3176 – MULTIPOTENT RAG1+ PROGENITORS EMERGE DIRECTLY FROM HEMOGENIC ENDOTHELIUM IN HUMAN PSC DERIVED HEMOPOIETIC ORGANOIDS

Abstract

Defining the ontogeny of the human adaptive immune system during early embryogenesis has implications for understanding childhood diseases including leukemias and autoimmune diseases. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cell genesis from pluripotent stem cell derived hematopoietic organoids. Under conditions favouring T-cell development, RAG1+ cells progressively up regulated a cohort of recognised T-cell associated genes, arresting development at the CD4+CD8+ stage. However, sort and re-culture experiments showed that early RAG1+ cells also possessed B-cell, myeloid and erythroid potential. Moreover, RNAseq analysis of the early RAG1+ cells indicated that, in addition to T-lineage genes, this population expressed genes associated with erythroid and myeloid lineages, as well as genes marking endothelium and hematopoietic progenitors. Consistent with this, imaging studies, flow cytometry and single cell RNAseq data showed that the first RAG1+ cells emerged directly from SOX17+ endothelial structures and co-expressed CD90, CDH5 and the endothelial marker, CAV1. These observations provide evidence for a wave of human T-cell development that originates directly from hemogenic endothelium via a RAG1+ intermediate with multilineage potential.