WO2021018296A1 - 一种通过体细胞重编程制备诱导多能干细胞的方法 - Google Patents

一种通过体细胞重编程制备诱导多能干细胞的方法 Download PDF

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WO2021018296A1
WO2021018296A1 PCT/CN2020/106327 CN2020106327W WO2021018296A1 WO 2021018296 A1 WO2021018296 A1 WO 2021018296A1 CN 2020106327 W CN2020106327 W CN 2020106327W WO 2021018296 A1 WO2021018296 A1 WO 2021018296A1
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cells
reprogramming
nanog
oct4
somatic cells
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French (fr)
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范靖
任芳
王安欣
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浙江霍德生物工程有限公司
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Priority to EP20847071.6A priority Critical patent/EP4008787A4/en
Priority to US17/597,970 priority patent/US20220325248A1/en
Priority to CN202080006440.1A priority patent/CN113454230B/zh
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Definitions

  • the invention belongs to the technical field of stem cells, and specifically relates to a method for preparing induced pluripotent stem cells through somatic cell reprogramming and induced pluripotent stem cells obtained therefrom.
  • the Yamanaka team invented a cocktail consisting of four genes Oct4, Sox2, Klf4 and c-Myc, which successfully reprogrammed terminally differentiated skin fibroblasts into induced pluripotent stem cells (induced pluripotent stem cells) through viral infection.
  • stem cells iPSCs
  • James Thomson used different four factor combinations (Oct4, Sox2, Nanog, and Lin28 combination) to successfully reprogram human fibroblasts into iPSCs through episomal plasmid transfection.
  • the above methods and techniques are relatively simple and stable, breaking through the moral and ethical limitations of using human embryonic stem cells in medicine, can solve the problem of immune rejection in cell transplantation therapy, and greatly expand the application potential of stem cell technology in clinical medicine.
  • the functional cells obtained from iPSCs technology and subsequent differentiation have great potential value in cell replacement therapy, pathogenesis research, and new drug screening.
  • TGF ⁇ can induce the expression of the EMT-related gene SNAIL in the early stage of reprogramming, which in turn induces EMT and hinders the reprogramming process.
  • TGF ⁇ receptor inhibitors can improve the efficiency of reprogramming and can be used to replace Sox2 and c in the Yamanaka four factors.
  • Cyclic AMP (cAMP) agonists can increase the expression of Oct4; glycogen synthase kinase (GSK) inhibitors can increase the expression of Nanog, thereby improving the reprogramming efficiency of specific cells.
  • GSK glycogen synthase kinase
  • Some methods even completely use chemical cocktails without involving any exogenously introduced transcription factors. However, this method has low reprogramming efficiency and is relatively time-consuming, and it is difficult to meet clinical needs. Some methods have not been used for reprogramming of human cells.
  • RNA or proteins can be reprogrammed to obtain iPSCs.
  • An object of the present invention is to provide a method for preparing induced pluripotent stem cells (iPSCs) through somatic cell reprogramming.
  • Another object of the present invention is to provide induced pluripotent stem cells (iPSCs) prepared by the above method.
  • iPSCs induced pluripotent stem cells
  • the inventors of the present invention found that by using only two introduced transcription factors as reprogramming inducing factors and three small molecule compounds as chemical inducers, it is possible to achieve simpler, more efficient, and more efficient treatment of somatic cells from different human sources than in the prior art.
  • the reprogramming method with wide application potential is successfully obtained iPSCs, thus completing the present invention.
  • the present invention provides a method for preparing induced pluripotent stem cells through somatic cell reprogramming, which includes the following steps: (1) Oct4 and Nanog are introduced as reprogramming inducing factors into somatic cells and then reprogrammed; 2) The partially or fully reprogrammed somatic cells obtained in step (1) are cultured in the presence of chemical inducers to obtain induced pluripotent stem cells (iPSCs), wherein the chemical inducers include TGF ⁇ receptor inhibitors, Cyclic AMP (cAMP) agonist and glycogen synthase kinase (GSK) inhibitor.
  • iPSCs induced pluripotent stem cells
  • the chemical inducers include TGF ⁇ receptor inhibitors, Cyclic AMP (cAMP) agonist and glycogen synthase kinase (GSK) inhibitor.
  • the TGF ⁇ receptor inhibitor is 616452, and/or the cyclic AMP (cAMP) agonist is Forskolin, and/or the glycogen synthase kinase (GSK) inhibitor is TD114-2.
  • cAMP cyclic AMP
  • GSK glycogen synthase kinase
  • Oct4 and Nanog as reprogramming inducing factors can be introduced into somatic cells in the form of their DNA, in the form of their RNA, or in the form of their protein products.
  • the somatic cell may be any cell known in the art, for example, skin-derived cells, blood-derived cells, urine-derived cells, liver cells, epithelial cells, Stomach cells, keratinocytes, etc.
  • the skin-derived cells may be skin fibroblasts.
  • the blood-derived cells may be erythroid progenitor cells.
  • the somatic cells are derived from humans, that is, human somatic cells.
  • the working concentration of the TGF ⁇ receptor inhibitor such as 616452 is 0.1-20 ⁇ M, more preferably 5-10 ⁇ M, and even more preferably 5 ⁇ M.
  • the working concentration of the cyclic AMP (cAMP) agonist such as Forskolin is 0.1-50 ⁇ M, more preferably 2-20 ⁇ M, still more preferably 10 ⁇ M.
  • the glycogen synthase kinase (GSK) inhibitor such as TD114-2 has a working concentration of 0.1-20 ⁇ M, more preferably 2-10 ⁇ M, and even more preferably 5 ⁇ M.
  • step (1) the vector containing the reprogramming inducing factors Oct4 and Nanog, respectively, is transformed into somatic cells by electrotransfection or chemical transfection, and then reprogramming is performed.
  • the transfection can be performed by electroporation.
  • the method of electroporation can be to add a reprogramming vector containing the inducing factors Oct4 and Nanog to the cell suspension, add the electroporation cup after mixing, and place the electroporation cup in the electroporation device to perform electroporation.
  • step (1) reprogramming is performed by infecting the somatic cell with a virus containing Oct4 and Nanog reprogramming inducing factors.
  • the virus is Sendai Virus.
  • the above-mentioned specific chemical inducer is added to the culture medium on the second day after the self-reprogramming inducer is introduced.
  • the present invention provides an induced pluripotent stem cell obtained by the method of the above-mentioned first aspect.
  • the present invention relates to a combination of two exogenously introduced inducers and three chemical inducers, and the combination is used for reprogramming somatic cells to prepare iPSCs.
  • the two exogenously introduced inducers are Oct4 And Nanog
  • the three chemical inducers are TGF ⁇ receptor inhibitors such as 616452, cyclic AMP (cAMP) agonists such as Forskolin, and glycogen synthase kinase (GSK) inhibitors such as TD114-2.
  • cAMP cyclic AMP
  • GSK glycogen synthase kinase
  • the Oct4 and Nanog can be introduced into somatic cells in the form of their DNA, RNA or protein.
  • the different forms of reprogramming inducing factors and the combination of three small molecular compounds as chemical inducers can significantly improve the reprogramming efficiency of human somatic cells and reduce the tumorigenicity of the obtained iPSCs.
  • 1A-C are bright field diagrams of iPSCs obtained by the method of Examples 1-3.
  • Figures 2A-E are the results of flow cytometry, showing the pluripotent cell markers expressed by iPSCs obtained by reprogramming skin fibroblasts.
