【作者】 吴月红；

【导师】 张涌； Prof.Peter Hornsby；

【作者基本信息】 西北农林科技大学 ， 发育生物学， 2010， 博士

【摘要】 诱导性多潜能干细胞(induced pluripotent stem cells,iPSCs )的研究尚处于初级阶段,仍存在许多技术问题,其中之一是细胞重编程效率太低。目前,很多提高体细胞重编程效率的方法都集中在细胞诱导之后的细胞重编程过程,或是不同的诱导方法,而对提高两个或多个病毒共感染细胞的效率方面尚未进行过系统全面的研究报道。本研究首先用高浓度聚凝胺浓缩病毒结合低速离心的方法感染与狨猴同源性相近的人成纤维细胞(PHF),优化逆转录病毒感染细胞的方案,并且应用小鼠(3T3)成纤维细胞进一步检测优化的感染细胞方法的可行性;其次,用优化的逆转录病毒感染细胞的方法将4种转录因子(hOct4,hSox2,hKlf4和hc-Myc)基因导入新生狨猴皮肤成纤维细胞,首次将其诱导成为miPSCs;同时建立了miPSCs的无饲养层培养体系,并且通过培养液中添加Rho-激酶抑制剂Y27632来促进miPSCs的单细胞的存活和增殖。1、分别以人成纤维细胞和3T3细胞为靶细胞,双嗜性包装细胞系Plat-A生产的表达绿色荧光蛋白pLEGFP-N1和表达红色荧光蛋白pMXs-dsRed2基因病毒按体积比为1:1(相同病毒滴度混合)的比例混合,比较未浓缩病毒的普通方法,优化的离心感染细胞的方法(spinoculation),超速离心浓缩病毒(10,000 g),高浓度聚凝胺(Polybrene)浓缩病毒( Flocculation )以及浓缩病毒结合离心感染细胞的方法(spinoculation/Flocculation)对共转染效率的影响,结果表明320μg/ml高浓度聚凝胺浓缩病毒(Flocculation)同低速离心感染细胞(spinoculation)相结合的方法将共转染率由普通方法的4.0±0.1%(PHF)和3.4±0.2%(3T3)分别提高到了53.0±0.6%(PHF)和35.0±0.4%(3T3)。2、用优化的逆转录病毒共转染的方法将逆转录病毒pMXs-hOct4,hSox2,hKlf4,hc-Myc 4种转录因子基因导入新生狨猴皮肤成纤维细胞(MF),用1mM丙戊酸的培养液处理12天。感染4×10~5 MF,获得大约100个iPSCs样克隆,克隆分离培养于MEF饲养层上。其中30个克隆能够扩大培养,并冷冻保存。从中挑选8个克隆作为进一步的生物学特性研究,具有典型胚胎干细胞的形态特征,碱性磷酸酶染色呈阳性;qPCR结果表明表达很高的Nanog,Oct4和Sox2 mRNAs而相应的载体基因保持沉默;具有正常的染色体核型;免疫细胞化学表明表达胚胎干细胞特异性标志Oct4以及阶段特异性胚胎表面抗原SSEA-4,肿瘤排斥抗原TRA-1-81;于严重联合性免疫缺陷小鼠(RAG2-/-,c-/-)体内形成具有内、中、外三个胚层不同组织结构的畸胎瘤。因此,所得miPSCs是完全重编程细胞。3、以MEF,STO分别制备条件培养液,比较了不同条件培养液对miPSCs的影响;同时,本试验以MEF为条件培养液比较了不同浓度血清(FBS)(10 %,15 %和20 %),不同浓度的条件培养液(35 %和17.5 %)以及不同浓度bFGF(20 ng/ml,100 ng/ml和150 ng/ml)对miPSCs的影响。通过qPCR的方法研究Oct4,Sox2,nanog表达的影响,于不同浓度FBS、条件培养液和bFGF中基因表达水平没有差异,并且同MEF为饲养层培养的miPSCs的基因表达水平没有差异;无饲养层条件下miPSCs碱性磷酸酶呈阳性;免疫细胞化学表明表达胚胎干细胞特异性标志Oct4以及表面抗原TRA-1-81和SSEA-3,SSEA-4;具有正常的核型;体外能够分化为神经元,严重联合性免疫缺陷小鼠(RAG2-/-,c-/-)体内形成具有三个胚层不同组织结构的畸胎瘤,说明无饲养层培养体系适用于miPSCs。4、用Accutase将miPSCs消化为单个细胞,通过将无饲养层培养液中添加10μmol/L Y27632,培养低密度miPSCs检验Y27632的影响,结果表明Y27632能够促进miPSCs存活,其克隆效率由5.0±0.4 %提高到33.0±6.7 % (P<0.01);提高冷冻复苏细胞的克隆效率到36.0±6.7 %(P<0.01);BrdU免疫荧光染色表明,Y27632能够促进miPSCs的增殖,BrdU阳性细胞数由27.0±2.1 %提高到了34.0±1.8 %(P<0.01);qPCR、免疫细胞化学表明,仍表达Nanog,Oct4和Sox2;具有正常的核型;能够体外分化为神经元,严重联合性免疫缺陷小鼠体内形成具有三个胚层组织的畸胎瘤,因此,10μmol/L Y27632培养条件下的miPSCs仍具有多能性。更多还原

