中科院上海生命科學(xué)院/上海交大醫(yī)學(xué)院健康科學(xué)研究所研究員金穎所率干細(xì)胞研究組與上海新華醫(yī)院的陳方教授合作，從孕婦產(chǎn)前診斷的羊水細(xì)胞中高效快速地建立了誘導(dǎo)多能干細(xì)胞,。
博士生李春亮等在金穎的指導(dǎo)下發(fā)現(xiàn)羊水細(xì)胞中一部分特殊類(lèi)群（即高表達(dá)NESTIN, VIMENTIN and GATA4,不表達(dá)OCT4, SOX2, NANOG and TRA-1-60）在病毒介導(dǎo)的四因子誘導(dǎo)下，感染后第二天發(fā)生形態(tài)上的劇烈變化,，第四天出現(xiàn)人胚胎干細(xì)胞類(lèi)似形態(tài)的克隆，第六天可以機(jī)械法挑選后進(jìn)行建系,。經(jīng)過(guò)統(tǒng)計(jì)發(fā)現(xiàn),，AKP陽(yáng)性的未分化克隆形成率最高可達(dá)到1.525%，有趣的是,，當(dāng)減少因子C-MYC后,，三因子同樣可以在第四天誘導(dǎo)出iPS克隆，只是AKP陽(yáng)性的未分化克隆形成率稍低,，從三個(gè)獨(dú)立的病人樣本中,，均能夠穩(wěn)定高效的誘導(dǎo)出iPS細(xì)胞,，提示這群細(xì)胞高效發(fā)生重編程具有普遍性。對(duì)建立的八株人類(lèi)誘導(dǎo)多能干細(xì)胞進(jìn)行進(jìn)一步的鑒定發(fā)現(xiàn),，這些細(xì)胞能夠長(zhǎng)期在體外穩(wěn)定傳代并保持46XX核型,，維持自我更新，蛋白和mRNA水平高表達(dá)全能性的標(biāo)志基因,，如OCT4, NANOG, SOX2, SSEA4, TRA-1-60等，AKP染色呈強(qiáng)陽(yáng)性,，甲基化PCR聯(lián)合測(cè)序法對(duì)OCT4的啟動(dòng)子進(jìn)行分析后發(fā)現(xiàn),，在未分化的iPS細(xì)胞和陽(yáng)性對(duì)照人胚胎干細(xì)胞中，OCT4的啟動(dòng)子呈現(xiàn)低甲基化,，而在供體羊水細(xì)胞和陰性對(duì)照人包皮成纖維細(xì)胞中則呈現(xiàn)高度甲基化,。STR分析結(jié)果證明這些誘導(dǎo)多能干細(xì)胞的確來(lái)自于對(duì)應(yīng)供體的羊水細(xì)胞，有趣的是,，全基因組表達(dá)譜掃描結(jié)果提示,，研究人員所得到的羊水細(xì)胞在表達(dá)譜相關(guān)性上和人胚胎干細(xì)胞非常相似，correlation coefficient達(dá)到0.8866,，這可能是它高效被重編程的原因之一,。誘導(dǎo)多能干細(xì)胞的主要應(yīng)用是分化和細(xì)胞移植，研究人員嘗試了體外和體內(nèi)的分化實(shí)驗(yàn),，在懸浮類(lèi)胚體和實(shí)驗(yàn)中,，科學(xué)家能得到典型的中、外,、內(nèi)胚層形態(tài)細(xì)胞,，RT-PCR檢測(cè)到了各個(gè)胚層標(biāo)志物的表達(dá)，定向誘導(dǎo)向神經(jīng)外胚層的分化中,，誘導(dǎo)35天后,，研究人員得到了高純度的神經(jīng)前體細(xì)胞。在體內(nèi)畸胎瘤實(shí)驗(yàn)中,，注射未分化的誘導(dǎo)多能干細(xì)胞到免疫缺陷小鼠三個(gè)月以后能夠得到良性的畸胎瘤,，切片分析能找到形態(tài)典型的肌肉，腸管,，神經(jīng)上皮,，軟骨，肺上皮等三胚層來(lái)源組織或者細(xì)胞,，而對(duì)照細(xì)胞即起始羊水細(xì)胞注射的各組均未發(fā)現(xiàn)畸胎瘤,。
綜上所述，該項(xiàng)研究第一次發(fā)現(xiàn)孕婦產(chǎn)前診斷的羊水細(xì)胞中高效快速建立了誘導(dǎo)多能干細(xì)胞,，重編程所發(fā)生所需要的時(shí)間（6天）為人類(lèi)誘導(dǎo)多能干細(xì)胞相關(guān)報(bào)道最短,，減少了在這個(gè)過(guò)程中細(xì)胞發(fā)生變異的可能,。也為重編程的機(jī)制研究以及基于新型技術(shù)探討重編程的研究提供了理想的細(xì)胞來(lái)源。
該成果于8月13日在線發(fā)表于國(guó)際權(quán)威雜志Human Molecular Genetics,。（生物谷Bioon.com）
金穎老師參加干細(xì)胞講座
生物谷推薦原始出處：
Human Molecular Genetics, doi:10.1093/hmg/ddp386
Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells
Chunliang Li1,4,#, Junmei Zhou5,6,#, Guilai Shi1,4, Yu Ma1,2, Ying Yang1,4, Junjie Gu1,2, Hongyao Yu1,4, Shibo Jin1,2, Zhe Wei1,4, Fang Chen5,6 and Ying Jin1,2,3,*
1 Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai JiaoTong University School of Medicine; 225 South Chongqing Road, Shanghai, 200025, China 2 Shanghai Stem Cell Institute, Shanghai JiaoTong University School of Medicine, Shanghai, China 3 Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai JiaoTong University School of Medicine, Shanghai, China 4 Graduate School of Chinese Academy of Sciences, Beijing, China 5 Department of Urology, Shanghai Children's Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China 6 Tissue Engineering Laboratory, Xinhua Hospital, Shanghai Institute for Pediatric Research, Shanghai JiaoTong University School of Medicine, Shanghai, China
Direct reprogramming of human somatic cells into pluripotency has broad implications in generating patient-specific induced pluripotent stem (iPS) cells for disease modeling and cellular replacement therapies. However, the low efficiency and safety issues associated with generation of human iPS cells have limited their usage in clinical settings. Cell types can significantly influence reprogramming efficiency and kinetics. To date, human iPS cells have been obtained only from a few cell types. Here, we report for the first time rapid and efficient generation of iPS cells from human amniotic fluid-derived cells (hAFDCs) via ectopic expression of four human factors: OCT4/SOX2/KLF4/C-MYC. Significantly, typical single iPS cell colonies can be picked up six days after viral infection with high efificiency. Eight iPS cell lines have been derived. They can be continuously propagated in vitro and express pluripotency markers such as AKP, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81, maintaining the normal karyotype. Transgenes are completely inactivated and the endogenous OCT4 promoter is adequately demethylated in the established iPS cell lines. Moreover, various cells and tissues from all three germ layers are found in embryoid bodies and teratomas respectively. In addition, microarray analysis demonstrates a high correlation coefficient between hAFDC-iPS cells and human embryonic stem cells, but a low correlation coefficient between hAFDCs and hAFDC-iPS cells. Taken together, these data identify an ideal human somatic cell resource for rapid and efficient generation of iPS cells, allowing us to establish human iPS cells using more advanced approaches and possibly to establish disease- or patient-specific iPS cells.
