浙江大学医学院干细胞与再生医学中心郭国骥教授课题组与浙大医学院段树民实验室及哈佛医学院Orkin实验室展开合作,首次提出多向转分化的概念,成功优化出小鼠成纤维细胞分别向胶质细胞、肌样细胞和脂肪细胞定向转分化的高效诱导条件,这项研究为再生医学的细胞来源问题提供了新的思路,该研究成果近期发表在了《Cell Research》上。
该课题组的韩晓平博士发现,利用小分子组合能将小鼠成纤维细胞同时转分化为来自三个胚层的多种成体细胞。免疫荧光染色、病理染色及电生理实验验证了转分化细胞的多样性和功能性。GFAP-GFP、AFP-GFP及OCT4-GFP报告基因细胞的相关实验揭示了小鼠成纤维转分化的动态过程。转录组、DNA甲基化以及组蛋白修饰等组学分析描绘了小分子组合诱导转分化过程的基因调控网络与染色质结构变化,鉴定了Sox2为这一过程的关键调控因子。最后,单细胞分析实验证实,小鼠成纤维细胞在处理第六天被激活成一种高异质性的混沌状态,进而在十多天后形成各种类型的成熟细胞。
文章指出:通过对单细胞基因表达检测,可以得到更多、更准确的关于转分化细胞的特异基因表达分子标记物。同时可以更有效地评估在化学药物刺激后,基因表达的变化情况,并通过对源自不同胚层的标记物进行分析,追踪转分化细胞的来源。
研究中作者利用Biomark系统和96.96 Dynamic Array芯片在单细胞水平对每个样本进行了多达96个基因位点的检测,聚类分析数据显示:特异的基因表达谱表明不同细胞组群其来源不同(图A)。t-SNE分析方法进一步显示了清晰的细胞分群数据(图B)。
(A) A hierarchical clustering heatmap showing single cell geneexpression analysis data of the iMT experiment. Non-treated MEFs andundifferentiated ESCs are also analyzed as controls. (B) t-SNE plot showing theiMT priming and fate decision pathway at single cell resolution.
相关阅读
Fluidigm公司Biomark HD高通量基因分析系统利用微流控技术,结合高度集成化的芯片,将反应控制在纳升级体系内,不仅节约了大量的试剂,更可利用FFPE等稀有样本甚至是单细胞样本完成定量PCR检测。利用该系统可对96个样本,单样本多达96个基因位点同时进行检测;开放式的体系设计,可根据不同研究目的灵活调整组合待测位点,大幅提高实验研究效率。 (http:// fluidigm.com/products/biomark-hd-system)
英文摘要
Recent advances have demonstrated the power of small molecules in promoting cellular reprogramming. Yet,the full potential of such chemicals in cell fate manipulation and the underlying mechanisms require further characterization. Through functional screening assays, we find that mouse embryonic fibroblast cells can be induced to trans-differentiate into a wide range of somatic lineages simultaneously by treatment with a combination of four chemicals. Genomic analysis of the process indicates activation of multi-lineage modules and relaxation of epigenetic silencing programs. In addition, we identify Sox2 as an important regulator within the induced network. Single cell analysis uncovers a novel priming state that enables transition from fibroblast cells to diverse somatic lineages. Finally, we demonstrate that modification of the culture system enables directional trans-differentiation towards myocytic, glial or adipocytic lineages. Our study describes a cell fate control system that may be harnessed for regenerative medicine.
参考文献:
A molecular roadmap for induced multi-lineagetrans-differentiation of fibroblasts by chemical combinations.
Han X, et al. Cell Res. 2017, 27(6):843.