  • A SSEA4;
  • B Tra-1-81;
  • C Tra-1-60;
  • D Oct4;
  • E Nanog.
  • Figures 3A-B show the results of (A) immunofluorescence staining and (B) qPCR identification of the three germ layers of iPSCs obtained by reprogramming skin fibroblasts.
  • 4A-C are bright field diagrams of iPSCs obtained by the methods of Examples 5-7.
  • Figures 5A-E are the results of flow cytometry, showing the pluripotent cell markers expressed by iPSCs obtained by reprogramming of erythroid progenitor cells.
  • A SSEA4;
  • B Tra-1-81;
  • C Tra-1-60;
  • D Oct4;
  • E Nanog.
  • Figures 6A-B show the results of (A) immunofluorescence staining and (B) qPCR identification of iPSCs derived from erythroid progenitor cell reprogramming.
  • Oct4 and Nanog are used as reprogramming inducing factors, and reprogramming is performed by introducing them into somatic cells.
  • Both Oct4 and Nanog are transcription factors that play an important role in maintaining pluripotency. It has been discovered in the prior art that a variety of transcription factors can be used to induce reprogramming of somatic cells into induced pluripotent stem cells, such as the aforementioned four-factor combination of Yamanaka, Oct4, c-Myc, Sox2 and Klf4, and James Thomson four-factor combination Oct4, Sox2, Nanog and Lin28, but the method of the present invention only uses Oct4 in Yamanaka four factors and Nanog in James Thomson four factors. In other words, the method of the present invention does not use transcription factors other than Oct4 and Nanog as reprogramming inducing factors.
  • Oct4 and Nanog can be introduced by methods known in the art for introducing transcription factors. Such methods include, but are not limited to, methods such as infecting somatic cells by introducing recombinant DNA vectors containing coding nucleotides, mRNA or RNA viruses to somatic cells to express Oct4 and Nanog inducing factors, or directly converting Oct4 and Nanog in protein form Introduced into somatic cells.
  • two reprogramming inducing factors are introduced into somatic cells in the form of DNA.
  • the nucleotide sequence encoding Oct4 and the nucleotide sequence encoding Nanog can be introduced into somatic cells.
  • the nucleotide sequence encoding Oct4 is a nucleotide sequence comprising SEQ ID NO: 6 or a nucleotide sequence consisting of SEQ ID NO: 6; or a nucleotide sequence comprising SEQ ID NO: 6; 6 has a nucleotide sequence that has at least 90% homology and encodes Oct4, or a nucleotide sequence consisting of a nucleotide sequence that has at least 90% homology with SEQ ID NO: 6 and encodes Oct4.
  • the nucleotide sequence encoding Nanog is a nucleotide sequence comprising SEQ ID NO: 15 or a nucleotide sequence consisting of SEQ ID NO: 15; or a nucleotide sequence comprising SEQ ID NO: 15 15 has a nucleotide sequence that has at least 90% homology and encodes Nanog, or a nucleotide sequence consisting of a nucleotide sequence that has at least 90% homology with SEQ ID NO: 15 and encodes Nanog.
  • the nucleotide sequence encoding Oct4 and the nucleotide sequence encoding Nanog can be placed in the same or different vectors. When placed in the same vector, the nucleotide sequence encoding Oct4 and the nucleotide sequence encoding Nanog may be under the control of the same or different regulatory sequences. The control sequence can be selected according to the type of target cell. In a specific embodiment, the nucleotide sequence encoding Oct4 and the nucleotide sequence encoding Nanog are placed in the same vector, such as pcDNA3.1.
  • the recombinant vector may also contain other elements, such as EBNA1 coding sequence and OriP sequence to improve the replication efficiency of the plasmid in the cell. In a specific embodiment, the sequence of the recombinant vector used in the present invention is shown in SEQ ID NO: 13 or SEQ ID NO: 24.
  • Methods of delivering a vector containing a target nucleotide into somatic cells are known in the art, including but not limited to electroporation, gene gun, lipofection, and calcium-mediated transfection. In a specific embodiment, electroporation is used.
  • the above-mentioned Oct4 and Nanog inducing factors can be expressed in vitro, and after obtaining the corresponding proteins, they can be introduced into differentiated cells, thereby achieving the purpose of the present invention.
  • Techniques for introducing proteins into cells are well known in the art, including but not limited to Tat-delivery and related techniques, electrotransfection (nuclear transfection), protein and cell ligand binding.
  • the DNA sequences of the above-mentioned Oct4 and Nanog inducing factors can be transcribed in vitro, and then the obtained mRNA can be directly introduced into differentiated cells to express in the cells to produce corresponding proteins. So as to achieve the purpose of the present invention.
  • Reprogramming can also be performed by infecting somatic cells with Sendai Virus containing RNA of two inducing factors.
  • Sendai Virus is a non-integrating virus that does not integrate into the genome of the infected cell and has relatively high safety.
  • TGF ⁇ receptor inhibitors TGF ⁇ receptor inhibitors
  • cyclic AMP (cAMP) agonists TGF ⁇ receptor inhibitors
  • GSK glycogen synthase kinase
  • iPSCs iPSCs are finally formed.
  • a TGF receptor inhibitor, a cyclic AMP (cAMP) agonist, and a glycogen synthase kinase (GSK) inhibitor are used.
  • the TGF ⁇ receptor inhibitor is 616452, and its chemical name is [2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5 -Naphthyridine](2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine, CAS No: 446859-33-2).
  • it is used at a working concentration of 0.1-20 ⁇ M, more preferably 5-10 ⁇ M, still more preferably 5 ⁇ M, such as being added to the culture medium at this concentration.
  • the cyclic AMP (cAMP) agonist is Forskolin, whose chemical name is [(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-3-vinyl-6,10,10b -Trihydroxy-3,4a,7,7,10a-Pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f]chromene-5- Ethyl]acetate ([(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-3-ethenyl-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1 -oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f]chromen-5-yl]acetate, CAS No: 66575-29-9).
  • it is used at a
  • the glycogen synthase kinase (GSK) inhibitor is TD114-2, and its chemical name is 6,7,9,10,12,13,15,16,18,19-decahydro-5 ,29:20,25-Dimethylene-26H-dibenzo[n,t]pyrrole[3,4-q][1,4,7,10,13,22]tetraoxadiazepine- 26,28(27H)-Diketone(6,7,9,10,12,13,15,16,18,19-Decahydro-5,29:20,25-dimetheno-26H-dibenzo[n,t] pyrrolo[3,4-q][1,4,7,10,13,22]tetraoxadiazacyclotetracosine-26,28(27H)-dione, CAS No: 436866-52-3).
  • it is used at a working concentration of 0.1-20 ⁇ M, more preferably
  • the medium used in the method of the present invention can be selected by those skilled in the art based on existing knowledge according to the type of cells to be cultured.
  • Example 1 Use of vectors encoding reprogramming factors to induce reprogramming of skin cells
  • PBS phosphate buffered solution
  • FBS fetal bovine serum
  • the medium can be changed.
  • the amount of DMEM medium is increased to 3mL, and the medium is changed every 1-3 days.
