Abstract
The spatio-temporal complexity of T cell differentiation from hematopoietic stem and progenitor cells (HSPCs) in the thymus has been challenging to recapitulate in vitro. While the OP9-DL1 monolayer co-culture system revolutionized thymopoiesis studies, serum-dependent monolayer systems can have inconsistent results. Our recently developed serum-free, 3D Artificial Thymic Organoid (ATO) system supports full in vitro T cell differentiation from human HSPCs and pluripotent stem cells; however, results using identical conditions with murine cells are suboptimal. Here, we demonstrate that culturing murine bone marrow (BM) HSPCs in a modified murine ATO system (M-ATO) consistently reproduces normal murine thymopoiesis. The M-ATO produces all T cell stages: from early thymic progenitors to mature T cell subsets, including TCRγδ+, single positive (SP8 and SP4) TCRαβ+, and FoxP3+ cells. RNA sequencing aligned M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATO-derived CD3+TCRαβ+ SP cells are mature, functional T cells that exhibit a broad TCR Vβ repertoire and respond to TCR activation. M-ATOs initiated with Rag1-/- marrow produced the same differentiation block as seen in the endogenous thymus, and the transgenic Notch reporter (TNR) mouse model revealed that M-ATOs induce Notch signaling patterns in developing thymocytes similar to that in the thymus. Thymopoiesis can be established in M-ATOs from a range of BM and or thymic populations, including Lineage negative c-Kit+Sca-1+ (LSK), SLAM+ hematopoietic stem cells (HSC), lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP) or early thymic progenitors (ETPs). The M-ATO is highly efficient at producing millions of T cells with a broad repertoire even when starting from purified single HSC or LSK cells. Thus, the M-ATO is a reproducible and efficient platform to study clonal murine T cell development and maturation from single HSCs and progenitors.
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