate that after induction into the naive state, the C-terminal peptide of the MSI1 protein can maintain the totipotency of cells after 25 cell passages, prolong the potential of cells to form blastoids, and maintain the human stem cell state.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the application of an MSI1 C-terminal protein short peptide in inducing and maintaining the original state of human embryonic stem cells. The MSI1 C-terminal protein short peptides are MSI1138-362 obtained by amplifying the human MSI1 gene shown in NM002442.4 with BamH1-Flag-MSI1V138-F / EcoRV-Flag-MSI1-R and / or MSI1272-362 obtained by amplifying the human MSI1 gene shown in NM002442.4 with BamH1-Flag-MSI1V138-F / EcoRV-Flag-MSI1-R. The sequence of BamH1-Flag-MSI1V138-F is shown in SEQ ID NO.21, the sequence of BamH1-Fag-MSI1V272-F is shown in SEQ ID NO.22, and the sequence of EcoRV-Flag-MSI1-R is shown in SEQ ID NO.20. The embryonic stem cells are human embryonic stem cell H9 cell line. Background Art

[0002] Stem cells have two completely different states of pluripotency: state (original state) and Primed state (initial state, or state to be activated). The two states of primed and primed differ in proliferation rate, molecular characteristics, X chromosome inactivation status and growth factor dependence.

[0003] Stem cells in the primed and primed states have the ability to differentiate into three germ layer cell lines in vitro culture or in teratoma experiments, but studies have shown that only Embryonic stem cells in this state are omnipotent and can form chimeric embryos. In the tetraploid blastocyst injection of stem cells, i.e., tetraploid compensation experiment (the gold standard for testing stem cell omnipotence), only Stem cells in the primed state can grow and develop into individuals, while stem cells in the primed state cannot develop into mature individuals in tetraploid compensation experiments and are also very likely to undergo apoptosis in chimeric embryo experiments. These phenomena indicate that Stem cells in the primed state have higher pluripotency than those in the primed state.

[0004] The formation of heterologous chimeras, such as human-pig chimera embryos, is a great hope for solving the problem of organ transplant donor sources in the future. However, most of the existing human pluripotent stem cells are in the primed state and must be effectively induced to state, can have potential clinical application value.

[0005] Currently, induction Human embryonic stem cells in this state still have insufficient induction efficiency, incompleteness and instability, and will lose their State characteristics and other issues.

[0006] Therefore, it is necessary to develop new methods to improve human embryonic stem cells State induction efficiency and State stability methods to generate more stable human embryonic stem cells.

[0007] Chinese patent application CN201310239565.0 discloses a method for differentiating human skin stem cells into primordial germ cells in vitro. The method involves mechanically isolating human skin stem cells, culturing them in vitro, and then using embryoid bodies to differentiate them. Following differentiation, the cells are induced for 14 days in vitro with cytokines such as bone morphogenetic protein-4, stem cell growth factor, epidermal growth factor, and basic fibroblast growth factor to obtain primordial germ cell-like cells that express early germ cell genes. Subsequently, porcine follicular fluid is used for induction. The method yields primordial germ cell-like cells that exhibit normal primordial germ cell morphology and specific molecular marker expression. Cytofluorimetric analysis of the induced primordial germ cell-like cells revealed the expression of Stra8 and Dazl, indicating the ability to undergo early meiosis.

[0008] Chinese patent application: CN201710500403.6 discloses a method for establishing the differentiation of induced pluripotent stem cells derived from familial hereditary premature ovarian failure into primordial genital ridge cells. This method uses adult cells from patients with familial hereditary premature ovarian failure, reprograms them into induced pluripotent stem cells in vitro, and then induces them to differentiate into primordial genital ridge cells, the precursor cells of oocytes.

[0009] However, there is no report on the application of the MSI1 C-terminal protein short peptide of the present invention in inducing and maintaining the primitive state of human embryonic stem cells. Summary of the Invention

[0010] The purpose of the present invention is to provide a MSI1 C-terminal protein short peptide in human embryonic stem cells to address the deficiencies of the existing technology. State induction and Application in state maintenance: All human embryonic stem cells used in the present invention are commercially available.

