WO2018000288A1 - 一种重编程因子组合在三维重编程体系中诱导多能干细胞的方法 - Google Patents

一种重编程因子组合在三维重编程体系中诱导多能干细胞的方法 Download PDF

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WO2018000288A1
WO2018000288A1 PCT/CN2016/087798 CN2016087798W WO2018000288A1 WO 2018000288 A1 WO2018000288 A1 WO 2018000288A1 CN 2016087798 W CN2016087798 W CN 2016087798W WO 2018000288 A1 WO2018000288 A1 WO 2018000288A1
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dimensional
loaded
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gene
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徐希明
邓纹纹
余青桐
余江南
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Jiangsu University
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Jiangsu University
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  • the invention relates to the field of biotechnology and tissue engineering, and relates to a gene delivery system, in particular to a reprogramming application based on a novel reprogramming factor combined in a three-dimensional system.
  • the present invention based on safety, replaces certain carcinogenic factors in the classical Yamanaka factor with a factor in the miRNA 302/367 family, and examines the reprogramming efficiency of the combination in a three-dimensional gene-loaded nanoparticle-collagen scaffold.
  • the cell reprogramming strategy was optimized.
  • the invention relates to the construction of a three-dimensional genetically loaded nanoparticle-collagen scaffold, and three-dimensional reprogramming of human umbilical cord stem cells.
  • the calcium phosphate-loaded genetic nanoparticles are prepared by reverse microemulsion method and fused with a three-dimensional collagen scaffold to form novel three-dimensional iPSCs. Induction system.
  • the invention applies the three-dimensional system to the reprogramming of somatic cells, evaluates the cell transfection efficiency and the cell reprogramming effect, and sets the traditional Yamanaka factor as the positive control under the same conditions, and the experimental results show that the three-dimensional gene transfer of the invention
  • the system is significantly superior to the two-dimensional gene delivery system in gene transfection efficiency; in addition, human-derived iPSCs induced by three-dimensional system appear iPSCs cell spheres on the 4th day after cell inoculation, and then embryoid body cell spheres are gradually formed.
  • HE staining, laser confocal and immunohistochemical analysis showed that human iPSCs induced by three-dimensional system have similar whole germ layer differentiation potential to embryonic stem cells.
  • the invention uses a chemical modification method to provide a somatic cell such as a human umbilical cord stem cell.
  • HUMSCs human umbilical cord stem cell.
  • hiPSCs human induced pluripotent stem cells
  • a method of combining reprogramming factors to induce pluripotent stem cells in a three-dimensional system comprising the steps of:
  • Step 4 Induction and amplification of iPSCs in a three-dimensional system:
  • the somatic cells may be HUMSCs cells.
  • the collagen scaffold prepared by the raw material provides a safe and stable three-dimensional culture environment for the somatic HUMSCs.
  • the calcium phosphate-loaded gene nanoparticle has good gene carrying capacity and is evenly distributed in the three-dimensional scaffold for directional reprogramming of somatic HUMSCs to iPSCs. Provides ample source of foreign genes and is capable of long-term release.
  • the expression of exogenous genes in the three-dimensional system was significantly higher than that of the two-dimensional system by RT-PCR.
  • iPSCs The results of iPSCs showed that the human iPSCs formed in the three-dimensional system have the ability to differentiate into the whole germ layer of embryonic stem cells. Stable amplification in vitro and passage to more than 20 generations. This method provides a safer pathway for iPSCs to further enrich the source of iPSCs.
  • Figure 1 is a scanning electron micrograph of a novel three-dimensional gene-loaded nanoparticle-collagen scaffold, in which black arrows indicate calcium phosphate-loaded gene nanoparticles.
  • Figure 2 shows the expression levels of Sox2, Oct4, NANOG, and SSEA4 by RT-PCR, in which CP/OSKM is a calcium phosphate-loaded classical Yamanaka factor nanoparticle, and CP/OS-miR is a calcium phosphate-loaded novel reprogramming factor nanoparticle.
  • Figure 3 shows the identification of iPSCs by immunofluorescence
  • Figure 4 shows the identification of three germ layer differentiation markers of iPSCs by immunofluorescence.
  • Figure 5 shows the reprogramming factor induced iPSCs generation timeline in a three-dimensional system.
  • DH5 ⁇ glycerol bacteria Najing Jitian Bio
  • human umbilical cord mesenchymal stem cells Jiangsu Beike Biotechnology Co., Ltd.
  • mouse embryonic fibroblasts Stem Cell Bank
  • NOD-SCID non-obese diabetic/severe combined immunodeficient mice
  • Non-viral plasmids OCT4, SOX2, and miR302-367 were purchased from Guangzhou Fuenergy Gene Co., Ltd.
  • Anti-OCT4, anti-SSEA-3, anti-SSEA-4, anti-TRA-1-81, anti-NANOG, anti- ⁇ -actin, anti- ⁇ III tubulin (above antibody provided by Abcam, USA); anti- AFP, anti-Tuj1, anti-Collagen II (three antibodies are provided by Santa Cruz, USA); goat anti-rabbit IgG-Cy3, type IV collagenase (provided by Sigma, USA); trypsin (provided by Biyuntian Biotechnology Research Institute) ; SYBR Premix Ex Taq enzyme, PrimeScript TM miRNA qPCR starter kit (TaKaRa provided Japan); basic fibroblast growth factor (of bFGF) (provided by the United States PeproTech Inc.)
