Transcriptional and protein-level profiling of engraftable human fetal liver (fl)-derived hematopoietic stem cells (HSCS)

We provide an in-depth characterization of engraftable fetal liver (FL) HSCs as these have been shown to possess superior engraftment potential compared to cord blood (CB) and bone marrow (BM) HSCs. We have single cell profiled 26,407 FL cells, of which 7,235 highly-enriched functional FL HSCs, both at the transcriptional and protein level to uncover the detailed molecular signature of engraftment potential. Overall design: Following dissociation of human FL, cells were divided into either CD34+-enriched cells or CD34- flowthrough cells via magnetic bead separation. The CD34- live cell fraction was further subdivided into GYPA+ and GYPA- via fluorescence activated cell sorting (FACS). From the CD34+-enriched fraction live CD34+ cells were sorted (CD34+bulk). In a separate sample, we further enriched this population using FACS based on GPI-80 expression (GPI-80+), a marker tightly linked to engraftment potential to focus our analysis on HSCs capable of long-term engraftment. Prior to sorting, cells were stained with a panel of oligo-tagged antibodies so that the antibody-derived tags (ADTs) corresponding to the cell surface markers present on each cell would also be captured in the sequencing data.

本研究对可植入胎肝（fetal liver, FL）造血干细胞（hematopoietic stem cells, HSCs）开展了深度表征——已有研究证实，这类造血干细胞的植入潜能优于脐血（cord blood, CB）与骨髓（bone marrow, BM）来源的造血干细胞。我们对26407个胎肝细胞进行了单细胞转录组与蛋白质组分析，其中包含7235个高度富集的功能性胎肝造血干细胞，旨在揭示植入潜能的精细分子特征。
实验整体设计如下：对人胎肝组织进行解离后，通过磁珠分选将细胞分为CD34阳性富集组分与CD34阴性流出组分。其中CD34阴性活细胞组分进一步通过荧光激活细胞分选（fluorescence activated cell sorting, FACS）分为GYPA阳性与GYPA阴性亚群。从CD34阳性富集组分中分选得到活的CD34阳性细胞（记为CD34+bulk）；在另一独立样本中，我们基于与植入潜能紧密相关的标志物GPI-80的表达，通过荧光激活细胞分选进一步富集该群体（记为GPI-80+），以将分析聚焦于具备长期植入潜能的造血干细胞。分选前，我们使用寡核苷酸标记抗体进行染色，使得对应细胞表面标志物的抗体衍生标签（antibody-derived tags, ADTs）也能被捕获至测序数据中。