[0011] To achieve the above object, the technical solution adopted by the present invention is:

[0012] In a first aspect, the present invention provides the use of a short peptide of the MSI1 C-terminal protein in preparing a kit for inducing embryonic stem cells into a naive state.

[0013] In a second aspect, the present invention provides the use of a short peptide of the MSI1 C-terminal protein in preparing a kit for maintaining the original state of embryonic stem cells.

[0014] Preferably, the embryonic stem cells comprise human embryonic stem cells.

[0015] In a third aspect, the present invention provides the use of a short peptide of the MSI1 C-terminal protein in preparing a kit for forming heterologous chimeric embryos.

[0016] Preferably, the heterologous chimeric embryo comprises a human-pig chimeric embryo.

[0017] In a fourth aspect, the present invention provides a method for increasing the naive state induction rate and the naive state stability of embryonic stem cells, characterized in that it comprises the step of using a short peptide of the MSI1 C-terminal protein.

[0018] Preferably, the MSI1 C-terminal protein short peptide can promote the conversion efficiency of embryonic stem cells to the naive state.

[0019] Preferably, the MSI1 C-terminal protein peptide can maintain the stability of the original state of stem cells for 25 generations.

[0020] The advantages of the present invention are:

[0021] It was first discovered that a short peptide from the C-terminal protein of MSI1 can promote the growth of human embryonic stem cells Conversion efficiency. The experimental results show that in the The MSI1 C-terminal protein short peptide can be maintained after 25 cell passages Cell totipotency, prolonged The potential of cells to form blastoids and maintain human stem cells status, to enhance human embryonic stem cells State induction efficiency and State stability provides a new method for generating more stable Human embryonic stem cells in this state have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Attachment Figure 1 The results demonstrate the successful construction of the human embryonic stem cell line H9-Clone8, which endogenously expresses the MSI1 C-terminal short peptide.

[0023] A) Schematic diagram of gene-edited human embryonic stem cells endogenously expressing the MSI1 C-terminal short peptide and detection primers;

[0024] B) Genomic PCR analysis confirmed the deletion of the MSI1 gene in the H9-Clone8 cell line;

[0025] C) Immunofluorescence detection of the N-terminus of MSI1 confirmed that the MSI1 expressed in the H9-Clone8 cell line lacked the N-terminus;

[0026] D) Immunofluorescence detection of the C-terminus of MSI1 confirmed that the MSI1 expressed in the H9-Clone8 cell line has a C-terminus.

[0027] Attachment Figure 2 It is confirmed that human embryonic stem cells endogenously expressing the MSI1 C-terminal protein short peptide have higher Induction efficiency, where:

[0028] A) Human embryonic stem cell line H9 was induced using 5i / L / A The results showed that both the wild-type human embryonic stem cell line H9-WT and the human embryonic stem cell line H9-Clone8 endogenously expressing the MSI1 C-terminal short peptide could be induced into state;

[0029] B) QPCR assay confirmed that the human embryonic stem cell line H9-Clone8 endogenously expressed the MSI1 C-terminal short peptide in After the state was induced, the stem cell pluripotency marker genes POU5F1, NANOG, KLF4 and The status marker genes TBX3 and DNMT3L had higher expression;

[0030] C) Cloning experiments confirmed that the human embryonic stem cell line H9-Clone8 endogenously expressing the MSI1 C-terminal short peptide has stronger cloning ability.

[0031] Attachment Figure 3 This indicates that the MSI1 C-terminal protein short peptide can improve Induced efficiency and prolonged The potential of cells to form blastocysts is maintained status, where:

[0032] A) Western blot analysis of the H9-Flag-MSI1 cell line overexpressing the full-length MSI1, the H9-Flag-MSI1V138 cell line overexpressing the MSI1 C-terminal peptide MSI1138-362, and the H9-Flag-MSI1V272 cell line overexpressing the MSI1 C-terminal peptide MSI1272-362. The results confirmed the successful construction of human embryonic stem cell lines overexpressing the MSI1 C-terminal peptide.

