2005 – RESTRAINT OF TGF-B SIGNALING IS NECESSARY FOR HEMATOPOIETIC STEM CELL FORMATION IN THE MOUSE EMBRYO.

Hematopoietic stem cells (HSCs) are used in the clinic to treat a variety of hematological diseases. We are currently unable to robustly or reliably produce HSCs ex vivo. Since all HSCs in adult bone marrow are formed in the embryo, understanding HSC ontogeny during embryonic development could improve strategies for de novo HSC generation. In the mouse embryo, hematopoietic stem and progenitor cells differentiate from hemogenic endothelial (HE) cells and accumulate within intra-aortic hematopoietic cluster (IAHC) cells in the aorta-gonad-mesonephros (AGM) region. IAHC cells are heterogenous and contain lympho-myeloid biased progenitors (LMPs), precursors to HSCs (pre-HSCs), and HSCs with LMPs comprising the majority of IAHC cells. Single cell RNA sequencing of IAHCs revealed LMPs and pre-HSCs are transcriptionally distinct. Smad7, which encodes a negative regulator of TFG-b/BMP signaling, was more highly expressed in pre-HSCs compared to LMPs. TGF-b signaling plays an essential role early in the specification of HE cells, but how the restraint of TGF-b signaling affects the specification or maturation of pre-HSCs later in hematopoietic development is unknown. Deleting Smad7 in HE cells at E9.5 using an endothelial-specific Cre did not impact the number of IAHC cells or LMPs formed but caused a complete loss of functional adult-repopulating HSCs in the AGM at E11.5 in Smad7 KO embryos. Limiting dilution transplants of E12.5 AGMs showed a 50% reduction in the number of adult-repopulating HSCs in Smad7 KO embryos, and recipients transplanted with Smad7 KO cells had an increased frequency of lymphoid-only engraftment versus multi-lineage engraftment compared to wild type. These data suggest restraint of TGF-b signaling is essential in either the specification of pre-HSCs or maturation of HSCs during embryonic development and may restrict formation of lymphoid-biased HSCs. Hematopoietic stem cells (HSCs) are used in the clinic to treat a variety of hematological diseases. We are currently unable to robustly or reliably produce HSCs ex vivo. Since all HSCs in adult bone marrow are formed in the embryo, understanding HSC ontogeny during embryonic development could improve strategies for de novo HSC generation. In the mouse embryo, hematopoietic stem and progenitor cells differentiate from hemogenic endothelial (HE) cells and accumulate within intra-aortic hematopoietic cluster (IAHC) cells in the aorta-gonad-mesonephros (AGM) region. IAHC cells are heterogenous and contain lympho-myeloid biased progenitors (LMPs), precursors to HSCs (pre-HSCs), and HSCs with LMPs comprising the majority of IAHC cells. Single cell RNA sequencing of IAHCs revealed LMPs and pre-HSCs are transcriptionally distinct. Smad7, which encodes a negative regulator of TFG-b/BMP signaling, was more highly expressed in pre-HSCs compared to LMPs. TGF-b signaling plays an essential role early in the specification of HE cells, but how the restraint of TGF-b signaling affects the specification or maturation of pre-HSCs later in hematopoietic development is unknown. Deleting Smad7 in HE cells at E9.5 using an endothelial-specific Cre did not impact the number of IAHC cells or LMPs formed but caused a complete loss of functional adult-repopulating HSCs in the AGM at E11.5 in Smad7 KO embryos. Limiting dilution transplants of E12.5 AGMs showed a 50% reduction in the number of adult-repopulating HSCs in Smad7 KO embryos, and recipients transplanted with Smad7 KO cells had an increased frequency of lymphoid-only engraftment versus multi-lineage engraftment compared to wild type. These data suggest restraint of TGF-b signaling is essential in either the specification of pre-HSCs or maturation of HSCs during embryonic development and may restrict formation of lymphoid-biased HSCs.