Fusion protein for inducing pluripotent stem cells and application method thereof

Abstract

Provided is a protein coded by a gene related to cell totipotency and a transcriptional activation domain of a mammalian YAP protein or a fusion protein of a segment with a transcriptional control activity, a coding nucleotide sequence, an expression vector and a composition thereof, as well as a method for inducing the pluripotent stem cells by using the fusion protein.

Claims

1. A method for reprogramming somatic cells into induced pluripotent stem cells, the method comprising the following steps: providing an OCT4-YAP.sup.TAD plasmid, a SOX2-YAP.sup.TAD plasmid, a NANOG-YAP.sup.TAD plasmid, a Klf4 plasmid, and a packaging plasmid, wherein the OCT4-YAP.sup.TAD plasmid comprises a nucleotide sequence set forth in SEQ ID NO:1, the SOX2-YAP.sup.TAD plasmid comprises a nucleotide sequence set forth in SEQ ID NO:3, the NANOG-YAP.sup.TAD plasmid comprises a nucleotide sequence set forth in SEQ ID NO:5, and the Klf4 plasmid comprises a nucleotide sequence set forth in SEQ ID NO:7; transfecting 293T cells respectively with the OCT4-YAP.sup.TAD plasmid, the SOX2-YAP.sup.TAD plasmid, the NANOG-YAP.sup.TAD plasmid, the Klf4 plasmid, and the packaging plasmid to obtain viruses, wherein the viruses respectively contain the nucleotide sequence set forth in SEQ ID NO:1, the nucleotide sequence set forth in SEQ ID NO:3, the nucleotide sequence set forth in SEQ ID NO:5, and the nucleotide sequence set forth in SEQ ID NO:7; transfecting the somatic cells with the viruses; culturing the transfected somatic cells; and screening the cultured somatic cells to obtain the induced pluripotent stem cells.

2. The method according to claim 1, wherein the OCT4-YAP.sup.TAD plasmid, the SOX2-YAP.sup.TAD plasmid, the NANOG-YAP.sup.TAD plasmid, and the Klf4 plasmid are constructed using a pMXs vector, and the pMXs vector has a nucleotide sequence set forth in SEQ ID NO:11.

3. The method according to claim 1, wherein the somatic cells comprise skin fibroblasts, blood cells and oral epithelial cells.

4. A method for reprogramming somatic cells into induced pluripotent stem cells, the method comprising the following steps: providing an OCT4-YAP.sup.TAD fusion protein, a SOX2-YAP.sup.TAD fusion protein, a NANOG-YAP.sup.TAD fusion protein, and a Klf4 protein, wherein the OCT4-YAP.sup.TAD fusion protein comprises an amino acid sequence set forth in SEQ ID NO:2, the SOX2-YAP.sup.TAD fusion protein comprises an amino acid sequence set forth in SEQ ID NO:4, the NANOG-YAP.sup.TAD fusion protein comprises an amino acid sequence set forth in SEQ ID NO:6, and the Klf4 protein comprises an amino acid sequence set forth in SEQ ID NO:8; treating the somatic cells with the OCT4-YAP.sup.TAD fusion protein, the SOX2-YAP.sup.TAD fusion protein, the NANOG-YAP.sup.TAD fusion protein, and the Klf4 protein; culturing the treated somatic cells; and screening the cultured somatic cells to obtain the induced pluripotent stem cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Hereinafter, the embodiments of the present invention are illustrated in conjunction with drawings, wherein:

(2) FIG. 1 shows a schematic diagram for constructing Oct4-YAP.sup.TAD, SOX2-YAP.sup.TAD, NANOG-YAP.sup.TAD vectors of the present invention;

(3) FIG. 2 shows a schematic diagram of time of forming pluripotent stem cells by induction of the OySyNyK method;

(4) FIG. 3 shows results of GFP positive clone counting, and comparison of efficiency of inducing iPS by OySyNyK method and efficiency of inducing iPS by OSNK method, wherein, the counting was carried out on the 12.sup.th day for OSNK method, while the counting was carried out on the 7.sup.th day for OySyNyk method;

(5) FIG. 4 shows proportions of GFP positive cells analyzed by flow cytometer, and the results show that OySyNyK induction method is more rapid and efficient than the OSNK induction method;