【Abstract】 Induced pluripotent stem cells ( iPSCs ) are adult cells that have been genetically reprogrammed to an embryonic stem cell–like state by being forced to express genes and factors for maintaining the properties of embryonic stem cells. iPSCs have the ability for self-renewal and differentiation into different kinds of cell types. Although additional research is needed, iPSCs are already an useful tool for drug development and modeling of diseases, hoping to use them in transplantation medicine. iPSCs hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Potentially, any mammalian species could serve as a translational model for this kind of autologous cell therapy, but nonhuman primates are particularly suited for such studies because of the aspects of their anatomy and physiology that they share with humans. Among nonhuman primates, the marmoset is especially relevant for biomedical research because of well developed models in this species for neurodegenerative disorders (Parkinson’s, Alzheimer’s and Huntington’s disease). Forms of experimental cell therapy have been developed for models of neural injury in the marmoset and the marmoset has been proposed as an ideal model for elucidation of safety and efficiency of new technologies in regenerative medicine.iPSCs have been derived from somatic cells of mouse, human, rhesus monkey, rat, pig, dog and rabbit. It’s another milestone in the history of stem cell development. But there is no report about marmoset iPSCs. iPSCs is still in their infancy, there are some problems to be solved, for example, low efficiency of reprogramming. So far, people focused on reprogramming or inducing methods, but there has been little work on optimizing conditions for coinfection with retroviral vectors.In this study, we opimized the methods for cell infection with higher concentration polybrene (Flocculation) combine with spinoculation by infecting human (HPF) and mouse (3T3) fibroblast. In addition, we derived marmoset iPSCs using the optimized retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc. Meanwhile, we established the feeder free condition of marmoset iPSCs, also Pho-kinase Y27632 promote the survival and proliferation of dissociated marmoset iPSCs.1. In this study, we used amphotropic retroviral vectors produced by the Plat-A cell line to investigate coinfection efficiency (defined as cells that are both red and green as a percentage of all cells infected) using green and red fluorescent proteins (pLEGFP-N1 and pMXs-dsRed2). Primary human fibroblasts and 3T3 cells were used as target cells. We tested the effect of spinoculation, concentration by centrifugation at 10,000g or by flocculation using Polybrene, concentration by Polybrene flocculation and optimized spinoculation on the coinfection rates. The results showed that unconcentrated vector preparations produced a coinfection rate of 4.0±0.1% (PHF) and 3.4±0.2% (3T3). Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation increased the coinfection rate to 53.0±0.6% (PHF) and 35.0±0.4% (3T3) separately.2. In this investigation, we produced marmoset iPSCs using optimized retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc. After transduction, we treated cells with 1mM Valproic acid (VPA) for 12 days. Starting with a population of 4×105 MF, we obtained around 100 colonies with iPSCs-like morphology. Of those, 30 were expanded sufficiently to be cryopreserved. From those, 8 were characterized in more detail. All the 8 clones fulfil the critical criteria for successful reprogramming: they exhibit typical iPSCs morphology; they are alkaline phosphatase positive; they express high levels of Nanog, Oct4 and Sox2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice (RAG2-/-, c-/-) these clones produce teratomas which have derivatives of all three germ layers (endoderm,α-fetoprotein; ectoderm,βIII-tubulin; mesoderm, smooth muscle actin). These experiments provide the evident that iPSCs technology can be adapted for use in the marmoset, as a future model of autologous cell therapy.3. In this study, we investigated the effect of condition medium produced from MEF and STO on marmoset iPSCs, feeder free condition can support marmoset iPSCs. meanwhile, we compared effect of different concentration of FBS (10%, 15%, 20%), condition medium (35% and 17.5%) and different concentration of bFGF (20 ng/ml,100 ng/ml and 150 ng/ml) on iPSCs. The results showed that mamoset iPSCs under different feeder free condition expressed the Oct4, Sox2, Nanog at the similar levels that were comparable to that under MEF feeder condition. They are alkaline phosphatase positive, they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice (RAG2-/-, c-/-) they produce teratomas that have derivatives of all three germ layers; Also, they differentiated to neurons in vitro.4. In this study, we investigated the effect of Y27632 on marmoset iPSCs. Y27632 significantly improved the efficiency of colony formation from single cells without changing pluripotent state and karyotype in feeder free culture for miPSCs. Dramatically increased cloning efficiency from 5.0±0.4 % to 33.0±6.7 %. In vitro, miPSCs gave rise to neurons. Measurement of proliferation by means of BrdU (bromodeoxyuridine) incoporation revealed that Y27632 promote miPSCs proliferation.更多还原