Daniel Michael G, Pereira Carlos-Filipe, Bernitz Jeffrey M, Lemischka Ihor R, Moore Kateri
Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai; The Graduate School of Biomedical Science, Icahn School of Medicine at Mount Sinai; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai.
Center for Neuroscience and Cell Biology, University of Coimbra.
J Vis Exp. 2016 Dec 16(118):54372. doi: 10.3791/54372.
This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34 cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.
本方案详细介绍了通过过表达转录因子（TFs）在小鼠胚胎成纤维细胞（MEFs）中诱导造血程序的方法。我们首先利用来自人CD34 - tTA/TetO - H2BGFP（34/H2BGFP）双转基因小鼠的MEFs设计了一个报告基因筛选。这些小鼠的CD34细胞用GFP标记H2B组蛋白，并在加入强力霉素（DOX）后停止标记。用候选TFs转导MEFs，然后观察GFP细胞的出现，这将表明获得了造血或内皮细胞命运。从18个候选TFs开始，通过组合排除过程，我们获得了一组能诱导最高比例GFP细胞的最小因子集。我们发现Gata2、Gfi1b和cFos是必需的，添加Etv6可提供最佳诱导效果。在重编程过程的不同时间点进行的一系列基因表达分析表明，这些细胞似乎经历了一个经历内皮向造血转变（EHT）的前体细胞阶段。因此，这种重编程过程在“培养皿”中模拟了发育性造血，允许在体外研究造血，并提供了一个平台来识别这一细胞特化过程的潜在机制。这种方法还为将这项工作转化到人类系统提供了一个框架，有望为血液疾病的治疗和研究中的许多应用生成患者特异性造血干细胞（HSCs）的替代来源。