  • the method for constructing the reprogramming vector is as follows: 1 Using plasmid (Addgen#20922) as a template, using KOD-Plus-Neo (TOYOBO#KOD-401) high-fidelity enzyme and primers F1/R1, F2/R2, F3/ R3 was amplified to obtain the EF-1 ⁇ promoter fragment, Oct4 coding sequence and Nanog coding sequence (the primer sequence and amplified sequence are shown in Table 1 below). 2Using pcDNA3.1(-) plasmid as template, using KOD-Plus-Neo (TOYOBO # KOD-401) high-fidelity enzyme and primer F4/R4 to amplify pcDNA3.1 fragment (see primer sequence and amplified sequence) Table 1) below.
  • the working concentration of small molecule compounds is as follows: the working concentration of TGF ⁇ receptor inhibitor 616452 is 5 ⁇ M, and the working concentration of cyclic AMP (cAMP) agonist Forskolin is 10 ⁇ M.
  • the working concentration of glycogen synthase kinase (GSK) inhibitor TD114-2 is 5 ⁇ M.
  • iPSC clones with cell morphology significantly different from skin fibroblasts can be observed (as shown in the upper panel of Figure 1A).
  • Single clones are selected to be inoculated into the feeder-free system for culture.
  • These iPSC clones with compact and clear borders formed by cells with smaller cell bodies have larger nuclei and higher nucleus-to-plasma ratio, which are typical iPSC morphology (As shown in the small picture at the bottom of Figure 1A).
  • Example 2 Use of vectors encoding reprogramming factors to induce reprogramming of skin cells
  • Example 2 Using the same operation steps as 1.1-1.9 in Example 1, fibroblasts derived from human skin tissue were used to perform the method of the present invention. The difference from Example 1 is that the construction of the reprogramming vector in step 1.8 is different. On the basis of the construct of Example 1, EBNA1 and OriP elements are added, so that the plasmid can be improved in somatic cells based on the EBNA1/OriP system.
  • the specific method of copying efficiency is as follows:
  • NEB#E2623 3Use NEBuilder HiFi DNA Assembly Bundle for Large Fragments (NEB#E2623) to connect pcDNA3.1 with EF-1 ⁇ promoter, Oct4 coding sequence, Nanog coding sequence, EBNA1 coding sequence and OriP sequence through homologous recombination, namely
  • the target vector is obtained, that is, pcDNA3.1-EF-Oct4-Nanog-EBNA1-OriP (SEQ ID NO: 24).
  • iPSC clones with a cell morphology significantly different from skin fibroblasts can be observed (as shown in the upper panel of Figure 1B).
  • Single clones are selected to be inoculated into the feeder-free system for culture.
  • These iPSC clones with compact and clear borders formed by cells with smaller cell bodies have larger nuclei and higher nucleus-to-plasma ratio, which are typical iPSC morphology (As shown in the small picture at the bottom of Figure 1B).
  • Example 3 Use of virus containing reprogramming inducing factor RNA to induce reprogramming of skin cells
  • fibroblasts derived from human skin tissue were used to perform the method of the present invention.
  • steps 1.7-1.9 in Example 1 the following step 3.7 is used to perform iPSC reprogramming on the virus using RNA containing reprogramming inducing factors.
  • the working concentration of small molecule compounds is as follows: the working concentration of TGF ⁇ receptor inhibitor 616452 is 5 ⁇ M, and the working concentration of cyclic AMP (cAMP) agonist Forskolin is 10 ⁇ M.
  • the working concentration of glycogen synthase kinase (GSK) inhibitor TD114-2 is 5 ⁇ M.
  • iPSC cell clones with cell morphology significantly different from skin fibroblasts can be observed (as shown in the upper panel of Figure 1C).
  • Single clones were selected and inoculated into the feeder-free system for culture. These clones formed by cells with smaller cell bodies are compact and have clear boundaries. The cells have larger nuclei and higher nuclear-to-cytoplasmic ratios, which are typical iPSC morphology (as shown in the figure). As shown in the small picture below 1C).
  • Example 4 Identification of iPSCs obtained by reprogramming of skin fibroblasts
  • the following molecular markers were analyzed on the obtained iPSCs by flow cytometry: SSEA4, Tra-1-81, Tra-1-60, Oct4 and Nanog.
  • SSEA4 Tra-1-81, Tra-1-60, Oct4 and Nanog.
  • the iPSCs obtained by the method of the present invention express the markers SSEA4, Tra-1-81, Tra-1-60, Oct4 and Nanog of human pluripotent stem cells, proving that the obtained cells have the characteristics of pluripotent stem cells .
  • the three germ layers were differentiated in vitro, and the expression of specific molecular markers in the three germ layers was detected by immunofluorescence staining and qPCR.
  • the results of immunofluorescence experiments using three markers showed that the obtained iPSCs can differentiate into cells of three germ layers.
  • Figure 3B shows the expression of specific molecular markers (endoderm: SOX17, mesoderm: MIXL1, ectoderm: PAX6) detected by qPCR, in which iPSCs highly express pluripotent stem cell molecular markers OCT4 and TRA-1 -81 (consistent with the results of the aforementioned flow cytometry analysis), and the three germ layers differentiated cells highly express the specific molecular markers of each germ layer (****p ⁇ 0.0001, use student's t-test). These results indicate that the iPSCs obtained are versatile.
  • Example 5 Use of vectors encoding reprogramming factors to induce reprogramming of peripheral blood cells
  • the reprogramming vector pcDNA3.1-EF-Oct4-Nanog (SEQ ID NO: 13) of factor Oct4 and Nanog mixed and added to the electroporation cup, placed the electroporation cup in the electroporation instrument, and performed electrotransformation at 820V, 20ms.
  • the medium used on the second day contains a combination of small molecule compounds.
  • the working concentration of small molecule compounds is as follows: the working concentration of TGF ⁇ receptor inhibitor 616452 is 5 ⁇ M, and the working concentration of cyclic AMP (cAMP) agonist Forskolin is 10 ⁇ M.
  • the working concentration of glycogen synthase kinase (GSK) inhibitor TD114-2 is 5 ⁇ M.
  • iPSC clones formed by adherent growth of cell clusters in suspension culture can be observed (as shown in the upper panel of Figure 4A). Single clones are selected to be inoculated into the feeder-free system for culture. These iPSC clones with compact and clear borders formed by cells with smaller cell bodies have larger nuclei and higher nucleus-to-plasma ratio, which are typical iPSC morphology (As shown in the small picture at the bottom of Figure 4A).
  • Example 6 Use of vectors encoding reprogramming factors to induce reprogramming of peripheral blood cells
  • the method of the present invention was performed using erythroid progenitor cells derived from human peripheral blood.
  • the reprogramming vector in step 4.6 uses the reprogramming vector pcDNA3.1-EF-Oct4-Nanog-EBNA1-OriP constructed as in Example 2 containing the inducing factors Oct4 and Nanog.
  • iPSC clones formed by adherent growth of cell clusters in suspension culture can be observed (as shown in the upper panel of Figure 4B). Single clones are selected to be inoculated into the feeder-free system for culture. These iPSC clones with compact and clear borders formed by cells with smaller cell bodies have larger nuclei and higher nucleus-to-plasma ratio, which are typical iPSC morphology (As shown in the small picture at the bottom of Figure 4B).
  • Example 7 Use of virus containing reprogramming inducing factor RNA to induce reprogramming of peripheral blood cells
  • the method of the present invention was performed using the same operation steps as in 5.1-5.5 in Example 5, using erythroid progenitor cells derived from human peripheral blood. Different from steps 4.6-4.7 in Example 4, the following step 6.6 is used to perform iPSC reprogramming on the virus using RNA containing reprogramming inducing factors.
