Unusual suspects: a surprise cast in making blood stem cells Free

In this edition of Blood, Palma et al¹ identify a novel set of genes whose expression is sufficient to confer hematopoietic stem cell (HSC) fate from murine embryonic stem cells (ESCs). Specifically, using a CRISPR activation screen during differentiation of ESCs, the authors uncovered 7 genes (Spata2, Aass, Dctd, Eif4enif1, Guca1a, Eya2, and Net1) collectively termed SADEiGEN, that together endow ESC-derived mesodermal populations with multilineage, serially engraftable hematopoietic repopulating capacity.

Remarkably, unlike earlier efforts to program HSCs,^2,3 which have largely relied on transcription factors as key drivers, SADEiGEN comprises an unanticipated group of genes encoding proteins with highly diverse functions. These include enzymes that regulate lysine degradation (Aass) and nucleotide metabolism (Dctd), a cofactor in the tumor necrosis factor signaling pathway that modulates cell death (Spata2), a calcium-binding protein that controls guanylate cyclase activity (Guca1a), a translation initiation factor shuttle protein that moderates protein synthesis (Eif4enif1), and a RhoA guanine nucleotide exchange factor that regulates cytoskeletal dynamics and cell migration (Net1). Eya2, encoding a protein tyrosine phosphatase involved in DNA damage response and cell cycle progression, represents the sole transcriptional coactivator of the group. Although Guca1a, Eya2, and Net1 have been identified in HSC gene expression profiles,^4,5 none have clearly established roles in HSC fate determination or overlap with HSC-reprogramming genes from previous studies.^2,3 

Why do the SADEiGEN factors differ so significantly from the “usual suspects” implicated in specifying HSC fate? The likely explanation lies in the distinct approach taken by Palma et al. The authors employed an unbiased, genome-wide CRISPR activation screen to upregulate endogenous gene expression, a strategy that contrasts with previous studies, which mostly relied on the exogenous introduction of selected HSC-associated transcription factors. Additionally, the authors transiently activated gene expression during a specific developmental window of ESC differentiation encompassing sequential precursors of hematopoietic fates from mesoderm through hemogenic endothelium. Notably, the resulting ESC-derived population capable of long-term, multilineage hematopoietic engraftment retains expression of the vascular endothelial growth factor receptor KDR/FLK1, a marker of hematoendothelial mesoderm that is typically downregulated upon hematopoietic fate commitment, suggesting relative immaturity of the engraftable ESC-derived population compared with the earliest engraftable populations found embryonically in vivo. This finding warrants further investigation, as the authors demonstrate the KDR⁺ population can go on to generate immunophenotypically mature HSCs both in vivo and in vitro, but it remains unclear if the in vitro generated HSC-like population retains the engraftment properties of its KDR⁺ predecessor.

By comparing transcriptional profiles from mouse and human single-cell data sets at equivalent embryonic stages with those of ESC-derived populations, the authors propose that SADEiGEN expression fosters conditions favoring intraembryonic mesoderm, the developmental origin of definitive HSCs. A subset of the SADEiGEN genes is broadly expressed in early intraembryonic mesoderm, whereas Eya2, Aass, and Guca1a are specifically upregulated in intraembryonic hematoendothelial precursors, implying stage-specific functions. Collectively, these findings suggest that the SADEiGEN factors may drive specific metabolic, signaling, and structural states that promote a sequential, intraembryonic-like mesodermal trajectory with enhanced HSC potential. In contrast, the default differentiation pathway of ESCs typically favors extraembryonic, yolk sac–like mesodermal fates that give rise to HSC-independent blood lineages.

The robust and reproducible in vitro generation of highly engraftable and durable HSCs remains a major challenge in regenerative medicine, with wide-ranging translational potential. The novel findings presented by Palma et al advance this goal by identifying a unique set of genes whose endogenous activation collaboratively establishes conditions favorable for HSC development during mesodermal differentiation of ESC. Critically, avoiding exogenous overexpression of transcription factors commonly implicated in leukemogenesis, this study may provide key insights into developing safer methods for HSC engineering from pluripotent cells for therapeutic applications. However, many important questions remain to be addressed in future studies. Most critically, the precise mechanism by which the SADEiGEN factors enhance HSC fate requires further exploration. Specifically, how do genes with such diverse functions coordinate mesodermal patterning to promote HSC emergence, and how does temporal regulation of their expression impact this process? Could fine-tuning their relative activation further enhance HSC output and repopulation capacity? And importantly, are these factors conserved in their ability to promote HSC development from human pluripotent stem cells? Addressing these questions could yield a deeper understanding of the sequential developmental stages and conditions required to prime mesoderm for HSC fates. Moreover, this knowledge could improve the robustness and efficiency of recent transgene-free approaches for generating HSCs from human pluripotent stem cells.^6,7 

Conflict-of-interest disclosure: B.H. declares no competing financial interests.