[0033] B) QPCR assay confirmed that human embryonic stem cells overexpressing the MSI1 C-terminal protein short peptide After induction, the stem cell pluripotency marker genes POU5F1 and NANOG The status marker genes DNMT3L and ZFP42 had higher expression;

[0034] C) Human embryonic stem cells in this state were passaged to passage 25 and then induced into blastocysts. The results showed that the control cell line H9-Vector and the cell line overexpressing full-length MSI1 H9-Falg-MSI1 could not form blastocysts, while the cell line H9-Flag-MSI1V138 overexpressing the MSI1 C-terminal protein short peptide MSI1138-362 and the cell line H9-Flag-MSI1V272 overexpressing the MSI1 C-terminal protein short peptide MSI1272-362 could still form blastocysts.

[0035] Attachment Figure 4 The human MSI1 gene and amino acid sequence are NCBI database ID: NM_002442.4. DETAILED DESCRIPTION

[0036] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention record, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the application's appended claims.

[0037] Example 1

[0038] 1 Methods and Results

[0039] (1) Gene editing to obtain human embryonic stem cells that endogenously express the MSI1 C-terminal protein short peptide

[0040] The gRNA sites used in gene editing are as follows Figure 1 As shown in A. Used Invitrogen TM The Precision gRNA Synthesis Kit (A29377) synthesizes gRNA targeting the MSI1 gene. The gRNA sequence is:

[0041] gRNA1:5'-TGGGGCGCGTCAGTCTCCAT-3'(SEQ ID NO.1)

[0042] gRNA2: 5'-CTCACCTCGGTGCCGGTTGG-3' (SEQ ID NO. 2).

[0043] Mix the synthesized gRNA and Cas9 protein according to the system

[0044] See Table 1 for components of tube 1 and Table 2 for components of tube 2

[0045] Table 1: gRNA1

[0046]

[0047] Table 2: gRNA2

[0048]

[0049] Mix the components in the tube evenly and place them at room temperature for 5-20 minutes to allow the gRNA to bind to the Cas9 protein. Then mix tube 1 and tube 2 and place them on ice for later use.

[0050] Digest human embryonic stem cells using Accutase (ThermoA1110501) for 5 minutes at 37°C. Pipette repeatedly to separate cells. Add two volumes of PBS to terminate digestion, transfer to a centrifuge tube, and centrifuge at 500g for 3 minutes. Resuspend the cells in 1 mL of PBS, wash once, and count. Take 2 × 10 5 Centrifuge the cells at 500g for 5 minutes. Carefully aspirate the supernatant as much as possible and add 10μL Resuspension Buffer R to resuspend the cells. Take 5μL of the resuspended cells and mix them with the gRNA-Cas9 protein complex. Use a 10μL electroporator to aspirate the cell-gRNA-Cas9 protein mixture and place it in the electroporation chamber. The electroporation parameters are 1200V / 20ms / 2plus (electroporation system is Neon, Thermo MPK5000). Transfer the electroporated cells to one well of a six-well plate containing preheated 10μM Y27632mTeSR1 culture medium. The next day, change to mTeSR1 culture medium without Y27632. After about 7 days, mark the circular single colony and use the pipette tip to scrape off a portion of the cells to extract the genome.

[0051] Verify genomic knockout using PCR. Primer sequences are:

[0052] Verify successful genome deletion primers:

[0053] Cut-Forward: 5'-CAGGGACCTGAGAGGGAAGA-3'(SEQ ID NO.3)

[0054] Cut-Reverse: 5'-ATCCAGCCCCACTCTCATCT-3'(SEQ ID NO.4)

[0055] The resulting PCR product was 950 bp or less in size.

[0056] Verify that the genome was not successfully deleted from the primers:

[0057] Uncut-Forward:5'-AAGGAGTGTCTGGTGATGCG-3'(SEQ ID NO.5)

[0058] Uncut-Reverse: 5'-AGAGTCACAGAAGCCACCG-3'(SEQ ID NO.6)

[0059] The resulting PCR product was 608 bp in size.

[0060] PCR identification results Figure 1 As shown in B.