  • the working concentration of small molecule compounds is as follows: the working concentration of TGF ⁇ receptor inhibitor 616452 is 5 ⁇ M, and the working concentration of cyclic AMP (cAMP) agonist Forskolin is 10 ⁇ M.
  • the working concentration of glycogen synthase kinase (GSK) inhibitor TD114-2 is 5 ⁇ M.
  • iPSC clones formed by adherent growth of cell clusters in suspension culture (as shown in the upper panel of Figure 4C) can be observed. Single clones are selected to be inoculated into the feeder-free system for culture. These iPSC clones with compact and clear borders formed by cells with smaller cell bodies have larger nuclei and higher nucleus-to-plasma ratio, which are typical iPSC morphology (As shown in the small picture at the bottom of Figure 4C).
  • Example 8 Identification of iPSCs obtained by reprogramming of erythroid progenitor cells
  • the following molecular markers were analyzed on the obtained iPSCs by flow cytometry: SSEA4, Tra-1-81, Tra-1-60, Oct4 and Nanog.
  • SSEA4 Tra-1-81, Tra-1-60, Oct4 and Nanog.
  • the iPSCs obtained by the method of the present invention express the markers SSEA4, Tra-1-81, Tra-1-60, Oct4 and Nanog of human pluripotent stem cells, proving that the obtained cells have characteristics of pluripotent stem cells .
  • the three germ layers were differentiated in vitro, and the expression of specific molecular markers in the three germ layers was detected by immunofluorescence staining and qPCR.
  • the results of immunofluorescence experiments using three markers showed that the obtained iPSCs can differentiate into cells of three germ layers.
  • Figure 6B shows the expression of specific molecular markers (endoderm: SOX17; mesoderm: MIXL1; ectoderm: PAX6) detected by qPCR, in which iPSCs highly express pluripotent stem cell molecular markers OCT4 and TRA-1 -81 (consistent with the results of the aforementioned flow cytometry analysis), and the three germ layers differentiated cells highly express the specific molecular markers of each germ layer (****p ⁇ 0.0001, use student's t-test). These results indicate that the iPSCs obtained are versatile.
  • the method for preparing iPSCs of the present invention reduces the transcription factors required for reprogramming to two, reduces tumorigenicity, and improves the transformation efficiency through the combination of three small molecule compounds.
  • the method of the present invention is simple, efficient, and easy to operate as a whole, and the iPSCs prepared by this method will be more suitable for clinical transformation and application.

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Abstract

提供了一种通过体细胞重编程制备诱导多能干细胞的方法以及由此得到的诱导多能干细胞。该方法包括将因子Oct4和Nanog作为重编程诱导因子导入体细胞后进行重编程；然后，将部分或完全重编程的体细胞培养在含有特定化学诱导剂的培养基中以得到诱导多能干细胞，该化学诱导剂包括TGFβ受体抑制剂、环AMP激动剂和糖原合成酶激酶抑制剂。

Description

一种通过体细胞重编程制备诱导多能干细胞的方法 技术领域
本发明属于干细胞技术领域，具体涉及一种通过体细胞重编程制备诱导多能干细胞的方法以及由此得到的诱导多能干细胞。
背景技术
2006年，Yamanaka团队发明了一种由Oct4、Sox2、Klf4和c-Myc四种基因构成的Cocktail，通过病毒感染的方式成功将终端分化的皮肤成纤维细胞重编程成为诱导多能干细胞(induced pluripotent stem cells，iPSCs)。一年后，James Thomson采用不同的四种因子组合(Oct4、Sox2、Nanog和Lin28组合)通过附加体质粒转染的方式成功将人的成纤维细胞重编程为iPSCs。以上方法技术相对简单和稳定，突破了在医学上使用人胚胎干细胞的道德伦理限制，可以解决细胞移植治疗中的免疫排斥问题，很大程度上拓展了干细胞技术在临床医学上的应用潜力。此外，iPSCs技术及后续分化得到的功能性细胞在细胞替代性治疗、发病机制的研究、新药筛选等方面具有巨大的潜在价值。