  • GAPDH upstream primer CGGAGTCAAC GGATTTGGTC GTAT 24
  • GAPDH downstream primer AGCCTTCTCC ATGGTGGTGA AGAC 24
  • OCT4 upstream primer ATGTGGTCCG AGTGTGGTTC 20
  • OCT4 downstream primer AAACCCTGGC ACAAACTCCA 20
  • NANOG upstream primer GAGATGCCTC ACACGGAGAC 20
  • NANOG downstream primer CTTTGGGACT GGTGGAAGAA 20
  • SSEA4 upstream primer TGGACGGGCA CAACTTCATC 20
  • Plasmid extraction kit (Promega, USA); LB medium (homemade); agarose, ethidium bromide, L-glutamine, PEI (25 kDa), dimethyl sulfoxide, ⁇ -mercaptoethanol, DAPI dye, Mitomycin C (Sigma, USA); Tris base, ampicillin, streptomycin, alkaline phosphatase assay kit (Biyuntian Biotechnology Research Institute); calcium chloride, paraformaldehyde (Zhongguo Group Chemical Reagent Co., Ltd.) ); MTT (3- (4,5- dimethyl-thiazol-2) -2,5-diphenyl tetrazolium bromide), Lipofectamine TM 2000, nonessential amino acids (NEAA), TRIzol reagent, Glycogen (Invitrogen, USA ); basal medium DMEM, basal medium DMEM/F12, knockout DMEM, fetal bovine serum, knockout serum substitute (GIBCO, USA); BD
  • Knockout DMEM medium was sequentially added with 20% serum substitute (Knockout SR), 2 mmol/L L-glutamine, 0.1 mmol/L ⁇ -mercaptoethanol, 1% non-essential amino acid, 100 U/ mL qing-streptomycin solution, 4 ng/mL human recombinant basic fibroblast growth factor, mix, place 4 ° C refrigerator, set aside.
  • Tris-HCl pH 8.8: 18.2 g of Tris base was dissolved in 100 mL of double distilled water, and concentrated hydrochloric acid was adjusted to pH.
  • Tris-HCl pH 6.8: 6.05 g of Tris base was dissolved in 100 mL of double distilled water, and concentrated hydrochloric acid was adjusted to Ph.
  • Tris-glycine electrophoresis buffer 94 g of glycine and 15.1 g of Tris base were dissolved in 950 mL of double distilled water, and then 50 mL of 10% SDS stock solution (w/v) was added.
  • Transmembrane buffer 5.8 g of Tris base, 2.9 g of glycine and 0.37 g of SDS were dissolved in 800 mL of double distilled water, and then 200 mL of methanol was added.
  • Triton X-100 0.2 mL Triton X-100 was dissolved in 100 mL of PBS.
  • Lysis solution 5 mmol/L MgCl 2 , 150 mmol/L NaCl, 1% Nonidet P-40 (NP-40) (v/v), 50 mmol/L Tris-HCl (pH 7.4), aprotinin 10 ⁇ g/mL, 1 mmol/L dithiothreitol (DTT), leupeptin 10 ⁇ g/mL and 1 mmol/L PMSF.
  • XW-80A vortex mixer (Jiangsu Qilin Medical Instrument Factory); Electrothermal constant temperature drying oven (Shanghai Yuejin Medical Instrument Factory); Ultra-low temperature high-speed centrifuge (Heraeus, Germany); Ultra-clean workbench (Suzhou purification plant); HH-S Digital thermostatic water bath (Jintan Medical Instrument Factory, Jiangsu City); THZ-82A constant temperature oscillator (Jintan Medical Instrument Factory, Jiangsu province); DY602S steady flow regulator electrophoresis instrument (Nanjing New Campus Biotechnology Research Institute); Gel Imager (Bio-Rad, USA); DHA/RNA/PROTEIN analysis (Shimadzu, Japan); JEM-2100 Transmission Electron Microscope (Japan Electronics); BI-90 Particle Size Distribution Instrument (Brookhaven, USA); ZEN3600 Potentiometer (UK) Malvern); inverted fluorescence microscope (Olympus, Japan); HB-1 heating plate (Wealtec, USA);
  • Example 1 Preparation of three-dimensional collagen-loaded gene nanoparticle scaffolds:
  • Igepal CO-520 was dissolved in cyclohexane to prepare an Igepal CO-520 29% Igepal CO-520/cyclohexane mixture;
  • microemulsion A Take a clean conical flask, add 25mL Igepal CO-520/cyclohexane mixture, then add 650 ⁇ L 0.01M calcium chloride solution to the net under magnetic stirring conditions. Conical flask, continue to stir for 2 min to form microemulsion A;
  • microemulsion B Take another clean conical flask, add 25mL Igepal CO-520/cyclohexane mixture, then add 650 ⁇ L of 0.06M disodium hydrogen phosphate and plasmid mixed solution 10 ⁇ L (including Oct4, Sox2) Each 2.5 ⁇ g, miR302/367 5 ⁇ g) was added to the net conical flask, and stirring was continued for 2 min to form microemulsion B;
  • microemulsion A was added dropwise to the microemulsion B while stirring, until the whole system was transparent and clear, and the polysaccharide-calcium phosphate hybrid nanoparticle microemulsion was obtained;
  • the ethanol solution containing the calcium phosphate-loaded gene nanoparticles was rotary evaporated at 37 ° C for 6 hours to remove the ethanol, and the concentrated solution was placed in a 12 kDa dialysis bag, and the pH 7.4 PBS was allowed to stand overnight at 4 ° C to obtain a calcium phosphate-loaded gene nanometer. Granule solution.