[0061] The cells that have successfully undergone deletion were picked out for further expansion and culture. Immunofluorescence staining was used to detect the expression of MSI1 protein in cells using antibodies that recognize the N-terminus of MSI1 protein (Abcam 52865) and antibodies that recognize the C-terminus of MSI1 protein (GeneTex GT2377). Figure 1 As shown in C and 1D: Compared with wild-type cells, the antibody detection that recognizes the N-terminus of MSI1 protein is negative, while the antibody detection that recognizes the C-terminus of MSI1 protein is positive, and the signal is localized in the cell nucleus.

[0062] (2) Detection of human embryonic stem cells endogenously expressing the MSI1 C-terminal protein short peptide Induction efficiency

[0063] Step 1:

[0064] Transfer human embryonic stem cells to feeder cells and culture them for two generations to allow the cells to adapt to the feeder culture environment. After the cells have adapted, aspirate the culture medium, add PBS to wash once, and use Accutase to digest the human embryonic stem cells in the feeder culture environment for 5 minutes at 37°C. Repeatedly blow to make single cells. Add twice the volume of PBS, transfer to a centrifuge tube, and centrifuge at 500g for 3 minutes. Remove the supernatant, add MEF culture medium to resuspend the cells, and transfer the cell suspension to a culture dish. Place it in a cell culture incubator and let it stand for 30 minutes to allow the feeder to adhere to the wall and remove the feeder cells. Take the supernatant of the culture dish, transfer it to a centrifuge tube, and centrifuge at 500g for 3 minutes. Resuspend the cells with mTeSR1 and count the cells. Transfer the cell suspension to a culture dish with feeder laid out one day in advance, 2×10 5 Cells / well of a 12-well plate, the culture medium is mTeSR1, and ROCK inhibitor Y27632 is added to the final concentration of 10μM. The next day, the mTeSR1 culture medium containing Y27632 is aspirated and replaced with 5i / L / A culture medium. The medium is changed every other day. There will be a peak of cell death during the induction period. The dead cells can be washed once with PBS. Dense spherical human embryonic stem cell clones can be seen around the 10th day. Cell clones with good morphology can be picked or directly subcultured and expanded. The clone morphology is as follows Figure 2A. The formula of 5i / L / A culture medium is shown in Table 3 below.

[0065] Table 3 Human embryonic stem cells Induction medium formula 5i / L / Amedium (50mL)

[0066]

[0067] Step 2:

[0068] Detection using RT-qPCR technology The expression level of marker genes in the state:

[0069] Using TRIzol TM RNA was extracted using the reagent (Thermo, 15596018). Uni All-in-One First-Strand cDNASynthesis SuperMix for qPCR(One-Step

[0070] cDNA was obtained by reverse transcription using the gDNARemoval (Beijing Quanshijin, AU341-02) kit. TM SYBR TM Green premix (Thermo, A25779) for QPCR detection The expression levels of status marker genes and the detection primer sequences are shown in Table 4, where GAPDH is the internal reference primer.

[0071] Table 4 Detection primer sequences

[0072]

[0073]

[0074] Test results such as Figure 2 As shown in B, human embryonic stem cells endogenously expressing the MSI1 C-terminal short peptide Induction, The marker gene has a higher expression. The crystal violet staining results are as follows Figure 2 The human embryonic stem cells endogenously expressing the MSI1 C-terminal short peptide shown in C have more clone formation. The above results show that human embryonic stem cells endogenously expressing the MSI1 C-terminal short peptide after gene editing have higher Induction efficiency.

[0075] (3) Construction of a human embryonic stem cell line overexpressing the MSI1 C-terminal protein peptide

[0076] Step 1: Construction of MSI1 C-terminal peptide overexpression vector

[0077] In this example, the overexpression vector is CS11-EF-MSC, which was purchased from Addgene and modified by replacing the original overexpressed gene with the multiple cloning site of PCDNA3.1 (purchased from Invitrogen).

[0078] In this example, primers specific for the MSI1 C-terminal short peptide were used to amplify the MSI1 fragment containing the Flag tag to express the Flag-fused MSI1 C-terminal short peptide recombinant protein.

