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我国掌握并发展能使体细胞重编程为多能干细胞的iPS技术
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近日，中科院广州生物医药与健康研究院华南干细胞与再生医学研究所在我国率先掌握并发展干细胞研究新技术，这项成果11月6日在自然子刊《细胞研究》上发表并引起广泛的关注。
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中科院广州生物医药与健康研究院的裴端卿小组率先在我国展开胚胎干细胞多能性维持与自我更新机制研究， 并在Oct4，Sox2， Nanog等转录因子在胚胎干细胞中的功能研究方面做出过一系列的工作。该小组的骨干成员秦大江等研究人员勇于探索，运用反转录病毒将Oct4，Sox2，Myc，和KLF4四个因子导入未经遗传修改的小鼠成纤维细胞，并将该类细胞去分化与重编程为与胚胎干细胞相似特征的干细胞。该类细胞具备胚胎干细胞所具备的特征，在国际上称为iPS (induced pluripotent stem cell)干细胞，中文的直译为“诱导多能干细胞”。 3 N$ m& t1 A4 @0 i+ g" C
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据文献报道，这项技术首先由日本科学家2006年发明。最近，美日科学家利用类似技术将人的成体细胞转化为诱导多能干细胞。 这些进展被认为是干细胞领域乃至整个生物学领域划时代的重大发现。除其在干细胞治疗与再生医学的应用价值外，也是人类揭示生物发育与疾病机制研究方法学上的突破，其前景广阔。
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: Q7 ?3 S* }2 R+ q 中科院广州生物研究院的这一项研究的创新点在于利用了未经修饰并且不带有选择标记的小鼠成体细胞，探索出了直接运用iPS 技术的新途径，并获取了近千分之三左右的高成功率。由于iPS技术还处于早期研发阶段，该技术的掌握与进一步创新将提升我国在该领域的研究水平。（广州分院）* O/ L- k1 o+ ]7 v% ~. E K
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Cell Research (2007) 17: 959–962. doi: 10.1038/cr.2007.92; published online 6 November 20078 I; k8 c' P; Q2 ^3 d* v
/ ~% }1 c9 p6 k$ G) O( z$ r! |" eDirect generation of ES-like cells from unmodified mouse embryonic fibroblasts by Oct4/Sox2/Myc/Klf4
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Dajiang Qin1, Wen Li1, Jin Zhang1 and Duanqing Pei1" v( N- n* B& S8 J
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1Stem Cell and Cancer Biology Group, Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, China.
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Correspondence: Duanqing Pei, Tel: +86-20-3229-0520 E-mail: pei_duanqing@gibh.ac.cn
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Dear Editor: A! x) @& d7 l9 k
, V1 U7 P/ j: K# P G1 cThe demonstration that four transcription factors, Oct4/Sox2/Myc/Klf4, can reprogram fibroblasts into ES-like cells or induced pluripotent stem cells (iPS cells) has generated tremendous interests not only in the field of stem cell biology, but also those related fields such as developmental biology and regenerative medicine 1, 2, 3, 4, 5. The advance has greatly improved the prospects of generating patient specific pluripotent stem cells for therapeutic purposes without therapeutic cloning, an approach with formidable technical as well as ethical challenges. The conceptual breakthrough of the iPS strategy is quite obvious, demonstrating for the first time that the reprogramming of somatic nuclei can be achieved through a rational combination of transcription factors with defined regulatory activities, in contrast to the ill defined reprogramming power of unfertilized eggs. However, the application of the iPS strategy is so far limited to mouse fibroblasts carrying engineered selection markers 3, 4. The need for selection using drug resistance or marker driven by Nanog- or Oct4- promoters would hamper not only its application in human therapy, but also any attempts to popularize this exciting experimental approach to other species such as primates. We reason that cells reprogrammed by Oct4/Sox2/Myc/Klf4 can be identified morphologically among the parental fibroblasts and the acquired pluripotent property should offer a growth advantage over their parental fibroblasts. We report here a simple protocol to generate iPS cells from unmodified mouse embryonic fibroblasts (MEF) by retroviral introduction of Oct4/Sox2/Myc/Klf4 without drug selection3, 6.7 C7 n& i$ e: u/ C/ Q6 b
1 s0 q+ n1 z! o; [6 }Mouse embryonic fibroblasts (MEFs) were isolated from wild type E13.5 embryos by standard protocol and cultured in DMEM supplemented with 10% FBS. Cells from passage 2 were infected with control retrovirus or a cocktail of retroviruses carrying Oct4, Sox2, Myc and Klf4 respectively at day 0 as illustrated in Figure 1A. Cells were observed and documented by photography for morphological changes as shown in Figure 1A for the time intervals and Figure 1B for culture morphology. It becomes apparent that MEFs infected with the four factors exhibit enhanced growth with morphological changes starting at Day 4 as shown in Figure 1B. Foci began to appear at Day 4 and became distinct at Day 9. At day 11, colonies as shown in Figure 1B were picked up, dissociated by trypsin digestion and plated into new culture dishes. The resulting colonies were documented as in Figure 1C with typical morphologies of mouse embryonic stem cells. To demonstrate that these cells possess characteristics of embryonic stem cells, we stained them for alkaline phosphatase activity. As shown in Figure 1D, colonies with stem cell morphology have strong alkaline phosphatase activity. Interestly, despite the various morphologies exhibited by the colonies in Figure 1D, all of them are AP positive, suggesting that the population of colonies we obtained may be at different pluripotent states. To assess the overall efficiency of reprogramming, we performed quantification based on morphology and AP activity. In a typical run of reprogramming experiment, we obtained a total of 932 colonies per 250 000 MEF cells at day 16 (Figure 1E). Among them, 53.9% of the colonies are morphologically similar to ES cells and 68.6% AP positive. The discrepancy between ES-like and AP positive colonies may reflect the diversity of clones generated by these four transcription factors without selection. Based on these data, we estimate that our colony forming efficiency is 0.37%. The estimated efficiency for AP positive colonies is 0.256%. Given the published efficiency of iPS being at 0.03% for Nanog- and Oct4- iPS cells based on drug resistance selection, the higher efficiency we obtained suggest that drug selection process eliminates about 80-90% of the colonies prematurely. As the reprogramming process takes more than 2 weeks to complete, early application of drug selection might have killed those cells that incorporated the four factors into their genomes, but did not have enough time to activate Nanog- or Oct4- driven drug resistance markers.
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1 `- Y# s5 P! y" b# J, ] Reprogramming of genetically unmodified MEF into ES-like cells. (A) Outline of the MEF reprogramming protocol. 2.5105 MEF cells were plated in two 100 mm dishes and were infected with Sox2, Klf4, c-Myc and Oct3/4 virus. Cells in dish 1(blue arrow) were passage on day 8 after infection and ES-like colonies form 4 days later. Cells in dish 2 (red arrow) were AP staining positive on day 14 and the colonies were counted. (B) Significant morphology changes observed on day 4 and ES-like colonies form on day 9 after MEF cells were infected with Sox2, Klf4, c-Myc and Oct3/4 virus. (C) Morphology of ES-like colonies on day 6 after passage. (D) AP positive colonies on day 14 post infection. (E) Estimated reprogramming efficiency of genetically unmodified MEF into AP positive ES-like cells 16 days post infection. (F) ES-like cell lines were established using the nonselective approach. Representative colonies from passage 6 cells were stained with anti-Rex1, Sox2 and SSEA1 antibodies and images were acquired through a Leica confocal system. |
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发表于 2009-6-28 20:59
发表于 2009-6-29 12:31
