RAG1+ multipotent progenitors emerge directly from hemogenic endothelium of human PSC derived haemopoietic organoids [single cell 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. Overall design: sinlge cell transcriptional profiling of RAG1+ cells generated early on (day 20 and 16) during air-liquid interface culture of haemopoietic organoids