In?Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells

摘要

We report a serum-free, 3D murine artificial thymic organoid (M-ATO) system that mimics normal murine thymopoiesis with the production of all T?cell stages, from early thymic progenitors to functional single-positive (CD8SP and CD4SP) TCRαβ and TCRγδ cells. RNA sequencing aligns M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATOs initiated with Rag1 sup?/?/sup marrow produce the same differentiation block as seen in the endogenous thymus, and Notch signaling patterns in M-ATOs mirror primary thymopoiesis. M-ATOs initiated with defined hematopoietic stem cells (HSCs) and lymphoid progenitors from marrow and thymus generate each of the downstream differentiation stages, allowing the kinetics of T?cell differentiation to be tracked. Remarkably, single HSCs deposited into each M-ATO generate the complete trajectory of T?cell differentiation, producing diverse TCR repertoires across clones that largely match endogenous thymus. M-ATOs represent a highly reproducible and efficient experimental platform for the interrogation of clonal thymopoiesis from HSCs.