RAG1+ multipotent progenitors emerge directly from hemogenic endothelium of human PSC derived haemopoietic organoids [bulk RNA-seq]

The Recombination Activation Gene, RAG1, expression of which presages T-cell receptor gene rearrangement, is a key marker of T-cell commitment. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cells genesis in the context of haemtopoietic organoids. We show that T-cell commitment occurs concomitantly with the emergence of blood cells from AGM-like haemogenic endothelium, predating the surface expression of CD5 and CD7. In this system, RAG1 marks an early haematopoietic progenitor emerging from SOX17+ endothelium, prior to down regulation of CD90 and VCAM and upregulation of the blood cell marker, CD45. Sort and re-culture experiments show that early RAG1+ cells possess T-cell, B-cell, myeloid and erythroid potential. However, under conditions that favor T-cell development, early RAG1+ cells progress to the CD4+CD8+CD3+ stage, vindicating their classification as bone fide T-cell progenitors. These observations suggest that like the zebrafish and mouse, humans can execute a non-HSC derived wave of T-cell development that includes a multipotent RAG1+ progenitor. Examination of gene expression (RNAseq) during various stages of T-lymphocyte differentiation using 2 pluripotent stem cell lines.

重组激活基因1（RAG1）的表达预示着T细胞受体基因重排的发生，是T细胞定向分化的关键标志物。本研究利用RAG1:GFP人类多能干细胞报告基因系，在造血类器官模型中探究了人类T细胞发生过程。研究发现，T细胞定向分化与主动脉-性腺-中肾（AGM）样生血内皮细胞产生血细胞同步发生，且早于CD5与CD7的表面表达。在该模型中，RAG1标记了一群源自SOX17+内皮细胞的早期造血祖细胞，该群细胞在CD90与VCAM表达下调、血细胞标志物CD45表达上调前即可被检测到。分选与复培养实验表明，早期RAG1+细胞具备T细胞、B细胞、髓系及红系分化潜能。然而在偏向T细胞发育的培养条件下，早期RAG1+细胞可发育至CD4+CD8+CD3+阶段，证实其可被归类为真正的T细胞祖细胞。本研究结果提示，与斑马鱼和小鼠模型一致，人类也存在非造血干细胞（Hematopoietic Stem Cell, HSC）来源的T细胞发育波，其中包含具有多向分化潜能的RAG1+祖细胞。本研究还利用2株多能干细胞系，对T淋巴细胞分化各阶段的基因表达情况进行了RNA测序（RNA-seq）分析。