  • Example 4 iPSCs induction
  • each well containing the inoculated cell scaffold was added DMEM medium containing 10% FBS, and continued to culture for 4 h;
  • the adult embryonic stem cell culture medium was changed, and the medium was changed every 2 days.
  • the cells were evenly dispersed in the scaffold; over time, on the fourth day (ie, on the 7th day of planting), the cell sphere began to appear; later, the embryoid body cells gradually formed. ball.
  • RNA extraction and qRT-PCR procedure After 4 transfections, the cells were washed once with PBS, total RNA was prepared using TRIzol reagent according to the instructions, and stored at -80 ° C until use. Total RNA was prepared using Takara's SYBR Premix Ex Taq, and the cDNA was diluted 20-fold with ddH 2 O, and then amplified using SybrGreen 2 ⁇ PCR Mix. Data were analyzed by SPSS statistical software for two-way analysis of variance, and T-test was used to examine the significant differences in the data. The results showed that in the three-dimensional system, after transfection with CP/OS-miR, the four iPSCs markers SOX2, OCT4, NANOG and SSEA3 were higher than the CP/OSKM transfection group at the transcriptional level.
  • the primary antibodies used included anti-TRA-1-81, anti-SSEA-3, anti-SSEA-4, anti-NANOG and anti-OCT4, and the secondary antibody was goat anti-rabbit IgG-Cy3.
  • Example 7 In vitro induction of differentiation into the three germ layers
  • mice alpha-fetoprotein AFP
  • mice The clonal cells were inoculated into a 24-well plate and cultured in an incubator. After adhering to the wall, the original medium was discarded and replaced with a neural cell medium (10 ng/mL was added to the DMEM/F12 medium). SHH, 10 ng/mL BDNF, 1 ⁇ mol/L RA), continued to culture for 1 day and then changed to Neurobasal cell culture medium (containing 10 ng/mL SHH, 10 ng/mL BDNF, 1 ⁇ mol/L RA, 2% B27), changing every 3 days. once. Control group: After the cells were attached, the solution was changed with Neurobasal medium containing 2% B27. Operate the same experimental group. Morphological changes of the cells were observed under an inverted microscope. On the 28th day after induction, murine anti-Tuj1 was used for immunofluorescence detection.