然而，随着iPSCs重编程技术的发展，通过外源引入转录因子进行iPSCs重编程的弊端和问题也逐渐凸显，如部分细胞重编程不完全、重编程效率低下、导入的原癌基因的致癌性等。因此，研究者们试图寻找四因子的替代者并尝试优化重编程系统，如替换具有致癌性的转录因子、添加提高重编程效率的转录因子或化合物，甚至完全摈弃转录因子而使用小分子化合物诱导体细胞重编程，从而在一定程度上避免潜在缺陷。
目前，多种小分子化合物已被发现能诱导多能干细胞的产生，这些小分子有的是通过抑制基因组甲基化作用，直接提高受体细胞被重编程的效率；有的则通过影响特定的信号通路，使重编程过程中产生的中间过渡型细胞和部分重编程的细胞转化为稳定的完全的诱导多能干细胞。比如TGFβ在重编程早期可诱导EMT相关基因SNAIL表达，进而诱导EMT，阻碍重编程进程；与此相反，TGFβ受体抑制剂可以提高重编程效率，并可用于替换Yamanaka四因子中的Sox2和c-Myc；环AMP(cAMP)激动剂能增加Oct4的表达；糖原合成酶激酶(GSK)抑制 剂能增加Nanog的表达，从而能提高特定细胞的重编程效率。有的方法甚至完全使用化学小分子混合物(Cocktail)而不涉及任何外源引入的转录因子。但这样的方法重编程效率较低，而且比较耗时，难以满足临床需求，有些方法尚未用于人的细胞的重编程。
迄今为止，研究人员已经在许多种不同类型的细胞上尝试了重编程技术，并取得了成功。包括利用四因子的基因、RNA或蛋白都能重编程得到iPSCs。
结合上述方法的特点，提供一种通过添加不同形式载体的重编程诱导因子以及小分子化合物的组合，从而能提高重编程效率，且安全高效的重编程人的体细胞得到iPSCs的制备方法具有重要意义。
发明内容
本发明的一个目的是提供一种通过体细胞重编程制备诱导多能干细胞(iPSCs)的方法。
本发明的另一个目的是提供一种通过上述方法制备的诱导多能干细胞(iPSCs)。
本发明的发明人发现通过仅仅使用两种导入的转录因子作为重编程诱导因子，配合三种小分子化合物作为化学诱导剂，能够对人体不同来源的体细胞实现比现有技术更加简单、高效、应用潜力广泛的重编程方法，成功获得iPSCs，由此完成了本发明。
因此，第一方面，本发明提供了一种通过体细胞重编程制备诱导多能干细胞的方法，包括如下步骤：(1)将Oct4和Nanog作为重编程诱导因子导入体细胞后进行重编程；(2)将步骤(1)获得的部分或完全重编程的体细胞在存在化学诱导剂的条件下培养以得到诱导多能干细胞(iPSCs)，其中，所述化学诱导剂包括TGFβ受体抑制剂、环AMP(cAMP)激动剂和糖原合成酶激酶(GSK)抑制剂。
优选地，所述TGFβ受体抑制剂是616452，和/或所述环AMP(cAMP)激动剂是Forskolin，和/或所述糖原合成酶激酶(GSK)抑制剂是TD114-2。
在本发明方法中，优选地，作为重编程诱导因子的Oct4和Nanog可以以其DNA的形式，以其RNA的形式，或者以其蛋白产物的形式导入体细胞中。
在本发明方法中，优选地，所述体细胞可以是本领域已知的任何细胞，例如， 皮肤来源的细胞，血液来源的细胞，也可以包括尿液来源的细胞，肝细胞，上皮细胞，胃细胞，角质细胞等。在一个更优选的实施方案中，所述皮肤来源的细胞可以是皮肤成纤维细胞。在另一个更优选的实施方案中，所述血液来源的细胞可以是红系祖细胞。
在本发明方法中，优选地，所述体细胞来源于人，即为人类体细胞。
在本发明方法中，优选地，所述TGFβ受体抑制剂如616452的工作浓度为0.1-20μM，更优选5-10μM，还更优选5μM。
在本发明方法中，优选地，所述环AMP(cAMP)激动剂如Forskolin的工作浓度为0.1-50μM，更优选2-20μM，还更优选10μM。
在本发明方法中，优选地，所述糖原合成酶激酶(GSK)抑制剂如TD114-2的工作浓度为0.1-20μM，更优选2-10μM，还更优选5μM。
在具体的实施方案中，在步骤(1)中通过将含有分别编码重编程诱导因子Oct4和Nanog的载体通过电转或化学转染的方式转入体细胞后进行重编程。例如，所述转染可以通过电转进行。其中，电转方式可为在细胞悬液中加入含有诱导因子Oct4和Nanog的重编程载体，混匀后加入电转杯中，将电转杯置于电转仪中，进行电转。
在另一个具体的实施方案中，在步骤(1)中通过将含有Oct4和Nanog重编程诱导因子的病毒感染所述体细胞以进行重编程。优选地，所述病毒为Sendai Virus。
在本发明方法中，优选地，自重编程诱导因子导入后第2天开始在培养基中添加上述特定化学诱导剂。
第二方面，本发明提供了一种诱导多能干细胞，其由上述第一方面的方法得到。
第三方面，本发明涉及外源导入的两种诱导因子和三种化学诱导剂的组合，该组合用于重编程体细胞以制备iPSCs的用途，所述外源导入的两种诱导因子为Oct4和Nanog，所述三种化学诱导剂为TGFβ受体抑制剂如616452，环AMP(cAMP)激动剂如Forskolin，糖原合成酶激酶(GSK)抑制剂如TD114-2组成。具体来说，所述Oct4和Nanog可以以其DNA、RNA或蛋白质的形式导入体细胞中。
在本发明中，重编程诱导因子的不同存在形式和作为化学诱导剂的三种小分 子化合物的组合能够显著提高人类体细胞的重编程效率，并降低所获iPSCs的成瘤性。
附图说明
图1A-C是实施例1-3的方法获得的iPSCs的明场图。
图2A-E是流式细胞仪的检测结果，显示皮肤成纤维细胞重编程获得的iPSCs表达的多能性细胞标志物。(A)SSEA4；(B)Tra-1-81；(C)Tra-1-60；(D)Oct4；(E)Nanog。
图3A-B显示了皮肤成纤维细胞重编程获得的iPSCs三胚层分化的(A)免疫荧光染色和(B)qPCR鉴定的结果。
图4A-C是实施例5-7的方法获得的iPSCs的明场图。
图5A-E是流式细胞仪的检测结果，显示红系祖细胞重编程获得的iPSCs表达的多能性细胞标志物。(A)SSEA4；(B)Tra-1-81；(C)Tra-1-60；(D)Oct4；(E)Nanog。
图6A-B显示了红系祖细胞重编程获得的iPSCs三胚层分化的(A)免疫荧光染色和(B)qPCR鉴定的结果。
发明详述
在本发明的方法中，仅使用了Oct4和Nanog作为重编程诱导因子，通过将其导入体细胞进行重编程。Oct4和Nanog均为转录因子，在维持多能性中发挥重要作用。现有技术中发现了多种转录因子可用于诱导体细胞重编程为诱导多能干细胞，例如前文所述的Yamanaka四因子组合中的Oct4、c-Myc、Sox2和Klf4及James Thomson四因子组合中的Oct4、Sox2、Nanog和Lin28，但本发明的方法仅使用Yamanaka四因子中的Oct4和James Thomson四因子中的Nanog。换言之，本发明的方法不使用除Oct4和Nanog之外的转录因子作为重编程诱导因子。
可以通过本领域已知用于导入转录因子的方法来导入Oct4和Nanog。这样的方法包括但不限于，通过导入包含编码核苷酸的重组DNA载体、mRNA或RNA的病毒感染体细胞等方法使体细胞表达Oct4和Nanog诱导因子，或者直接将Oct4和Nanog以蛋白质的形式导入体细胞中。
在本发明方法的一个实施方案中，将两种重编程诱导因子以DNA的形式导入体细胞中。具体来说，可以将编码Oct4的核苷酸序列和编码Nanog的核苷酸序列导入体细胞中。在具体的实施方案中，所述编码Oct4的核苷酸序列为包含SEQ ID NO:6的核苷酸序列或由SEQ ID NO:6组成的核苷酸序列；或者为包含与SEQ ID NO:6具有至少90％同源性并且编码Oct4的核苷酸序列，或由与SEQ ID NO:6具有至少90％同源性并且编码Oct4的核苷酸序列组成的核苷酸序列。在具体的实施方案中，所述编码Nanog的核苷酸序列为包含SEQ ID NO:15的核苷酸序列或由SEQ ID NO:15组成的核苷酸序列；或者为包含与SEQ ID NO:15具有至少90％同源性并且编码Nanog的核苷酸序列，或由与SEQ ID NO:15具有至少90％同源性并且编码Nanog的核苷酸序列组成的核苷酸序列。
