Restraint of TGF-β signaling by SMAD7 is necessary for the maturation of hematopoietic stem cells in the mouse embryo Free

Hematopoietic stem and progenitor cells (HSPCs) in the mouse embryo arise from a subset of arterial hemogenic endothelial cells which bud into the lumen of the major vessels, forming intra-arterial hematopoietic cluster (IAHC) cells – a heterogenous population of non-HSC derived progenitors, the precursors of HSCs (pre-HSCs), and HSCs (Kissa and Herbomel 2010, Zhu, Gao et al. 2020). The first HSCs arise at embryonic day (E) 11.5 in the mouse embryo– almost 4 days after the onset of hematopoiesis and after hundreds of committed progenitors have already emerged (de Bruijn, Speck et al. 2000, Rybtsov, Sobiesiak et al. 2011, Gordon-Keylock, Sobiesiak et al. 2013, Rybtsov, Ivanovs et al. 2016). At E11.5, there are an estimated 65 pre-HSCs and 1 functional HSC (Rybtsov, Ivanovs et al. 2016). The rarity of these cells poses a significant challenge for identifying signals or genes required for their formation. We previously performed single-cell transcriptional profiling of endothelial cells, hemogenic endothelial cells, and HSPCs during hematopoietic development and found that a negative regulator of TGF-β signaling, Smad7, was specifically upregulated in pre-HSCs, suggesting that inhibition of TGF-β signaling may be necessary to generate pre-HSCs or HSCs. TGF-β signaling plays an essential role early in the specification of hemogenic endothelial cells, but how the restraint of TGF-β signaling affects the generation of pre-HSCs or their maturation into HSCs later in hematopoietic development is unknown.

Deleting Smad7 at E9.5 using an endothelial-specific Cre (Smad7 KO) does not impact the number of IAHC cells or non-HSC-derived progenitors but results in a complete loss of functional adult-repopulating HSCs in the aorta-gonad-mesonephros region (AGM) in E11.5 Smad7 KOembryos. Limiting dilution transplants of E12.5 AGMs, which normally contain approximately 2-3 HSCs, revealed an 85% reduction in HSCs in Smad7 KOembryos compared to wild type embryos (0.52 HSCs/AGM Smad7 KO vs 2.56 HSCs/AGM WT, p=5.3e-6). HSPCs eventually migrate from the AGM to the fetal liver (FL). We observed a 60% reduction in HSCs in the E12.5 FL (11.0 HSCs/FL Smad7 KO vs 26.2 HSCs/FL WT, p=0.0167), suggesting that the reduction in HSCs in the AGM of Smad7 KO embryos is not the result of precocious migration of HSCs to the FL.

Pre-HSCs are defined functionally as cells that cannot directly engraft an irradiated adult but can be matured ex vivo into adult repopulating cells. We performed a limiting dilution pre-HSC maturation assay by sorting IAHC cells from E11.5 embryos and found Smad7 KO IAHC cells had an 85% reduction in functional pre-HSCs (1:181 WT vs 1:1084 Smad7 KO, p=2.59e-5). We performed scRNA-seq profiling of wild type and Smad7 KO endothelial and IAHC cells at E11.5 to determine whether the generation of pre-HSCs was affected by loss of SMAD7. scRNA-seq revealed that the loss of SMAD7 increased the proportion of pre-HSCs relative to other hematopoietic cells by approximately 2-fold (p=0.003), indicating that pre-HSC formation was not affected and suggesting instead that pre-HSC to HSC maturation may be impaired. We directly examined the role of SMAD7 in pre-HSC maturation by deleting Smad7 with a ubiquitously expressed Rosa26^CreERT after cells were sorted from E11.5 embryos. Ex vivo deletion of SMAD7 in pre-HSCs resulted in an 87% decrease in functional LT-HSCs (1:252 WT vs 1:1075 Smad7 KO, p=0.004), indicating that inhibiting TGF-β signaling is necessary for the proper maturation of pre-HSCs into HSCs but not their formation from endothelial cells.

In summary, we identified the first signaling pathway in hematopoietic development that specifically promotes the maturation of HSCs from their embryonic precursors (pre-HSCs). Several groups have successfully demonstrated the ability to produce HSCs from iPSC cultures (Piau, Brunet-Manquat et al. 2023, Ng, Sarila et al. 2024). Understanding the signals required in the specification and maturation of HSCs in vivo during development, and when they should be applied and inhibited, will inform efforts to optimize HSC production from iPSCs.