(6) FIG. 5 shows GFP fluorescence pictures, which are used for comparison of different speeds and efficiencies of iPS induction by the conventional OSNK method and iPS induction by the OySyNyK method of the present invention;

(7) FIG. 6 shows results of alkaline phosphatase staining (NAP), and the results indicate that on the 7.sup.th day the OySyNyK method shows very high efficiency in iPS induction, while the OSNK method induces the generation of few iPS clones;

(8) FIG. 7 shows that the clones formed by induction of the OySyNyK method have a morphology after line establishment and passage similar to morphology of mES, and good state can be maintained after long-term of passage. Figure A shows the morphology of iPS clone of the 1.sup.st passage after line establishment, and Figure B shows the morphology of iPS clone of the 10.sup.th passage after line establishment.

(9) FIG. 8 shows expression identification of mRNA level of induced pluripotent stem cells, and as shown in the figure, the OySyNyK method can more rapidly and more efficiently induce the expression of endogenous pluripotent factors OCT4, SOX2, NANOG than the OSNK method;

(10) FIG. 9 shows expression identification of mRNA level of induced pluripotent stem cells, and as shown in the figure, the OySyNyK method can more rapidly and more efficiently induce the expression of endogenous pluripotent factors Eras, Dax1 than the OSNK method;

(11) FIG. 10 shows using the iPSc successfully induced by the OySyNyK method of the present invention to line establishment and passage, and in the 6 cell lines for line establishment as shown in the figure, exogenously expressed inducing genes are all silent;

(12) FIG. 11 shows the iPS induction carried out by the OySyNyK method of the present invention, in which bisulfite sequencing method can be used to detect the promoter regions of Oct4, Nanog cell factors change on the 3.sup.rd day from epigenetic inhibition state into activity expression state; FIG. A shows MEF sample; FIG. B shows FACS screened GFP positive cells of the 3.sup.rd day sample of iPS induction by OySyNyK method; FIG. C shows FACS screened GFP positive cells of the 5.sup.th day sample of iPS induction by OySyNyK method; FIG. D shows line establishment cell sample of iPS induction by OySyNyK method; and FIG. F shows mES positive control sample.

(13) FIG. 12 shows the generation of teratoma on immunodeficient mice injected with OySyNyK-iPSc of the present invention, in which HE stain exhibits structure of 3 blastoderms;

(14) FIG. 13 shows the generation of chimeric mice (FIG. A) by blastula injection of OySyNyK-iPSc of the present invention, and the generation of germline transmission mice (FIG. B).

DETAILED DESCRIPTION OF THE INVENTION

(15) The present invention is illustrated as follows by referring to specific examples. Those skilled in the art would understand that these examples are merely used for illustrating the present invention, rather than restricting the protection scope of the present invention in any way.

(16) Unless specifically pointing out, all reagents used in the following examples are of analytically pure grade, and commercially available.

(17) Embodiment 1: Induction of Pluripotent Stem Cells by Using OySyNyK Method

(18) 1.1 Main Reagents and Materials

(19) HEK293T culture media formula: high-glucose DMEM culture media, which was added with 10% fetal bovine serum as well as penicillin 100 U/ml and streptomycin 100 g/ml in final concentrations.

(20) MEF culture media formula: high-glucose DMEM culture media, which was added with 10% fetal bovine serum, 0.055 mM -mercaptoethanol, 2 mM L-glutamine, 0.1 mM nonessential amino acids, as well as penicillin 100 U/ml and streptomycin 100 g/ml in final concentrations.

(21) iPSC culture media formula: high-glucose DMEM culture media, which was added with 10% fetal bovine serum, 0.055 mM -mercaptoethanol, 2 mM L-glutamine, 0.1 mM nonessential amino acids, as well as penicillin 100 U/ml, streptomycin 100 g/ml, 50 g/ml vitamin C (Sigma), and LIF 1000 U/ml in final concentrations.