编码Oct4的核苷酸序列和编码Nanog的核苷酸序列可以置于相同或不同的载体中。当置于相同载体中时，编码Oct4的核苷酸序列和编码Nanog的核苷酸序列可以处于相同或不同的调控序列的控制下。调控序列可以根据目标细胞的类型进行选择。在具体的实施方案中，将编码Oct4的核苷酸序列和编码Nanog的核苷酸序列置于相同的载体中，如pcDNA3.1。在重组载体中还可以包含其它元件，例如包含EBNA1编码序列和OriP序列以提高质粒在细胞内的复制效率。在具体的实施方案中，本发明使用的重组载体的序列如SEQ ID NO:13或SEQ ID NO:24所示。
将包含目标核苷酸载体递送至体细胞中的方法是本领域已知的，包括但不限于电穿孔、基因枪、脂质体转染、钙介导的转染。在具体的实施方案中，使用电穿孔。
在本发明方法中，可以在体外表达上述Oct4和Nanog诱导因子，获得相应的蛋白质后，导入到分化的细胞中，从而实现本发明的目的。将蛋白质导入到细胞中的技术在本领域中是公知的，包括但不限于Tat-delivery及相关技术，电转(核转染)，蛋白和细胞配体结合。
本领域的普通技术人员也可以理解，可以将上述Oct4和Nanog诱导因子的DNA序列在体外进行转录，然后将获得的mRNA直接导入到分化的细胞中，使其在细胞中表达产生相应的蛋白，从而实现本发明的目的。也可通过用包含两种诱导因子的RNA的Sendai Virus感染体细胞来进行重编程。Sendai Virus是一种 非整合性病毒，不会整合到被感染细胞的基因组中，具有相对较高的安全性。
在向体细胞中转入了重编程诱导因子Oct4和Nanog之后，使细胞在存在TGFβ受体抑制剂、环AMP(cAMP)激动剂和糖原合成酶激酶(GSK)抑制剂中的一种或多种作为化学诱导剂的条件下培养，最终形成iPSCs。在优选的实施方案中，使用一种TGFβ受体抑制剂、一种环AMP(cAMP)激动剂和一种糖原合成酶激酶(GSK)抑制剂。
在优选的实施方案中，TGFβ受体抑制剂是616452，其化学名称为[2-(3-(6-甲基吡啶-2-基)-1H-吡唑-4-基)-1,5-萘啶](2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine，CAS No:446859-33-2)。优选地，其以0.1-20μM，更优选5-10μM，还更优选5μM的工作浓度使用，如以该浓度加入到培养基中。
在优选的实施方案中，环AMP(cAMP)激动剂是Forskolin，其化学名称为[(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-3-乙烯基-6,10,10b-三羟基-3,4a,7,7,10a-五甲基-1-氧代-5,6,6a,8,9,10-六氢-2H-苯并[f]色烯-5-基]乙酸酯([(3R,4aR,5S,6S,6aS,10S,10aR,10bS)-3-ethenyl-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2H-benzo[f]chromen-5-yl]acetate,CAS No:66575-29-9)。优选地，其以0.1-50μM，更优选2-20μM，还更优选10μM的工作浓度使用，如以该浓度加入到培养基中。
在优选的实施方案中，糖原合成酶激酶(GSK)抑制剂是TD114-2，其化学名称为6,7,9,10,12,13,15,16,18,19-十氢-5,29:20,25-二甲烯-26H-二苯并[n,t]吡咯[3,4-q][1,4,7,10,13,22]四氧杂二氮杂四环素-26,28(27H)-二酮(6,7,9,10,12,13,15,16,18,19-Decahydro-5,29:20,25-dimetheno-26H-dibenzo[n,t]pyrrolo[3,4-q][1,4,7,10,13,22]tetraoxadiazacyclotetracosine-26,28(27H)-dione，CAS No:436866-52-3)。优选地，其以0.1-20μM，更优选2-10μM，还更优选5μM的工作浓度使用，如以该浓度加入到培养基中。
在本发明的方法中使用的培养基可以根据所培养的细胞的类型由本领域技术人员基于已有的知识进行选择。
实施例
为了便于理解本发明，下面将通过具体实施例进一步对本发明的技术方案进行说明。本领域的普通技术人员可以理解本发明并不限于所述实施例，并且本领域的普通技术人员可以基于说明书的教导对实施例进行修改。这些修改同样包含在本发明由后附的权利要求所定义的本发明的范围内。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
实施例1.使用编码重编程因子的载体诱导皮肤细胞的重编程
1.1将来源于人的皮肤组织放入培养皿中，用4℃预冷的磷酸盐缓冲溶液(PBS)快速反复漂洗4次。
1.2用灭菌后的眼科剪和手术刀处理皮肤组织，使白色皮下脂肪去除，拨离表皮及皮下组织，留下真皮层，得到预处理的皮肤组织。将预处理的皮肤组织转移到培养基中后，切成小块，得到边缘整齐的皮肤组织块。
1.3取3mL的胎牛血清(FBS)均匀加到6孔细胞培养板的每孔后，放入皮肤组织块，放置培养箱孵育0.5-1h，使组织粘在培养板底部。
1.4在6孔细胞培养板中每孔加入1mL含有20％(v/v)FBS的DMEM(Gibco)培养基，放回培养箱培养。
1.5显微镜下观察到有成纤维细胞爬出时即可换液，将DMEM培养基的量增加至3mL，每隔1-3天更换培养基。
1.6待成纤维细胞在每个培养孔中汇合到孔的边缘时，弃旧培养基，PBS洗2遍，加入1mL的0.25％胰酶-EDTA(GIBCO)，细胞培养箱内放置约4～6min，待细胞变圆脱落、漂浮时，立即加入含有20％(v/v)FBS的DMEM培养基终止消化，将细胞轻轻吹起后转移至15mL离心管，200g离心3min，吸弃上清液，加入细胞培养基混匀，转移细胞至培养瓶中，置于细胞培养箱中继续培养。
1.7当第2代皮肤成纤维细胞汇合率达到80％-90％，弃旧培养基，细胞用PBS洗两遍后，加入3mL 0.25％的胰酶-EDTA(Gibco)消化至细胞分散，加入含有20％(v/v)FBS的DMEM培养基终止消化，将细胞轻轻吹起后转移至15mL离心管，200g离心3min，弃上清，加入适量PBS重悬细胞，血球计数板计数。
1.8取约8×10 5皮肤成纤维细胞，200g离心3min，PBS洗2遍，OPTI-MEM(Gibco)洗一遍，弃上清，按Celetrix Kit试剂盒说明书，加入相应电转 液试剂，重悬细胞，在细胞悬液中加入含有诱导因子Oct4和Nanog的重编程载体，混匀后加入电转杯中，将电转杯置于电转仪中，以430V，30ms，进行电转。
其中，所述重编程载体构建方法如下：①以质粒(Addgen#20922)为模板，用KOD-Plus-Neo(TOYOBO#KOD-401)高保真酶和引物F1/R1、F2/R2、F3/R3分别扩增得到EF-1α启动子片段、Oct4编码序列和Nanog编码序列(引物序列和扩增后的序列见下表1)。②以pcDNA3.1(-)质粒为模板，用KOD-Plus-Neo(TOYOBO #KOD-401)高保真酶和引物F4/R4扩增得到pcDNA3.1片段(引物序列和扩增得到的序列见下表1)。③用NEBuilder HiFi DNA Assembly Bundle for Large Fragments (NEB#E2623)，通过同源重组的方式将pcDNA3.1与EF-1α启动子、Oct4编码序列和Nanog编码序列连接，使Oct4和Nanog的编码序列在EF-1α启动子的调控下，即得到目的载体，即pcDNA3.1-EF-Oct4-Nanog(SEQ ID NO:13)。
