Ansary Stem Cell Institute, Division of Regenerative Medicine, Department of Medicine, Weill Cornell Medicine (WCM), New York, NY, USA.
Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine and Infertility, Weill Cornell Medicine (WCM), New York, NY, USA.
Nat Protoc. 2018 Dec;13(12):2758-2780. doi: 10.1038/s41596-018-0060-3.
The ability to generate hematopoietic stem cells (HSCs) in vitro would have an immeasurable impact on many areas of clinical practice, including trauma, cancer, and congenital disease. In this protocol, we describe a stepwise approach that converts adult murine endothelial cells (ECs) to HSCs, termed 'reprogrammed ECs into hematopoietic stem and progenitor cells' (rEC-HSPCs). The conversion, which is achieved without cells transitioning through a pluripotent state, comprises three phases: induction, specification, and expansion. Adult ECs are first isolated from Runx1-IRES-GFP; Rosa26-rtTa mice and maintained in culture under EC growth factor stimulation and Tgfβ inhibition. In the first (induction) phase of conversion (days 0-8), four transcription factors (TFs)-FosB, Gfi1, Runx1, and Spi1 (FGRS)-are expressed transiently, which results in endogenous Runx1 expression. During the second (specification) phase (days 8-20), endogenous Runx1 FGRS-transduced ECs commit to a hematopoietic fate and no longer require exogenous FGRS expression. Finally, the vascular niche drives robust proliferation of rEC-HSPCs during the expansion phase (days 20-28). The resulting converted cells possess a transcriptomic signature and long-term self-renewal capacity indistinguishable from those of adult HSCs. In this protocol, we also describe functional in vitro and in vivo assays that can be used to demonstrate that rEC-HSPCs are competent for clonal engraftment and possess multi-lineage reconstitution potential, including antigen-dependent adaptive immune function. This approach thus provides a tractable strategy for interrogating the generation of engraftable hematopoietic cells, advancing the mechanistic understanding of hematopoietic development and HSC self-renewal.
体外生成造血干细胞（HSCs）将对许多临床实践领域产生不可估量的影响，包括创伤、癌症和先天性疾病。在本方案中，我们描述了一种逐步的方法，将成年鼠内皮细胞（ECs）转化为 HSCs，称为“重编程 ECs 为造血干细胞和祖细胞”（rEC-HSPCs）。这种转化是在细胞不经过多能状态的情况下实现的，包含三个阶段：诱导、特化和扩增。首先从 Runx1-IRES-GFP；Rosa26-rtTa 小鼠中分离成年 ECs，并在 EC 生长因子刺激和 TGFβ抑制下进行培养。在转化的第一（诱导）阶段（第 0-8 天），表达四种转录因子（TFs）-FosB、Gfi1、Runx1 和 Spi1（FGRS）-，导致内源性 Runx1 表达。在第二（特化）阶段（第 8-20 天），内源性 Runx1 FGRS 转导的 EC 开始向造血命运分化，不再需要外源性 FGRS 表达。最后，血管龛在扩增阶段（第 20-28 天）驱动 rEC-HSPCs 的大量增殖。得到的转化细胞具有与成年 HSCs 相同的转录组特征和长期自我更新能力。在本方案中，我们还描述了可用于证明 rEC-HSPCs 具有克隆植入能力和多谱系重建潜力（包括抗原依赖性适应性免疫功能）的体外和体内功能测定。因此，该方法为研究可植入造血细胞的生成提供了一种可行的策略，推进了对造血发育和 HSC 自我更新的机制理解。