(22) 1.2 Experimental Methods

(23) Retroviruses were prepared by a conventional method known in the art, in which pMXs retroviral vector (purchased from Addgene) plasmids were separately Oct4-Yap.sup.TAD(Oy), Sox2-Yap.sup.TAD (Sy), Nanog-Yap.sup.TAD (Ny), Klf4 (K) and packaging plasmid, each in amount of 11 were used to transfect HEK293T cells by calcium phosphate precipitation method. After transfection for 12 h, fresh culture media was used for replacement. After transfection for 48 h, viral supernatant was collected, and filtered with 0.45 m PVDF filter. OCT4-GFP MEF (primary passage embryo fibroblasts as prepared from OCT4-GFP transgenic mice (purchased from Jackson Laboratory) with pregnancy for 13.5 days) was inoculated 16 h ahead in density of 510.sup.4 on a 12-well plate. The 4 kinds of viruses were mixed in a ratio of 1:1:1:1, and added with polybrene in final concentration of 8 g/ml, and infection was carried out by using a viral load of 2 ml per well. After infection for 24 h, iPS culture media were used for replacement, and this time was defined as the 0.sup.th hour. After about 246 hr, expression of single cell OCT4-GFP started, and OCT4-GFP positive iPSC clone started to appear on the 3.sup.rd day. The counting of iPSC was carried out or the monoclones were selected for passage on the 6.sup.th or 7.sup.th day.

(24) Embodiment 2: Induction of Pluripotent Stem Cells by Using OSNK Method

(25) HEK293T culture media formula: high-glucose DMEM culture media, which was added with 10% fetal bovine serum as well as penicillin 100 U/ml and streptomycin 100 g/ml in final concentrations.

(26) MEF culture media formula: high-glucose DMEM culture media, which was added with 10% fetal bovine serum, 0.055 mM -mercaptoethanol, 2 mM L-glutamine, 0.1 mM nonessential amino acids, as well as penicillin 100 U/ml and streptomycin 100 g/ml in final concentrations.

(27) iPSC culture media formula: high-glucose DMEM culture media, which was added with 10% fetal bovine serum, 0.055 mM -mercaptoethanol, 2 mM L-glutamine, 0.1 mM nonessential amino acids, as well as penicillin 100 U/ml, streptomycin 100 g/ml, 50 g/ml vitamin C (Sigma), and LIF 1000 U/ml in final concentrations.

(28) Retrovirus packaging: pMXs retroviral vector (purchased from Addgene) plasmids (which were separately Oct4 (O), Sox2 (S), Nanog (N), Klf4(K)), and Ecopac packaging plasmid, each in amount of 11 g, were used to transfect HEK293T cells by calcium phosphate precipitation method. After transfection for 12 h, fresh culture media was used for replacement. After transfection for 48 h, viral supernatant was collected, and filtered with 0.45 m PVDF filter. OCT4-GFP MEF was inoculated 16 h in advance in density of 510.sup.4 on a 12-well plate. The 4 kinds of viruses were mixed in a ratio of 1:1:1:1, and added with polybrene in final concentration of 8 g/ml, and infection was carried out by using a viral load of 2 ml per well. After infection for 24 h, iPS culture media were used for replacement, and this time was defined as the 0.sup.th hour. The expression of OCT4-GFP was observed on about the 4.sup.th day, OCT4-GFP positive iPSC clone started to appear on the 7.sup.th day, and the counting of iPS clones was carried out or monoclones were selected for passage between the 12.sup.th and 14.sup.th day.

(29) Embodiment 3: Morphological Structures and GFP Expression Identification of the Induced Pluripotent Stem Cells of the OySyNyK Method and the OSNK Method

(30) GFP reporter plasmids in which OCT4-GFP was driven to express by Oct4 promoter were integrated into genome of transgenic mice, which were used to indicate endogenous Oct4 gene expression and were an important index for iPS pluripotency.

(31) As shown in FIG. 5, the conventional OSNK induced pluripotent stem cells shows low efficiency and long time-consuming, in which a small amount OCT4-GFP cell expression started on about the 5.sup.th day, and obvious iPS clones were not observed on the 7.sup.th day. On the contrary, in the method of the present invention, OCT4-GFP expression appeared on the 1.sup.st day, primary formation of iPS clones started on the 3.sup.rd day, and a large amount of iPS clones in good state were formed on the 7.sup.th day. After line establishment and passages, the iPS clones formed by the method of the present invention showed no significant morphological difference in comparison with the iPS clones produced by induction of the conventional OSNK method.

