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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.

During steady-state hematopoiesis, the monocytic lineage, which includes macrophages, osteoclasts, monocytes, and monocyte-derived dendritic cells (moDCs), arises from a progenitor hierarchy in bone marrow. In humans, multipotent myeloid progenitors such as the granulocyte-monocyte progenitor (GMP) are proximal in this hierarchy, however the distal stages of monocytopoieis are unclear. We report here identification of a human clonogenic, monocytic-committed progenitor with a lineage negative, CD34+ CD38+ CD45RA+ CD123int CD115+ phenotype, which was developmentally conserved across bone marrow, cord blood, and fetal liver. Ex vivo analysis demonstrated a high cloning efficiency, and monocytic but not granulocytic CFU potential. Differentiation in the presence of M-CSF resulted in exclusive production of functional M2-like macrophages, whereas cultures modified with RANKL and GM-CSF/IL-4 resulted in highly enriched cultures of functional osteoclasts and moDCs, respectively. Transplantation into immunodeficient mice reconstituted bone marrow, splenic, pulmonary, and hepatic macrophage subsets, as well as circulating CD14+ monocytic cells. Of note, GMP depleted of CD115+ cells could regenerate the monocytic progenitor phenotype in vitro and in vivo, consistent with a step-wise program of lineage restriction during monocytopoiesis. Identification of the steady-state human monocytic progenitor offers opportunities for therapeutic expansion or modification of the monocytic lineage.