SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells.

PU.1 (SPI1) is pivotal in hematopoiesis, yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Comparing human in vivo and in vitro EHT transcriptomes revealed SPI1's regulatory role. Knocking down SPI1 during in vitro EHT led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis, we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells, respectively - as downstream targets of SPI1. Overexpressing KLF1 or LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically, KLF1 overexpression restores myeloid lineage potential, while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1-LYL1 axis in the regulatory network in in vivo EHT. Taken together, our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT, potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs).

Cross-species transcriptomics reveals bifurcation point during the arterial-to-hemogenic transition.

Hemogenic endothelium (HE) with hematopoietic stem cell (HSC)-forming potential emerge from specialized arterial endothelial cells (AECs) undergoing the endothelial-to-hematopoietic transition (EHT) in the aorta-gonad-mesonephros (AGM) region. Characterization of this AECs subpopulation and whether this phenomenon is conserved across species remains unclear. Here we introduce HomologySeeker, a cross-species method that leverages refined mouse information to explore under-studied human EHT. Utilizing single-cell transcriptomic ensembles of EHT, HomologySeeker reveals a parallel developmental relationship between these two species, with minimal pre-HSC signals observed in human cells. The pre-HE stage contains a conserved bifurcation point between the two species, where cells progress towards HE or late AECs. By harnessing human spatial transcriptomics, we identify ligand modules that contribute to the bifurcation choice and validate CXCL12 in promoting hemogenic choice using a human in vitro differentiation system. Our findings advance human arterial-to-hemogenic transition understanding and offer valuable insights for manipulating HSC generation using in vitro models.

PEAR1 is a potential regulator of early hematopoiesis of human pluripotent stem cells.

Hemogenic endothelial (HE) cells are specialized endothelial cells to give rise to hematopoietic stem/progenitor cells during hematopoietic development. The underlying mechanisms that regulate endothelial-to-hematopoietic transition (EHT) of human HE cells are not fully understand. Here, we identified platelet endothelial aggregation receptor-1 (PEAR1) as a novel regulator of early hematopoietic development in human pluripotent stem cells (hPSCs). We found that the expression of PEAP1 was elevated during hematopoietic development. A subpopulation of PEAR1+ cells overlapped with CD34+ CD144+ CD184+ CD73- arterial-type HE cells. Transcriptome analysis by RNA sequencing indicated that TAL1/SCL, GATA2, MYB, RUNX1 and other key transcription factors for hematopoietic development were mainly expressed in PEAR1+ cells, whereas the genes encoding for niche-related signals, such as fibronectin, vitronectin, bone morphogenetic proteins and jagged1, were highly expressed in PEAR1- cells. The isolated PEAR1+ cells exhibited significantly greater EHT capacity on endothelial niche, compared with the PEAR1- cells. Colony-forming unit (CFU) assays demonstrated the multilineage hematopoietic potential of PEAR1+ -derived hematopoietic cells. Furthermore, PEAR1 knockout in hPSCs by CRISPR/Cas9 technology revealed that the hematopoietic differentiation was impaired, resulting in decreased EHT capacity, decreased expression of hematopoietic-related transcription factors, and increased expression of niche-related signals. In summary, this study revealed a novel role of PEAR1 in balancing intrinsic and extrinsic signals for early hematopoietic fate decision.