[0079] The primers used are as follows:

[0080] BamH1-Flag-MSI1:

[0081] 5'-AAAGGATCCATGGATTACAAGGATGACGACGATAAGATGGAGACTG ACGCGCCC-3'(SEQ IDNO.19)

[0082] EcoRV-Flag-MSI1-R:

[0083] 5'-AAAGATATCTCACTTATCGTCGTCATCCTTGTAATCGTGGTACCCATT GGTGAAGGCTG-3'(SEQ ID NO.20)

[0084] The PCR product size is 1161 bp

[0085] BamH1-Flag-MSI1V138-F:

[0086] 5'-AAAGGATCCATGGATTACAAGGATGACGACGATAAGCTGATGTTCG ACAAAACCACC-3'(SEQID NO.21)

[0087] EcoRV-Flag-MSI1-R:

[0088] 5'-AAAGATATCTCACTTATCGTCGTCATCCTTGTAATCGTGGTACCCATT GGTGAAGGCTG-3'(SEQ ID NO.20)

[0089] The PCR product size is 756 bp

[0090] BamH1-Flag-MSI1V272-F:

[0091] 5'-AAAGGATCCATGGATTACAAGGATGACGACGATAAGGTGGAATGTAAGAAAGCTCAGCCAA-3'(SEQ ID.22)

[0092] EcoRV-Flag-MSI1-R:

[0093] 5'-AAAGATATCTCACTTATCGTCGTCATCCTTGTAATCGTGGTACCCATTGGTGAAGGCTG-3'(SEQID 20)

[0094] The PCR product size is 627 bp

[0095] Human embryonic stem cell RNA was extracted using conventional methods and then purified using Invitrogen TM RNA was reverse transcribed into cDNA using the SuperScript IV First-Strand Synthesis System. Using the cDNA as a template, fragments of 1125, 720, and 591 bp in length were amplified by PCR. These fragments were digested with the restriction endonucleases BamHI and EcoRV and constructed into the CS11-EF-MSC vector. Finally, the CS11-EF-MSI1V138 and CS11-EF-MSI1V272 vectors were constructed to express Flag-MSI1, Flag-MSI1V138, and Flag-MSI1V272 fusion proteins.

[0096] Step 3: Packaging lentivirus

[0097] Transfect 293FT cells with packaged lentivirus. Transfection of 293FT cells in a 100mm dish is used as an example. Remove 293FT cells at approximately 90%-100% confluency and remove the MEF culture medium. Wash once with 5mL of PBS. Add 3mL of 0.05% trypsin-EDTA to digest at 37°C for 5 minutes. Pellet until single cells are obtained. Transfer to a 15mL centrifuge tube, add 6mL of MEF-neutralized trypsin, and centrifuge at 500g for 5 minutes.

[0098] Resuspend the cells in MEF culture medium at a ratio of 1:2 for cell passage.

[0099] On the second day, the cell density was about 80%, at which point the cells were stretched and suitable for transfection and virus preparation.

[0100] To obtain high-quality virus using the Lipofectamine 3000 transfection system, add the following components as shown in Table 5.

[0101] Table 5 Composition of each tube

[0102]

[0103]

[0104] Mix tube A and tube B and let it stand at room temperature for 10 minutes.

[0105] Add the transfection mixture dropwise into the cell culture dish and gently mix the culture medium.

[0106] 6 h after transfection, the medium was changed to Lentivirus packing medium (DMEM, 5% FBS, 1×GlutaMAX, 100 mM Sodium Pyruvate).

[0107] 24 h after transfection, the first batch of supernatant containing virus was collected and placed at 4°C.

[0108] 52 h after transfection, the second batch of supernatant containing virus was collected and mixed with the supernatant collected in the first wave.

[0109] The supernatant was centrifuged at 3000 g for 10 min and filtered through a 0.45 μm syringe filter to remove cell debris.

[0110] One third of the volume of the supernatant was added with 40% PEG8000, and the mixture was placed on a rotary mixer and mixed at 4°C overnight.

[0111] Centrifuge at 16000g for 1 hour at 4°C, remove the supernatant, and resuspend the viral pellet in 500 μL of DMEM / F12. Aliquot the viral solution and freeze at -80°C.