1.9电转结束后，取出电转杯，迅速将细胞悬液吸出加入铺好Matrigel(Corning)的细胞培养板内，培养板中提前加入2mL含20％(v/v)FBS的DMEM培养基。第2天，更换为2～3mL TeSR-E7培养基(Stem Cell Technologies)，37℃细胞培养箱静置培养。此后每天进行全量换液。其中第2天开始使用的培养基中含有小分子化合物组合，小分子化合物的工作浓度如下：TGFβ受体抑制剂616452的工作浓度为5μM，环AMP(cAMP)激动剂Forskolin的工作浓度为10μM，糖原合成酶激酶(GSK)抑制剂TD114-2的工作浓度为5μM。
重编程约12天后，可观察到细胞形态明显区别于皮肤成纤维细胞的iPSC克隆(如图1A上方小图所示)。挑选单克隆接种至feeder-free体系下进行培养，这些由胞体较小的细胞形成的克隆紧凑、边界清晰的iPSC克隆，细胞具有较大的细胞核、较高的核质比，为典型的iPSC形态(如图1A下方小图所示)。
实施例2.使用编码重编程因子的载体诱导皮肤细胞的重编程
使用与实施例1中1.1-1.9相同的操作步骤，使用来源于人皮肤组织的成纤维细胞进行本发明的方法。与实施例1中不同的是，步骤1.8的重编程载体的构建不同，在实施例1的构建体的基础上，增加了EBNA1和OriP元件，从而可以基于EBNA1/OriP体系来提高质粒在体细胞内的复制效率，具体方法如下：
①以质粒(Addgen#20922)为模板，用KOD-Plus-Neo(TOYOBO#KOD-401)高保真酶和引物F5/R5、F6/R6、F7/R7、F8/R8、F9/R9分别扩增得到EF-1α启动子片段、Oct4编码序列、Nanog编码序列、EBNA1编码序列和OriP序列(引物序列和扩增后的序列见下表1)。②以pcDNA3.1(-)质粒为模板，用KOD-Plus-Neo(TOYOBO#KOD-401)高保真酶和引物F10/R10扩增得到pcDNA3.1片段(引物序列和扩增得到的序列见下表2)。③用NEBuilder HiFi DNA Assembly Bundle for Large Fragments(NEB#E2623)，通过同源重组的方式将pcDNA3.1与EF-1α启动子、Oct4编码序列、Nanog编码序列、EBNA1编码序列和OriP序列连接，即得到目的载体，即pcDNA3.1-EF-Oct4-Nanog-EBNA1-OriP(SEQ ID NO:24)。
重编程约12天后，可观察到细胞形态明显区别于皮肤成纤维细胞的iPSC克隆(如图1B上方小图所示)。挑选单克隆接种至feeder-free体系下进行培养，这些由胞体较小的细胞形成的克隆紧凑、边界清晰的iPSC克隆，细胞具有较大的细胞核、较高的核质比，为典型的iPSC形态(如图1B下方小图所示)。
实施例3.使用包含重编程诱导因子RNA的病毒诱导皮肤细胞的重编程
使用与实施例1中1.1-1.6中相同的操作步骤，使用来源于人皮肤组织的成纤维细胞进行本发明的方法。与实施例1中的步骤1.7-1.9不同，采用如下步骤3.7对其使用含有重编程诱导因子的RNA的病毒进行iPSC重编程。
3.7当第2代皮肤成纤维细胞汇合率达到80％-90％，将含有Oct4和Nanog重编程诱导因子的Sendai Virus，与细胞混合培养2天后，弃掉培养基，加入2mL的20％(v/v)FBS的DMEM培养基培养，每隔一天进行换液。培养5天后，加入1mL的0.25％胰酶-EDTA(GIBCO)，细胞培养箱内放置约4～6min，待细胞变圆脱落、漂浮时，加入到Vitronectin(Gibco)包被的细胞培养板培养。其中第2天开始使用的培养基中含有小分子化合物组合，小分子化合物的工作浓度如下：TGFβ受体抑制剂616452的工作浓度为5μM，环AMP(cAMP)激动剂Forskolin的工作浓度为10μM，糖原合成酶激酶(GSK)抑制剂TD114-2的工作浓度为5μM。
重编程约12天后，可观察到细胞形态明显区别于皮肤成纤维细胞的iPSC细胞克隆(如图1C上方小图所示)。挑选单克隆接种至feeder-free体系下进行培养， 这些由胞体较小的细胞形成的克隆紧凑、边界清晰，细胞具有较大的细胞核、较高的核质比，为典型的iPSC形态(如图1C下方小图所示)。
实施例4.皮肤成纤维细胞重编程获得的iPSCs的鉴定
使用包括流式细胞术、免疫荧光染色和定量PCR(qPCR)在内的不同方法对通过重编程皮肤成纤维细胞获得的iPSCs进行了表征和鉴定。
通过流式细胞术对获得的iPSCs进行了如下分子标志物的分析：SSEA4、Tra-1-81、Tra-1-60、Oct4和Nanog。如图2所示，通过本发明方法获得的iPSCs表达人多能干细胞的标志物SSEA4、Tra-1-81、Tra-1-60、Oct4和Nanog，证明了获得的细胞具有多能干细胞的特征。
为验证获得的iPSCs的全能性，对其在体外进行了三胚层分化，并使用免疫荧光染色和qPCR检测了三个胚层的特异性分子标志物表达情况。如图3A所示，使用三种标志物(内胚层：SOX17；中胚层：CDX2；外胚层：PAX6)的免疫荧光实验结果显示，获得的iPSCs可以分化得到三个胚层的细胞。图3B显示了qPCR检测三个胚层的特异性分子标志物(内胚层：SOX17、中胚层：MIXL1、外胚层：PAX6)的表达情况，其中iPSCs高表达多能干细胞分子标志物OCT4、TRA-1-81(与前述流式细胞仪分析结果一致)，而三胚层分化的细胞高表达各个胚层的特异性分子标志物(****p<0.0001，使用student’s t-test)。这些结果说明获得的iPSCs具有全能性。
实施例5.使用编码重编程因子的载体诱导外周血细胞的重编程
5.1取10mL来源于人的外周血，使用RosetteSep TM和SepMate TM试剂盒(Stem Cell Technologies)富集并扩增红系祖细胞。具体为将10mL血液从采血管里转移至普通离心管中，加入50μL RosetteSep TM Human Progenitor Cell Basic Pre-Enrichment Cocktail，充分混匀，室温静置10min。
5.2在SepMate离心管中沿着中心孔分别加入3.5mL Lymphoprep TM，室温孵育10min后，加入10mL PBS+2％FBS，充分混匀，分别将5mL血液沿着SepMate离心管壁，1200g离心10min，小心将黄色上清迅速倒入新的普通离心管中(注意不要混入底部杂细胞)，300g离心8min。
5.3离心完毕后，弃去上清液，加入0.5mL StemSpan TM SFEM II培养基重悬计细胞总数，按5×10 6/mL的密度取2mL细胞分别接种到6孔板内，于37℃、5％CO 2细胞培养箱中培养。
5.4第1天：将细胞悬液转移至新的6孔板中，以去除贴壁的杂细胞，每孔补加入0.5mL StemSpan TM SFEM II培养基，于37℃、5％CO 2细胞培养箱中培养。
5.5第2天、第4天、第6天和第8天：分别将细胞悬液收集到离心管里，400g离心5min，弃上清，加入StemSpan TM SFEM II培养基重悬，吹打3-4次后，以2mL体积分别接种至新的6孔板每孔中，充分摇匀，于37℃、5％CO 2细胞培养箱中培养。
5.6第9天：红系祖细胞数量明显增加，并在第10天时迅速富集，当细胞数量达到2×10 6可以进行电转。将细胞悬液收集，400g离心5min，弃上清弃上清，按Celetrix Kit试剂盒说明书，加入相应电转液试剂，重悬细胞，在细胞悬液中加入如实施例1中所构建的含有诱导因子Oct4和Nanog的重编程载体pcDNA3.1-EF-Oct4-Nanog(SEQ ID NO:13)，混匀后加入电转杯中，将电转杯置于电转仪中，以820V,20ms，进行电转。