(32) Embodiment 4: Alkaline Phosphatase Staining Identification of Induced Pluripotent Stem Cells of the OySyNyK Method and the OSNK Method

(33) Alkaline phosphatase staining was performed by using kits of Millipore, and has specific steps as follows:

(34) Cell culture solution was drawn off, moistened and washed with PBS once, fixed with PFA for 1-2 min. Fixing solution was drawn off, TBST was used for moistening and washing once. To each well of 12-well plate, 1 ml of alkaline phosphatase reagent was added, after 10-15 min of standing away from light at room temperature, staining solution was drawn off, PBS buffer solution was used for moistening and washing once, and the cells were finally stored in PBS solution.

(35) As shown in FIG. 6, on the 7.sup.th day, the iPS induced by the OSNK method showed no significant staining, while the iPS induced by the method of the present invention showed a large amount of alkaline phosphatase staining positive clones.

(36) Embodiment 5: Typical Pluripotent Stem Cell Growth Characteristics of Most of the iPS Generated by Induction of the OySyNyK Method

(37) FIG. 7 showed that after line establishment and passages, the iPS clones generated by induction of the OySyNyK method could well maintain clonal morphology similar to that of embryonic stem cells, and typical clonal morphology was still maintained after long-term of culture (consecutive 10 passages).

(38) Embodiment 6: Expression Identification of mRNA Levels of the Induced Pluripotent Stem Cells of the OySyNyK Method and the OSNK Method

(39) In the procedures for induction of iPS by the OSNK method and the OySyNyK method, cells were collected on the designated days, and lysed by Trizol, then RNA was extracted. 2 g of RNA was taken to perform inverse transcription to generate cDNA, then real-time PCR analysis was performed.

(40) Primer sequences are as follows:

(41) TABLE-US-00001 OCT4: (SEQIDNO:12) Forwarddirection: 5-TAGGTGAGCCGTCTTTCCAC-3 (SEQIDNO:13) Reversedirection: 5-GCTTAGCCAGGTTCGAGGAT-3 SOX2: (SEQIDNO:14) Forwarddirection: 5-AGGGCTGGGAGAAAGAAGAG-3 (SEQIDNO:15) Reversedirection: 5-CCGCGATTGTTGTGATTAGT-3 NANOG2: (SEQIDNO:16) Forwarddirection: 5-ATCCCTTCCCTCGCCATCAC-3 (SEQIDNO:17) Reversedirection: 5-GGCATTGATGAGGCGTTCC-3 Dax1 (SEQIDNO:18) Forwarddirection: 5-TGCTGCGGTCCAGGCCATCAAGAG-3 (SEQIDNO:19) Reversedirection: 5-GGGCACTGTTCAGTTCAGCGGATC-3 Eras (SEQIDNO:20) Forwarddirection: 5-TGCCTACAAAGTCTAGCATCTTG-3 (SEQIDNO:21) Reversedirection: 5-CTTTTACCAACACCACTTGCAC-3 GAPDH: (SEQIDNO:22) Forwarddirection: 5-AGTCAAGGCCGAGAATGGGAAG-3 (SEQIDNO:23) Reversedirection: 5-AAGCAGTTGGTGGTGCAGGATG-3

(42) As shown in FIG. 8, in comparison with the OSNK method, the OySyNyK method could more rapidly and efficiently induce the expression of endogenous pluripotent factors OCT4, SOX2, NANOG; as shown in FIG. 9, in comparison with the OSNK method, the OySyNyK method could more rapidly and efficiently induce the expression of endogenous pluripotent factors Eras, Dax1.

(43) Embodiment 7: Tests of In Vitro Formation of Teratoma

(44) After feeder was removed from iPS cells of induction and line establishment by the OySyNyK method, the cells in amount of 2*10.sup.6 were injected to SCID naked mice (purchased from Vital River) at upper part of hind leg, teratoma tissues were collected after about 2 months and subjected to hematoxylin and eosin staining. As shown in FIG. 12, the teratoma generated by the iPS of induction and line establishment by the OySyNyK method had structure of 3 blastoderms, which confirmed the totipotency of the iPS cell line.