[0112] Step 3: Viral infection of human embryonic stem cells

[0113] Aspirate the mTeSR1 medium and wash once with PBS. Digest the hESCs with Accutase for 5 minutes at 37°C. Pellet the cells repeatedly until they are single cells. Add two volumes of PBS to terminate the digestion. Transfer the cells to a centrifuge tube and centrifuge at 500g for 3 minutes.

[0114] Resuspend the cells in mTeSR1 culture medium and count them.

[0115] Take 2×10 5 The cells were seeded into 24-well plates pre-coated with Matrigel. Y27632 was added to the culture medium at a final concentration of 10 μM to prevent cell apoptosis.

[0116] Add 100 μL of virus solution and mix well with the cells. After 12 h, change the solution to remove dead cells.

[0117] Western blot was used to detect overexpression. Figure 3The fusion proteins shown in A can all be overexpressed in human embryonic stem cells.

[0118] (4) Detection of human embryos overexpressing the MSI1 C-terminal protein peptide State induction efficiency

[0119] Refer to the method of step 1 of method (2) to induce the cells to Status, then refer to method (2) step 2 for detection The expression level of the status marker gene. Figure 3 As shown in B, human embryonic stem cells overexpressing Flag-MSI1V138 and Flag-MSI1V272 have higher expression levels than wild-type cells. This indicates that overexpressing Flag-MSI1V138 and Flag-MSI1V272, which are short peptides of MSI1 C-terminus, can improve the expression of human embryonic stem cells. Induction efficiency.

[0120] (5) For advanced algebra (25 generations) Blastocyst-like induction of human embryonic stem cells

[0121] Method (4) was used to overexpress the MSI1 C-terminal short peptide Human embryonic stem cells were passaged for 25 generations. Accutase was then used to digest the cells and break the cell clumps into single cells. After centrifugation, the pellet was resuspended and counted in blastocyst-like induction medium (DMEM / F12, 1×N2, 1×B27+, 1.5mM PD0325901, 1mM A83-01, 10mM Y-27632). 200 cells were transferred per well into a U-bottom low-adsorption 96-well plate and centrifuged at 400g for 3 minutes. Blastocyst-like formation was observed after 48 hours of culture in a cell culture incubator. Figure 3 C shows wild type and overexpression of MSI1 full-length protein Human embryonic stem cells cannot form blastocysts, while cells overexpressing the MSI1 C-terminal peptide Human embryonic stem cells can form blastocysts. This indicates that the MSI1 C-terminal protein can stabilize human embryonic stem cells. state, so that it still has pluripotency after 25 generations

[0122] 2 Conclusion

[0123] Results Figure 1-3 .in, Figure 1 This showed that the human embryonic stem cell line H9-Clone8 endogenously expressing the MSI1 C-terminal short peptide was successfully constructed. Figure 2 This indicates that human embryonic stem cells endogenously expressing the MSI1 C-terminal peptide have a higher Induction efficiency. Figure 3This indicates that the MSI1 C-terminal protein short peptide can improve Induced efficiency and prolonged The potential of cells to form blastocysts is maintained status.

[0124] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principles of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. SEQUENCE LISTING <110> Shanghai Tongji Hospital <120> Application of MSI1 C-terminal protein short peptide in inducing and maintaining the naive state of human embryonic stem cells <130> / <160> twenty two <170> PatentIn version 3.3 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 tggggcgcgt cagtctccat 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 ctcacctcgg tgccggttgg 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 cagggacctg agagggaaga 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 atccagcccc actctcatct 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <400> 5 aaggagtgtc tggtgatgcg 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <400> 6 agagttcaca gaagccaccg 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 ctaggcaaac ccaccccact 20 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <400> 8 ttcagcaaac acctgctgga c 21 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <400> 9 gatgctcacc ccaccttctt 20 <210> 10 <211> twenty one <212> DNA <213> Artificial sequence <400> 10 tttctcacct gtgtgggttc g 21 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <400> 11 agagtggtga cggagacagg 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <400> 12 aagcgatcaa gcagcgacta 20 <210> 13 <211> twenty three <212> DNA <213> Artificial sequence <400> 13 gaggctaaag aactttggga tca 23 <210> 14 <211> 19 <212> DNA <213> Artificial sequence <400> 14 catttcgggg tcggcctta 19 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <400> 15 tctcaagctc cgtttcaccc 20 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <400> 16 acttgtcctt acatggggcg 20 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <400> 17 tcggagtcaa cggatttggt 20 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <400> 18 ttcccgttct cagccttgac 20 <210> 19 <211> 54 <212> DNA <213> Artificial sequence <400> 19 aaaggatcca tggattacaa ggatgacgac gataagatgg agactgacgc gccc 54 <210> 20 <211> 59 <212> DNA <213> Artificial sequence <400> 20 aaagatatct cacttatcgt cgtcatcctt gtaatcgtgg tacccattgg tgaaggctg 59 <210> 21 <211> 57 <212> DNA <213> Artificial sequence <400> 21 aaaggatcca tggattacaa ggatgacgac gataagctga tgttcgacaa aaccacc 57 <210> 22 <211> 61 <212> DNA <213> Artificial sequence <400> 22 aaaggatcca tggattacaa ggatgacgac gataaggtgg aatgtaagaa agctcagcca 60 a 61

