Hadland Brandon K, Varnum-Finney Barbara, Nourigat-Mckay Cynthia, Flowers David, Bernstein Irwin D
Clinical Research Division, Fred Hutchinson Cancer Research Center; Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Washington School of Medicine;
Clinical Research Division, Fred Hutchinson Cancer Research Center.
J Vis Exp. 2018 May 8(135):56973. doi: 10.3791/56973.
The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.
在胚胎发育过程中研究造血干细胞（HSC）起源的能力受到早期胚胎中HSC前体稀少以及缺乏功能性鉴定单个假定HSC前体长期多谱系植入潜力的检测方法的限制。在此，我们描述了一种能够在单细胞水平上分离和鉴定经过功能验证的HSC前体的方法。首先，我们利用索引分选来记录每个单独分选细胞的精确表型参数，使用表型标记的组合来富集HSC前体，并使用额外的标记进行实验分析。其次，将每个经过索引分选的细胞与来自主动脉-性腺-中肾（AGM）区域的血管生态位基质共培养，该基质支持未植入的HSC前体在移植实验中成熟为具有多谱系、长期植入潜力的功能性HSC。这种方法能够将克隆造血前体的表型特性与其功能植入潜力或其他特性（如转录谱）相关联，为在单细胞水平上详细分析HSC前体发育提供了一种手段。