  • mice The clonal cells were inoculated into a 24-well plate and cultured in an incubator. After adhering to the wall, the original medium was discarded and replaced with 10 ng/mL TGFb-1, 20 ng/mL bFGF, 10 -8. DMEM medium with M dexamethasone and 10% FBS was changed every 3 days. Control group: After the cells were attached, the cells were changed with DMEM medium containing 10% FBS, and the other operations were the same as those of the experimental group. Morphological changes of the cells were observed under an inverted microscope. On the 28th day after the induction, murine anti-type II collagen was used for immunofluorescence detection, and the operation was referred to in Example 5.

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Abstract

提供了一种重编程因子组合在三维体系中诱导多能干细胞的方法，所述方法采用胶原支架与磷酸钙纳米粒作为基因载体构建三维非病毒基因传递系统，可用于人源体细胞的多能重编程研究。

Description

一种重编程因子组合在三维重编程体系中诱导多能干细胞的方法 技术领域
本发明涉及生物技术和组织工程领域，涉及基因传递系统，具体涉及一种基于新型重编程因子组合在三维体系中的重编程应用。
发明背景
目前，通过引入外源基因(包括DNA和miRNA)进行细胞重编程的方法仍然占据细胞重编程的主导地位。经典的Yamanaka转录因子组合Oct4、Sox2、Klf4和c-Myc已经在多个种属(如小鼠、人、大鼠、猪等)、[参见：Cao,X.,Deng,W.,Qu,R.,Yu,Q.,Li,J.,Yang,Y.,Cao,Y.,Gao,X.,Xu,X.,Yu,J.Non‐viral co‐delivery of the four yamanaka factors for generation of human induced pluripotent stem cells via calcium phosphate nanocomposite particles[J].Advanced Functional Materials,2013,23(43):5403-5411；Li,W.,Wei,W.,Zhu,S.,Zhu,J.,Yan,S.,Lin,T.,Hao,E.,Hayek,A.,Deng,H.,Sheng,D.Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors[J].Cell Stem Cell,2009,4(1):16–19；Jai-Hee,M.,June Seok,H.,Suhyun,K.,Jihyun,K.,Jihye,H.,Phil Jun,K.,Aeree,K.,Hyun Ok,K.,Kwang Youn,W.,Byung Sun,Y.Two-step generation of induced pluripotent stem cells from mouse fibroblasts using id3and oct4[J].Journal of Molecular Cell Biology,2012,4(1):59-62；Okita,K.,Hong,H.,Takahashi,K.,Yamanaka,S.Generation of mouse-induced pluripotent stem cells with plasmid vectors[J].Nature Protocol,2010,5(3):418-28]多种细胞(如小鼠胚胎成纤维细胞、小鼠和人的皮肤成纤维细胞、人皮肤角质细胞、人脐带间充质细胞、人睾丸细胞等)重编程中得到应用[参见：Jeong Beom,K.,Holm,Z.,Guangming,W.,Luca,G.,Kinarm,K.,Vittorio,S.,Araúzo-Bravo,M.J.,David,R.,Dong Wook,H.,Martin,Z.Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors[J].Nature,2008,454(7204):646-650；Aoi,T.,Yae,K.,Nakagawa,M.,Ichisaka,T.,Okita,K.,Takahashi,K.,Chiba,T.,Yamanaka,S.Generation of pluripotent stem cells from adult mouse liver and stomach cells[J].Science,2008,321(5889):699-702；Stadtfeld,M.,Brennand,K.,Hochedlinger,K.Reprogramming of pancreatic beta cells into induced pluripotent stem cells[J].Current Biology,2008,18(12):890-894；Hanna,J., Markoulaki,S.,Schorderet,P.,Carey,B.W.,Beard,C.,Wernig,M.,Creyghton,M.,Steine,E.J.,Cassady,J.P.,Foreman,R.Direct reprogramming of terminally differentiated mature b lymphocytes to pluripotency[J].Cell,2008,133(2):250–264；Eminli,S.,Foudi,A.M.Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells[J].Nature Genetics,2009,41(9):968-976]，足见Yamanaka因子对于细胞重编程研究影响深远。除此之外，人们还发现了Nanog或Lin28等可与Yamanaka因子组合一起，或者替代Yamanaka因子组合中的一些因子，也能够成功诱导iPSCs生成[参见:Fatima,C.,Sch？Ler,H.R.Nanog:A new recruit to the embryonic stem cell orchestra[J].Cell,2003,113(113):551-552]。
近年来，科学家们在研究重编程机理的过程中发现的一些非编码miRNA，特别是miRNA302/367家族，对细胞重编程的过程有显著的促进作用[参见：Liao,B.,Bao,X.,Liu,L.,Feng,S.,Zovoilis,A.,Liu,W.,Xue,Y.,Cai,J.,Guo,X.,Qin,B.Microrna cluster 302–367enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition[J].Journal of Biological Chemistry,2011,286(19):17359-17364；Zhang,Z.,Xiang,D.,Heriyanto,F.,Gao,Y.,Qian,Z.,Wu,W.-S.Dissecting the roles of mir-302/367cluster in cellular reprogramming using tale-based repressor and talen[J].Stem Cell Reports,2013,1(3):218-225]。基于这些发现，本发明从安全性出发，以miRNA302/367家族中的一种因子代替经典Yamanaka因子中的某些致癌因子，于三维载基因纳米粒-胶原支架中考察该组合的重编程效率，优化了细胞重编程策略。
发明内容
本发明涉及三维载基因纳米粒-胶原支架的构建，人源脐带干细胞的三维重编程两部分，通过反向微乳法制备磷酸钙载基因纳米粒并与三维胶原支架融合，形成了新型三维iPSCs诱导体系。本发明将该三维体系应用于体细胞的重编程，并对细胞转染效率、细胞重编程效果进行评价，同时设置相同条件下传统Yamanaka因子作为阳性对照，实验结果表明：本发明的三维基因传递系统在基因转染效率方面显著优于二维基因传递系统；此外，通过三维体系诱导生成的人来源iPSCs自细胞接种后第4天开始出现iPSCs细胞球，此后逐渐形成拟胚体状细胞球。经HE染色，激光共聚焦，免疫组化分析，表明经三维体系诱导生成的人源iPSCs具有类似于胚胎干细胞的全胚层分化潜能。
本发明采用了化学修饰的方法，提供了一种将体细胞如人源脐带干细胞 (HUMSCs)定向重编程为人源诱导多能干细胞(hiPSCs)的高效无毒的三维基因传递系统。
本发明的技术方案如下：
一种重编程因子组合在三维体系中诱导多能干细胞的方法，它包括如下步骤：
步骤1.胶原支架的制备：
取适量2～5％胶原溶液于支架制备的聚四氟乙烯模具中(68×38×5mm3)，先在4℃放置0.5～1h，再迅速放在-80℃冰箱预冻3h以上,然后冻干；于烘箱内热固化8～10h之后，用无水乙醇浸泡24h，再以10～100％浓度梯度的乙醇溶液洗涤，双蒸水洗至无醇味；再次冻干，切成大小为5×5×3mm3的小块，于超净台进行紫外灭菌备用；
步骤2.磷酸钙纳米粒的制备：
配制体积分数为20～30％的Igepal CO-520/环己烷混合液并以此为溶剂，以0.01～0.05M氯化钙溶液，0.03～0.08M磷酸氢二钠和含Oct4、Sox2、Klf4、c-Myc、miR302a、miR302b、miR302或miR302-367中的三种质粒各2.5μg的质粒混合溶液10μL为原料，通过反相微乳法，得到磷酸钙载基因纳米粒微乳；再经柱层析，旋转蒸发及透析等步骤，得到多磷酸钙载基因纳米粒溶液；
步骤3.三维载基因纳米粒‐胶原支架的制备：
取5×5×3mm3的支架样品放入24孔板的板孔中以无血清DMEM培养基使支架充分润湿；吸除多余的培养基；调整磷酸钙载基因纳米粒的浓度，使每100μL纳米粒溶液中含0.8～1.5μg总质粒；向每一块润湿之后5×5×3mm3的支架上滴加100μL磷酸钙载基因纳米粒溶液后冷冻干燥，得到三维载基因纳米粒-胶原支架；
步骤4.三维体系中的iPSCs诱导和扩增：
取步骤3得到的5×5×3mm3三维载基因纳米粒-胶原支架并以无血清DMED浸润；将200μL体细胞悬液(5～8×104个细胞/支架)加入浸润后的支架，在完全培养基中培养3～5天后，换成人胚胎干细胞培养基，每2～3天换液一次，扩增诱导多能干细胞。
上述的方法，所述的体细胞可以是HUMSCs细胞。
有益效果
选取不含致癌基因的重编程因子组合确保iPSCs的安全性，以天然I胶原为 原料制备的胶原支架为体细胞HUMSCs提供了安全稳定的三维培养环境，磷酸钙载基因纳米粒具有良好的基因携载能力，且均匀分布于三维支架中，为体细胞HUMSCs向iPSCs的定向重编程提供了充足的外源基因来源并能够长效释放。经RT‐PCR评价，三维体系中外源基因的表达量显著高于二维体系；经iPSCs相关鉴定，结果表明，三维体系中形成的人源iPSCs具有类似于胚胎干细胞的全胚层分化能力，且能够与体外稳定扩增、传代至20代以上。该方法为iPSCs提供了更为安全的诱导途径，进一步丰富了iPSCs的来源。