5.7电转结束后，取出电转杯，迅速将细胞悬液吸出加入Matrigel包被(Corning)的细胞培养板内，培养板中提前加入StemSpan TM SFEM II培养基。第2天，每孔补加1mL StemSpan TM SFEM II培养基，37℃细胞培养箱静置培养。第3天和第5天，每孔补加1mL ReproTeSR培养基(Stem Cell Technologies)。第7天，可见有细胞开始贴壁生长，弃掉旧培养基，此后每天每孔更换为2mL ReproTeSR培养基。其中第2天开始使用的培养基中含有小分子化合物组合，小分子化合物的工作浓度如下：TGFβ受体抑制剂616452的工作浓度为5μM，环AMP(cAMP)激动剂Forskolin的工作浓度为10μM，糖原合成酶激酶(GSK)抑制剂TD114-2的工作浓度为5μM。
重编程约12天后，可观察到悬浮培养的细胞团贴壁生长形成的iPSC克隆(如图4A上方小图所示)。挑选单克隆接种至feeder-free体系下进行培养，这些由胞体较小的细胞形成的克隆紧凑、边界清晰的iPSC克隆，细胞具有较大的细胞核、较高的核质比，为典型的iPSC形态(如图4A下方小图所示)。
实施例6.使用编码重编程因子的载体诱导外周血细胞的重编程
使用与实施例5中5.1-5.7相同的操作步骤，使用来源于人外周血的红系祖细胞进行本发明的方法。与实施例4不同的是，步骤4.6的重编程载体使用如实施例2中所构建的含有诱导因子Oct4和Nanog的重编程载体pcDNA3.1-EF-Oct4-Nanog-EBNA1-OriP。
重编程约12天后，可观察到悬浮培养的细胞团贴壁生长形成的iPSC克隆(如图4B上方小图所示)。挑选单克隆接种至feeder-free体系下进行培养，这些由胞体较小的细胞形成的克隆紧凑、边界清晰的iPSC克隆，细胞具有较大的细胞核、较高的核质比，为典型的iPSC形态(如图4B下方小图所示)。
实施例7.使用包含重编程诱导因子RNA的病毒诱导外周血细胞的重编程
使用与实施例5中5.1-5.5中相同的操作步骤，使用来源于人外周血的红系祖细胞进行本发明的方法。与实施例4中的步骤4.6-4.7不同，采用如下步骤6.6对其使用含有重编程诱导因子的RNA的病毒进行iPSC重编程。
7.6第9天：红系祖细胞数量明显增加，将细胞悬液收集，400g离心5min，弃上清，加入含有以RNA形式存在的Oct4和Nanog重编程诱导因子的病毒Sendai Virus，与细胞混合培养2天后，弃掉培养基，加入2mL ReproTeSR培养基。此后每天每孔更换2mL ReproTeSR培养基。其中第2天开始使用的培养基中含有小分子化合物组合，小分子化合物的工作浓度如下：TGFβ受体抑制剂616452的工作浓度为5μM，环AMP(cAMP)激动剂Forskolin的工作浓度为10μM，糖原合成酶激酶(GSK)抑制剂TD114-2的工作浓度为5μM。
重编程约12天后，可观察到悬浮培养的细胞团贴壁生长形成的iPSC克隆(如图4C上方小图所示)。挑选单克隆接种至feeder-free体系下进行培养，这些由胞体较小的细胞形成的克隆紧凑、边界清晰的iPSC克隆，细胞具有较大的细胞核、较高的核质比，为典型的iPSC形态(如图4C下方小图所示)。
实施例8.红系祖细胞重编程获得的iPSCs的鉴定
使用包括流式细胞术、免疫荧光染色和定量PCR(qPCR)在内的不同方法对通过重编程红系祖细胞获得的iPSCs进行了表征和鉴定。
通过流式细胞术对获得的iPSCs进行了如下分子标志物的分析：SSEA4、Tra-1-81、Tra-1-60、Oct4和Nanog。如图5所示，通过本发明方法获得的iPSCs表达人多能干细胞的标志物SSEA4、Tra-1-81、Tra-1-60，Oct4和Nanog，证明了获得的细胞具有多能干细胞的特征。
为验证获得的iPSCs的全能性，对其在体外进行了三胚层分化，并使用免疫荧光染色和qPCR检测了三个胚层的特异性分子标志物表达情况。如图6A所示，使用三种标志物(内胚层：SOX17；中胚层：CDX2；外胚层：PAX6)的免疫荧光实验结果显示，获得的iPSCs可以分化得到三个胚层的细胞。图6B显示了qPCR检测三个胚层的特异性分子标志物(内胚层：SOX17；中胚层：MIXL1；外胚层：PAX6)的表达情况，其中iPSCs高表达多能干细胞分子标志物OCT4、TRA-1-81(与前述流式细胞仪分析结果一致)，而三胚层分化的细胞高表达各个胚层的特异性分子标志物(****p<0.0001，使用student’s t-test)。这些结果说明获得的iPSCs具有全能性。
表1.PCR扩增产物
Figure PCTCN2020106327-appb-000001
表2：PCR扩增产物
Figure PCTCN2020106327-appb-000002
Figure PCTCN2020106327-appb-000003
本发明的制备iPSCs的方法减少重编程所需的转录因子至2个，降低了成瘤性，并通过3个小分子化合物的组合提高转化效率。本发明的方法整体上简便高效、易于操作，用这种方法制备的iPSCs将更适合于临床转化和应用。
以上所述仅是本发明的优选实施方式，应当指出，对于熟悉本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (11)

  1. 一种通过体细胞重编程制备诱导多能干细胞的方法，包括如下步骤：
    (1)将因子Oct4和Nanog作为重编程诱导因子导入体细胞后进行重编程；
    (2)将步骤(1)获得的部分或完全重编程的体细胞在存在化学诱导剂的条件下培养以得到诱导多能干细胞(iPSCs)，其中，所述化学诱导剂包括TGFβ受体抑制剂、环AMP激动剂和糖原合成酶激酶抑制剂。
  2. 根据权利要求1所述的方法，其中重编程诱导因子Oct4和Nanog以其核酸的形式，或者以其蛋白产物的形式导入体细胞中。
  3. 根据权利要求1所述的方法，其中重编程诱导因子Oct4和Nanog以其DNA的形式，以其mRNA的形式，或者以其蛋白产物的形式导入体细胞中。
  4. 根据权利要求1所述的方法，其中所述TGFβ受体抑制剂是616452，和/或所述环AMP激动剂是Forskolin，和/或所述糖原合成酶激酶抑制剂是TD114-2。
  5. 根据权利要求1至4中任一项所述的方法，其中，
    TGFβ受体抑制剂616452的工作浓度为0.1-20μM，更优选5-10μM，还更优选5μM；和/或
    环AMP激动剂Forskolin的工作浓度为0.1-50μM，更优选2-20μM，还更优选10μM；和/或
    糖原合成酶激酶抑制剂TD114-2的工作浓度为0.1-20μM，更优选2-10μM，还更优选5μM。
  6. 根据权利要求1所述的方法，其中，所述体细胞选自皮肤来源的细胞，血液来源的细胞，尿液来源的细胞，肝细胞，上皮细胞，胃细胞，或者角质细胞。
  7. 根据权利要求1至6中任一项所述的方法，其中，所述体细胞来源于人。
  8. 根据权利要求1所述的方法，其中，在步骤(1)中通过将含有分别编码重编程诱导因子Oct4和Nanog的核苷酸序列的重组载体转入体细胞后进行重编程。
  9. 根据权利要求1所述的方法，其中，在步骤(1)中通过将含有分别编码重编程诱导因子Oct4和Nanog的病毒感染体细胞后进行重编程。
  10. 一种诱导多能干细胞，其通过权利要求1-9中任一项所述的方法得到。
  11. 重编程诱导因子和化学诱导剂的组合，其用于重编程体细胞以获得iPSCs 的用途，所述重编程诱导因子为Oct4和Nanog，所述化学诱导剂为TGFβ受体抑制剂、环AMP激动剂和糖原合成酶激酶抑制剂。
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