Claims

1. Use of a vector overexpressing a short peptide of the MSI1 C-terminal protein in a kit for preparing a kit for inducing embryonic stem cells into a naive state, characterized in that: The MSI1 C-terminal protein short peptides are two MSI1 C-terminal protein short peptides, MSI1 138-362 obtained by amplification using BamH1-Flag-MSI1V138-F / EcoRV-Flag-MSI1-R and / or MSI1272-362 obtained by amplification using BamH1-Flag-MSI1V272-F / EcoRV-Fag-MSI1-R, using the human MSI1 gene shown in NM002442.4 as a template; the BamH1-Flag-MSI1V138-F sequence is shown in SEQ ID NO.21, the BamH1-Fag-MSI1V272-F sequence is shown in SEQ ID NO.22, and the EcoRV-Flag-MSI1-R sequence is shown in SEQ ID NO.20; and the embryonic stem cells are human embryonic stem cell H9 cell line.

2. Use of a vector overexpressing a short peptide of the MSI1 C-terminal protein in the preparation of a kit for maintaining the original state of embryonic stem cells, characterized in that: The MSI1 C-terminal protein short peptides are two MSI1 C-terminal protein short peptides, MSI1 138-362 obtained by amplification using BamH1-Flag-MSI1V138-F / EcoRV-Flag-MSI1-R and / or MSI1272-362 obtained by amplification using BamH1-Flag-MSI1V272-F / EcoRV-Fag-MSI1-R, using the human MSI1 gene shown in NM002442.4 as a template; the BamH1-Flag-MSI1V138-F sequence is shown in SEQ ID NO.21, the BamH1-Fag-MSI1V272-F sequence is shown in SEQ ID NO.22, and the EcoRV-Flag-MSI1-R sequence is shown in SEQ ID NO.20; and the embryonic stem cells are human embryonic stem cell H9 cell line.

3. A method for increasing the induction rate of embryonic stem cells in the naive state and improving the stability of the naive state, characterized in that: The method includes constructing a human embryonic stem cell line overexpressing an MSI1 C-terminal protein short peptide; the MSI1 C-terminal protein short peptide is amplified using the human MSI1 gene shown in NM002442.4 as a template, and two MSI1 C-terminal protein short peptides, MSI1 138-362 obtained by amplification using BamH1-Flag-MSI1V138-F / EcoRV-Flag-MSI1-R and / or MSI1272-362 obtained by amplification using BamH1-Fag-MSI1V272-F / EcoRV-Fag-MSI1-R; the BamH1-Flag-MSI1V138-F sequence is shown in SEQ ID NO. 21, the BamH1-Fag-MSI1V272-F sequence is shown in SEQ ID NO. 22, and the EcoRV-Flag-MSI1-R sequence is shown in SEQ ID NO. 20; and the embryonic stem cell is a human embryonic stem cell H9 cell line.

4. The method according to claim 3, characterized in that The MSI1 C-terminal protein short peptide can promote the conversion efficiency of embryonic stem cells to the original state.

5. The method according to claim 3, characterized in that The MSI1 C-terminal protein short peptide can maintain the stability of the original state of stem cells for 25 generations.

Images