Induction of a hemogenic program in mouse fibroblasts

Definitive hematopoiesis emerges during embryogenesis via an endothelial-to-hematopoietic transition. We attempted to induce this process in mouse fibroblasts by screening a panel of factors for hemogenic activity. We identified a combination of four transcription factors, Gata2, Gfi1b, cFos, and Etv6 that efficiently induces endothelial-like precursor cells with the subsequent appearance of hematopoietic cells. The precursor cells express a human CD34 reporter, Sca1 and Prominin1 within a global endothelial transcription program. Emergent hematopoietic cells possess nascent/specifying hematopoietic stem cell gene expression profiles and cell surface phenotypes. After transgene silencing and reaggregation culture, the specified cells generate hematopoietic colonies in vitro. Thus, we have shown that a simple combination of transcription factors is sufficient to induce a complex, dynamic and multi-step developmental program in vitro. These findings provide insights into the specification of definitive hemogenesis and a platform for future development of patient-specific stem/progenitor cells as well as more differentiated blood products. Overall design: mRNA-seq profiling on populations generated after transduction with Gata2, Gfi1b, cFos and Etv6 at day 20 and day 35.

定型造血（definitive hematopoiesis）在胚胎发生过程中通过内皮向造血转化（endothelial-to-hematopoietic transition）产生。本研究尝试在小鼠成纤维细胞中诱导该过程，通过筛选一组因子以鉴定其造血生成活性。我们最终筛选得到由四个转录因子组成的组合：Gata2、Gfi1b、cFos及Etv6，该组合可高效诱导类内皮前体细胞，并随之产生造血细胞。这类前体细胞在整体内皮转录程序中，表达人源CD34报告基因、Sca1与Prominin1。新生造血细胞具有处于形成/特化阶段的造血干细胞基因表达谱及细胞表面表型。在经转基因沉默及细胞重聚集培养后，经特化的细胞可在体外形成造血集落。由此我们证实，仅需一组简单的转录因子组合，即可在体外诱导出复杂、动态且多步骤的发育程序。本研究结果为阐明定型造血生成的特化机制提供了新见解，同时为未来开发患者特异性干细胞/祖细胞以及更成熟的血液制品搭建了研究平台。总体实验设计：对转染Gata2、Gfi1b、cFos及Etv6后第20天和第35天获取的细胞群进行信使RNA测序（mRNA-seq）转录组分析。