(45) Embodiment 8: Silence of Retrovirus Exogenous Gene Expression

(46) The samples of 6 iPS cell lines obtained by induction of the OySyNyK method and line establishment and passage were taken on the 3.sup.rd day after the OySyNyK viruses infected MEF, the MEF cells were subjected to Trizol lysis, RNA was extracted, 2 g of RNA was subjected to inverse transcription to generate cDNA, then real-time PCR analysis was performed. The primer sequences were as follows:

(47) TABLE-US-00002 OCT4: (SEQIDNO:24) Forwarddirection: 5-GGGTGGACCATCCTCTAGAC-3 (SEQIDNO:25) Reversedirection: 5-CCAGGTTCGAGAATCCAC-3 SOX2: (SEQIDNO:26) Forwarddirection: 5-GGGTGGACCATCCTCTAGAC-3 (SEQIDNO:27) Reversedirection: 5-GGGCTGTTCTTCTGGTTG-3 NANOG: (SEQIDNO:28) Forwarddirection: 5-GGGTGGACCATCCTCTAGAC-3 (SEQIDNO:29) Reversedirection: 5-GGCATTGATGAGGCGTTCC-3 KLF4: (SEQIDNO:30) Forwarddirection: 5-GGGTGGACCATCCTCTAGAC-3 (SEQIDNO:31) Reversedirection: 5-GCTGGACGCAGTGTCTTCTC-3 GAPDH: (SEQIDNO:32) Forwarddirection: 5-AGTCAAGGCCGAGAATGGGAAG-3 (SEQIDNO:33) Reversedirection: 5-AAGCAGTTGGTGGTGCAGGATG-3

(48) As shown in FIG. 10, in the 6 iPS cell lines obtained by induction of the OySyNyK method and line establishment and passage, all exogenously expressed Oy, Sy, Ny, K were of expression silencing state.

(49) Embodiment 9: Rapid Demethylation of Oct4 and Nanog Promoters During iPS Induction of the OySyNyK Method

(50) Genomes of various samples were extracted, then treated with bisulfite. This test used the CpGenome Turbo Bisulfite Modification Kit of Millipore for treatment of samples. The products were subjected to PCR of Oct4 and Nanog promoter regions, the PCR products were subjected to blunt-end ligation with pEASY-T3 vector (purchased from Transgen), 10 clones were randomly selected for sequencing. The primer information was as follows:

(51) TABLE-US-00003 DNAmethylationanalysisofNANOGpromoter Forwarddirection: (SEQIDNO:34) 5-GATTTTGTAGGTGGGATTAATTGTGAATTT-3 Reversedirection: (SEQIDNO:35) 5-ACCAAAAAAACCCACACTCATATCAATATA-3 DNAmethylationanalysisofOCT4promoter: Forwarddirection: (SEQIDNO:36) 5-ATGGGTTGAAATATTGGGTTTATTTA-3 Reversedirection: (SEQIDNO:37) 5-CCACCCTCTAACCTTAACCTCTAAC-3

(52) The analysis of cytosine methylation states of Oct4, Nanog promoter regions showed that, in comparison with the conventional OSNK induction method, the OySyNyK method could bring about rapid demethylation in promoter regions of Oct4, Nanog within a time period as shorter as 1-2 days, and change them from expression inhibitory state into high-level expression active state.

(53) Embodiment 10: Use of the iPS Induced by OySyNyK Method in Generation of Chimeric Mice and Germline Transmission

(54) The iPS induced by the OySyNyK method was injected into 3.5 days blastulas of ICR mice, then blastulas were transplanted into uteruses of surrogacy female mice. In the produced off-spring mice, there were chimeric mice with mixed coat colors, then the chimeric male mice were mated with wild-type ICR female mice, and there were pure black mice among the produced off-spring mice.

(55) As shown in left side of the figure, the iPS induced by the OySyNyK method could successfully produce chimeric mice. As shown in right side of the figure, the produced chimeric mice could successfully perform germline transmission. These indicate that the iPS induced by the OySyNyK method had good pluripotency.