Single-Cell Analysis Identifies Distinct Stages of Human Endothelial-to-Hematopoietic Transition

Abstract

During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT). To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34 cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context.

Human haematopoietic stem cell development: from the embryo to the dish

“…The low level of SOX17 in haematogenic cells is consistent with literature reporting the downregulation of SOX17 during the EHT (Clarke et al, 2013;Nobuhisa et al, 2014). Similarly, single-cell analysis of hPSCderived cells documented a downregulation of SOX17 at the EHT point (Guibentif et al, 2017). …”

Induction of human hemogenesis in adult fibroblasts by defined factors and hematopoietic coculture

“…These TFs (labeled in blue, red, green, or purple) are found in the literature and support acquisition of a more hematopoietic TF landscape [6,57,58] (Fig. TFs seen to be upregulated in E9.5 HE and E9.5 artery HE are shown in red [58], and hematopoietic TFs upregulated as hPSC-derived CD34 + cells undergo mesodermal specification, EHT, and eventual blood production are shown in green [6]. Notably, this analysis demonstrates clear upregulation of MAZ, RCOR1, and ZKSCAN1 in each 3GF population.…”

“…Hierarchical clustering of GGF and 3GF cells, again after removal of dimension 1 to correct for batch effects, shows a close relationship between D25 CD49f + CD34 + cells and, interestingly, clustering of GGF D25 CD49f + CD34 À and 3GF D15 CD49f + CD34 + cells (Fig. These TFs (labeled in blue, red, green, or purple) are found in the literature and support acquisition of a more hematopoietic TF landscape [6,57,58] (Fig. Previous data show that the GGF D25 CD49f + CD34 À population clusters most closely resemble known HSCs from published work [54], suggesting that the 3GF D15 CD49f + CD34 + population may most closely resemble endogenous HSCs in the 3GF reprogramming system.…”

Transcriptional networks in acute myeloid leukemia

“…Heptad‐bound genes are enriched in pathways related to blood development and bind and regulate their own regulatory elements such that they form an interconnected circuit . The heptad circuit appears to be established in hemogenic endothelium; ERG in particular, and also FLI1 are downregulated once cells transit beyond the HSC stage, and analysis of chromatin accessibility at stem cell enhancer regions for the heptad genes suggests that the heptad circuit is shut down once blood cells differentiate beyond the progenitor stage . However, heptad binding is aberrantly maintained or reactivated in some AMLs .…”

“…ERG (V-ets avian erythroblastosis virus E26 oncogene homologue), a TF required for definitive hematopoiesis and ongoing HSC function, [189][190][191] is expressed in the earliest blood progenitors, then downregulated during normal blood development. [192][193][194] ERG can be translocated in malignancies including AML, [195][196][197] and is often dysregulated in cytogenetically normal AML where high expression predicts poor patient outcome. 198,199 ERG overexpression has also been directly implicated in leukemogenesis.…”