附图说明
图1为新型三维载基因纳米粒‐胶原支架扫描电镜图,其中黑色箭头指示磷酸钙载基因纳米粒。
图2为RT‐PCR检测Sox2,Oct4,NANOG,SSEA4的表达水平，其中CP/OSKM为磷酸钙载经典Yamanaka因子纳米粒，CP/OS‐miR为磷酸钙载新型重编程因子纳米粒
图3为免疫荧光鉴定iPSCs标志物
图4为免疫荧光鉴定iPSCs三胚层分化标志物
图5为三维体系中重编程因子诱导iPSCs生成时间轴
以下实施例所采用器材：
1菌株、细胞株和小鼠
DH5α甘油菌(南京基天生物)；人脐带间充质干细胞(江苏省北科生物科技有限公司)；小鼠胚胎成纤维细胞(上海中科院干细胞库)；非肥胖糖尿病/重度联合免疫缺陷小鼠(NOD-SCID)(扬州大学比较医学中心)
2质粒
非病毒质粒OCT4、SOX2、以及miR302-367购买于广州复能基因有限公司
3抗体、酶和细胞因子
Anti-OCT4、anti-SSEA-3、anti-SSEA-4、anti-TRA-1-81、anti-NANOG、anti-β-actin、anti-βIII tubulin(以上抗体由美国Abcam公司提供)；anti-AFP、anti-Tuj1、anti-Collagen II(三种抗体由美国Santa Cruz提供)；羊抗兔IgG-Cy3、IV型胶原酶(由美国Sigma提供)；胰蛋白酶(碧云天生物技术研究所提供)；SYBR Premix Ex Taq酶、PrimeScriptTMmiRNA qPCR starter kit(日本TaKaRa提供)；碱性成纤维生长因子(bFGF)(由美国PeproTech公司提供)
4寡聚核苷酸链
引物合成(上海生工生物工程有限公司合成)
引物序列：
GAPDH上游引物：CGGAGTCAAC GGATTTGGTC GTAT 24
GAPDH下游引物：AGCCTTCTCC ATGGTGGTGA AGAC 24
SOX2上游引物：GCCCTGCAGT ACAACTCCAT 20
SOX2下游引物：GACTTGACCA CCGAACCCAT 20
OCT4上游引物：ATGTGGTCCG AGTGTGGTTC 20
OCT4下游引物：AAACCCTGGC ACAAACTCCA 20
NANOG上游引物：GAGATGCCTC ACACGGAGAC 20
NANOG下游引物：CTTTGGGACT GGTGGAAGAA 20
SSEA4上游引物：TGGACGGGCA CAACTTCATC 20
SSEA4下游引物：GGGCAGGTTC TTGGCACTCT 20
5其它试剂
质粒抽提试剂盒(美国Promega)；LB培养基(自制)；琼脂糖、溴化乙锭、L-谷氨酰胺、PEI(25kDa)、二甲基亚砜、β-巯基乙醇、DAPI染料、丝裂霉素C(美国Sigma)；Tris碱、氨苄青霉素、链霉素、碱性磷酸酶测定试剂盒(碧云天生物技术研究所)；氯化钙、多聚甲醛(国药集团化学试剂有限公司)；MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四唑溴盐)、LipofectamineTM2000、非必需氨基酸(NEAA)、TRIzol试剂、Glycogen(美国Invitrogen)；基础培养基DMEM、基础培养基DMEM/F12、knockout DMEM、胎牛血清、knockout血清替代品(美国GIBCO)；BD Matrigel基质胶(美国BD公司)；发光剂(ECL-plus)(瑞典Amersham Biosciences)；PVDF膜(瑞士Roche)
6溶液和培养基的配制
(1)完全培养基：低糖DMEM，加入10％FBS和100U/mL青-链霉素溶液。
(2)人胚胎干细胞培养液：Knockout DMEM培养基中依次加入20％血清替代物(Knockout SR)，2mmol/L左旋谷氨酰胺，0.1mmol/Lβ-巯基乙醇，1％非必需氨基酸，100U/mL青-链霉素溶液，4ng/mL人类重组碱性成纤维细胞生长因子，混匀，放置4℃冰箱，备用。
(3)磷酸盐缓冲液(PBS)1L配方(pH7.4)：取NaCl 8.00g，KCl 0.20g，Na2HPO4·12H2O 3.5g，KH2PO4 0.2g，加入800mL双蒸水，调pH至7.4，定容至1000mL，分装，高压灭菌，4℃保存。
(4)30％丙烯酰胺：29g丙烯酰胺和1g N,N’-亚甲双丙烯酰胺溶于100mL双蒸 水中，过滤备用。
(5)Tris-HCl(pH 8.8)：18.2g Tris碱溶于100mL的双蒸水中,浓盐酸调pH。
(6)Tris-HCl(pH 6.8)：6.05g Tris碱溶于100mL的双蒸水中,浓盐酸调Ph。
(7)2×SDS-PAGE Loading Buffer：20％甘油(v/v)，0.2％溴酚蓝(w/v)，4％SDS(w/v)，200mmol/L DTT，100mmol/L Tris-HCl(pH6.8)。
(8)5×Tris-甘氨酸电泳缓冲液：94g甘氨酸和15.1g Tris碱溶于950mL双蒸水中，再加入50mL 10％的SDS贮存液(w/v)。
(9)转膜缓冲液：5.8g Tris碱，2.9g甘氨酸和0.37g SDS溶于800mL双蒸水中，再加入200mL甲醇。
(10)10％SDS贮存液：100g电泳级SDS溶解在1000mL双蒸水中，加热至68℃助溶，浓盐酸调节pH至7.2。
(11)10×TBS：80gNaCl，30gTris和2gKCl溶解于1000mL双蒸水中，浓盐酸调节pH至7.4。
(12)4％多聚甲醛：0.04g多聚甲醛溶解在1mL PBS中。
(13)0.2％TritonX-100：0.2mL TritonX-100溶解在100mL PBS。
(14)0.1％：0.01g BSA溶解在10mL TBS-T中。
(15)裂解液：5mmol/L MgCl2，150mmol/L NaCl，1％Nonidet P-40(NP-40)(v/v)，50mmol/L Tris-HCl(pH 7.4)，aprotinin 10μg/mL，1mmol/L二硫苏糖醇(dithiothreitol，DTT)，leupeptin 10μg/mL和1mmol/L PMSF。
7仪器
XW-80A旋涡混合器(江苏海门市麒麟医用仪器厂)；电热恒温干燥箱(上海跃进医疗器械厂)；超低温高速离心机(德国Heraeus)；超净工作台(苏州净化厂)；HH-S数显恒温水浴锅(江苏省金坛市医疗仪器厂)；THZ-82A恒温振荡器(江苏省金坛市医疗仪器厂)；DY602S稳流稳压电泳仪(南京新校园生物技术研究所)；凝胶成像仪(美国Bio-Rad)；DHA/RNA/PROTEIN analysis(日本岛津)；JEM-2100透射电镜(日本电子)；BI-90粒径分布仪(美国Brookhaven)；ZEN3600电位仪(英国Malvern)；倒置荧光显微镜(日本Olympus)；HB-1加热板(美国Wealtec)；二氧化碳培养箱(美国Thermo)；酶标仪(美国Molecular Devices)；Nikon TE 2000PFS荧光显微镜(日本Nikon)；Western blot蛋白电泳仪(美国Bio-Rad公司)；漩涡振荡器(上海第一医学院仪器厂)；电子分析天平(上海第一医学院仪器厂)
实施例1：三维胶原载基因纳米粒支架的制备：
(1)取适量2％胶原溶液于支架制备的聚四氟乙烯模具中(68×38×5mm3)，先在4℃放置0.5h，再迅速放在-80℃冰箱预冻3h以上,然后冻干；
(2)于120℃烘箱内热固化10h；
(3)之后，用无水乙醇浸泡24h，再以95％，85％，75％，65％，55％，30％，15％浓度梯度的乙醇溶液洗涤，最后用大量双蒸水洗涤，直至无醇味；
(4)再次冻干，切成大小为5×5×3mm3的小块，于超净台进行紫外灭菌3h，备用。热固化之后的过程均保证无菌操作。
实施例2：磷酸钙载基因纳米粒的制备：
(1)将Igepal CO-520溶解于环己烷中制备Igepal CO-520体积分数为29％的Igepal CO-520/环己烷混合液；
(2)微乳A的制备：取一干净锥形瓶，加入25mL Igepal CO-520/环己烷混合液，然后将650μL 0.01M氯化钙溶液，在磁力搅拌的条件下，一起加入到净锥形瓶，继续搅拌2min，形成微乳A；
(3)微乳B的制备：另取一干净锥形瓶，加入25mL Igepal CO-520/环己烷混合液，然后将650μL 0.06M磷酸氢二钠和质粒混合溶液10μL(含Oct4、Sox2、各2.5μg,miR302/367 5μg)一起加入到净锥形瓶，继续搅拌2min，形成微乳B；
(4)在4℃条件下，将微乳A逐滴加入到微乳B，边加边搅拌，直至整个体系透明澄清，得到多糖-磷酸钙混杂纳米粒微乳；
(5)加5mL pH＝7.0的无水乙醇对磷酸钙混杂纳米粒微乳进行稀释；
(6)然后上硅胶色谱柱，先用无水乙醇洗脱环己烷及游离的DNA，然后用含有5×10-4mM NaCl的70％乙醇溶液洗脱多糖-磷酸钙混杂纳米粒，分别收集不同时间点的洗脱夜，HPLC法测定其DNA含量；
(7)将含有磷酸钙载基因纳米粒的乙醇溶液在37℃旋转蒸发6h除去乙醇，浓缩液置于12kDa的透析袋中，pH7.4的PBS中4℃过夜，即得到磷酸钙载基因纳米粒溶液。
实施例3：三维载基因纳米粒-胶原支架的制备
(1)取5×5×3mm3的支架样品放入24孔板的板孔中，每孔加入0.5mL无血清DMEM培养基，让支架充分润湿；
(2)0.5h之后，吸除多余的培养基；
(3)调整磷酸钙载基因纳米粒的浓度，使每100μL纳米粒溶液中含0.8μg总质粒；
(4)向每一块润湿之后5×5×3mm3的支架上滴加100μL磷酸钙载基因纳米粒溶液，必须缓慢、少量多次的滴加，使纳米粒溶液与支架充分接触、吸附；
(5)然后，冷冻干燥，得到三维载基因纳米粒-胶原支架。
实施例4：iPSCs诱导
(1)每一块5×5×3mm3的三维载基因纳米粒-胶原支架用200μL无血清DMEM培养基润湿；
(2)制备体细胞人脐带间充质干细胞的细胞悬液，将200μL细胞悬液(5×104个细胞/支架)少量多次滴加到润湿的支架上；
(3)接种细胞的支架于37℃、5％CO2的培养箱中放置0.5h；
(4)然后，每个放有接种细胞支架的孔中加入含10％FBS的DMEM培养基，继续培养4h；
(5)在上述培养基中培养3天之后，换成人胚胎干细胞培养基，每2天换液一次。在第2天的时候，细胞呈均匀分散的状态分布在支架内；随着时间推移，第4天(即种植细胞第7天)时，开始出现细胞球；以后逐渐形成拟胚体状的细胞球。
实施例5：qRT‐PCR
总RNA的提取和qRT-PCR过程：转染4次之后，细胞用PBS清洗一次，用TRIzol试剂按照说明书的要求制备总RNA，并保存于-80℃，备用。总RNA用Takara公司的SYBR Premix Ex Taq制备cDNA，cDNA经ddH2O稀释20倍后，采用SybrGreen2×PCR Mix进行扩增。数据采用SPSS统计学软件进行双因素方差分析，T检验考察数据显著性差异。结果表明：在三维体系中，经CP/OS-miR转染后，四种iPSCs标志物SOX2，OCT4，NANOG以及SSEA3在转录水平上均高于CP/OSKM转染组。
实施例6：免疫荧光
采用免疫荧光显色进行多能性标记因子的检测，具体操作步骤：
(1)将细胞用PBS洗一次，然后用4％多聚甲醛室温固定30min；
(2)然后用0.2％Triton X-100处理30min，接着在封闭液(0.1％BSA)中封 闭1h；
(3)将处理后的细胞与稀释于封闭液的一抗于4℃孵育过夜，或者37℃孵育1h；
(4)用PBS洗3次后，加入稀释于封闭液的二抗于37℃避光孵育1h；
(5)用PBS洗3次后，加入0.1μg/mL DAPI溶液(用PBS配制)室温避光孵育20-30min；
(6)用PBS洗3次后，于荧光显微镜下观察并拍照。
使用的一抗包括anti-TRA-1-81、anti-SSEA-3、anti-SSEA-4、anti-NANOG和anti-OCT4，二抗为羊抗兔IgG-Cy3。
实施例7：体外诱导向三胚层的分化
(1)将三维体系中的iPSCs细胞用IV型胶原酶于37℃孵育30min，用含血清的培养基终止反应，轻轻吹打，收集细胞悬液；
(2)1500rpm，离心5min，弃上清，细胞用胚胎干细胞培养基重悬成细胞悬液，然后转移到低黏附性的6孔板中，让细胞团聚，悬浮生长；
(3)37℃培养3天之后，1500rpm，离心5min，收集细胞团，重复(1)的操作，用将细胞接种到用BD Matrigel基质胶(用DMEM培养基稀释50倍)处理的24孔板中；
(4)待细胞贴壁之后，进行三胚层的诱导分化：
①肝细胞的诱导分化
实验组：将克隆细胞球接种到24孔板中，放入培养箱中培养，待贴壁后弃掉原有培养基，换为肝细胞培养基(在DMEM培养基中加入HGF 20ng/mL、FGF4 10ng/mL、青霉素100U/mL、链霉素100U/mL、地塞米松10-8M、FBS 2％)，每3天换液一次。对照组：待细胞贴壁后用含10％FBS的DMEM培养基换液，其它操作同实验组。倒置显微镜下观察细胞的形态变化。在诱导后的第28天，用鼠抗甲胎蛋白(Alpha Fetal Protein，AFP)进行免疫荧光检测。
②神经细胞的诱导分化
实验组：将克隆细胞球接种到24孔板中，放入培养箱中培养，待贴壁后弃掉原有培养基，换为神经细胞培养基(在DMEM/F12培养基中加入10ng/mL SHH、10ng/mL BDNF、1μmol/L RA)，继续培养l天后换成Neurobasal细胞培养基(含有10ng/mL SHH、10ng/mL BDNF、1μmol/L RA、2％B27)，每3天换液一次。对照组：待细胞贴壁后用含2％B27的Neurobasal培养基换液，其它 操作同实验组。倒置显微镜下观察细胞的形态变化。在诱导后的第28天，采用鼠抗Tuj1进行免疫荧光检测。
③软骨细胞诱导分化
实验组：将克隆细胞球接种到24孔板中，放入培养箱中培养，待贴壁后弃掉原有培养基，换为含有10ng/mL TGFb-1、20ng/mL bFGF、10-8M地塞米松、10％FBS的DMEM培养基，每3天换液一次。对照组：待细胞贴壁后用含10％FBS的DMEM培养基换液，其它操作同实验组。倒置显微镜下观察细胞的形态变化。在诱导后的第28天，采用鼠抗II型胶原蛋白进行免疫荧光检测，操作参考实施例5。免疫荧光结果显示：内胚层标志AFP、中胚层标记II型胶原蛋白(Collagen II)以及外胚层标志βIII微管蛋白(Tuj1)的表达都非常明显，证明本实验生产的iPSCs具有类似于胚胎干细胞的多向分化潜能。

Claims (5)

  1. 一种重编程因子组合在三维体系中诱导多能干细胞的方法，其特征是它包括如下步骤：
    步骤1.胶原支架的制备：
    取适量2～5％胶原溶液于支架制备的聚四氟乙烯模具中(68×38×5mm3)，先在4℃放置0.5～1h，再迅速放在-80℃冰箱预冻3h以上,然后冻干；于烘箱内热固化8～10h之后，用无水乙醇浸泡24h，再以10～100％浓度梯度的乙醇溶液洗涤，双蒸水洗至无醇味；再次冻干，切成大小为5×5×3mm3的小块，于超净台进行紫外灭菌备用；
    步骤2.磷酸钙纳米粒的制备：
    配制体积分数为20～30％的Igepal CO-520/环己烷混合液并以此为溶剂，以0.01～0.05M氯化钙溶液，0.03～0.08M磷酸氢二钠和质粒混合溶液10μL为原料，通过反相微乳法，得到磷酸钙载基因纳米粒微乳；再经柱层析，旋转蒸发及透析等步骤，得到多磷酸钙载基因纳米粒溶液；
    步骤3.三维载基因纳米粒‐胶原支架的制备：
    取5×5×3mm3的支架样品放入24孔板的板孔中以无血清DMEM培养基使支架充分润湿；吸除多余的培养基；调整磷酸钙载基因纳米粒的浓度，使每100μL纳米粒溶液中含0.8～1.5μg总质粒；向每一块润湿之后5×5×3mm3的支架上滴加100μL磷酸钙载基因纳米粒溶液后冷冻干燥，得到三维载基因纳米粒-胶原支架；
    步骤4.三维体系中的iPSCs诱导和扩增：
    取步骤3得到的5×5×3mm3三维载基因纳米粒-胶原支架并以无血清DMED浸润；将200μL体细胞悬液加入浸润后的支架，在完全培养基中培养3～5天后，换成人胚胎干细胞培养基，每2～3天换液一次，扩增诱导多能干细胞。
  2. 根据权利要求1所述的方法，其特征是：步骤1所述的聚四氟乙烯模具的规格为68×38×5mm3。
  3. 根据权利要求1所述的方法，其特征是：步骤2中所述的质粒混合溶液是含Oct4、Sox2、Klf4、c-Myc、miR302a、miR302b、miR302或miR302-367中的任意三种质粒各2.5μg的质粒混合溶液。
  4. 根据权利要求1所述的方法，其特征是：所述的体细胞悬液加入浸润后的支架是每个支架含5～8×104个细胞。
  5. 根据权利要求1-4任一所述的方法，其特征是：所述的体细胞是HUMSCs细胞
PCT/CN2016/087798 2016-06-30 2016-06-30 一种重编程因子组合在三维重编程体系中诱导多能干细胞的方法 Ceased WO2018000288A1 (zh)

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