Investigating the emergence of hematopoietic stem cells in a mouse embryo

From the Hadland Lab, Clinical Research Division

During the process of embryogenesis, hematopoietic stem cells (HSCs)—cells with the ability to generate the entirety of cells involved in hematopoiesis and that have self-renewal capabilities—develop. The hemogenic endothelium is comprised of specialized endothelial cells responsible for developing these HSCs. However, prior to their emergence, other progenitors, such as multipotent progenitor cells, arise first to assist the developing embryo. Previous research has hypothesized that these multipotent progenitor cells may have a role to play in the generation of HSCs. However, this has yet to be confirmed. Members of the Hadland Laboratory in the Fred Hutch Clinical Research Division sought to better understand this and to further elucidate the emergence of HSCs in a recent study by utilizing mouse embryos, single-cell index sorting and single-cell RNA-sequencing. Dr Hadland described how findings from this research, recently published in Cell Reports, “contribute new insights into the origin of HSCs from hemogenic endothelial precursors during embryonic development.”

Previous research from the authors focused on the role of HSCs within a specific region of the embryonic mesoderm during the period when HSCs have been determined to have transplantable potential. They identified a subset of hemogenic precursors that co-express two proteins, EPCR and CD61 and possess the ability for continuous HSC activity. Building on this earlier research with single-cell index sorting methodology, they established that most clonal hemogenic precursors did not have the capacity to produce HSCs, rather they more commonly produced hematopoietic progenitor cells. Thus, the group sought to identify robust markers for distinguishing hemogenic endothelium with HSC potential. Having previously identified CXCL12, canonical ligand of CXCR4, as essential in the development of HSCs, the team thus postulated CXCR4 would be differentially expressed in this population. Indeed, CXCR4 was found to be expressed on the rare subset of hemogenic precursors with the capability of generating HSCs. To further examine this finding, they confirmed the long-term multilineage capability of CXCR4 expressing HSCs in an in vivo model. 

Hematopoietic stem cells can be characterized by cell surface marker CXCR4 and expression of arterial specific transcriptional signatures.
Hematopoietic stem cells can be characterized by cell surface marker CXCR4 and expression of arterial specific transcriptional signatures. Image provided by Dr Hadland.

Next, the authors attempted to define the lineage capacity of hematopoietic progenitor cells who do not have HSC potential. By undertaking co-culture and colony forming unit assays, they identified a subset of cells which were lineage restricted and lacked the necessary components for HSC evolution. These data “provide evidence for a revised paradigm of developmental hematopoiesis in which multilineage hematopoietic progenitors initially arise from hemogenic endothelium independently of self-renewing HSCs, which is likely important to provide blood cells to meet the immediate needs of the developing embryo,” explained Dr Hadland. 

Lastly, with the goal of identifying transcriptional expression patterns in CXCR4 expressing hemogenic endothelium with HSC potential, the group undertook single-cell RNA-sequencing experiments. Utilizing computational approaches, they separated CXCR4 positive and negative cells into clusters and assessed expression patterns. CXCR4 positive clusters were determined to have significantly higher gene-set scores for mature arterial endothelial cells when compared to CXCR4 negative clusters. Further, differential expression of specific genes was observed between the two cell populations, with genes associated with dormancy and self-renewal highly expressed in the CXCR4 positive group. “The study suggests an essential role for genes tied to arterial endothelial transcriptional programs in establishing the HSC competence of hemogenic endothelium by promoting transient metabolic and mitotic dormancy, delaying hematopoietic differentiation, and enhancing HSC self-renewal,” said Dr Hadland. “These programs are likely essential to expand the HSC pool during development to meet the needs for life-long adult hematopoiesis,” he continued.

Data generated from this study have provided valuable insights into the field of developmental hematopoiesis. “These findings have important implications for long-standing efforts to generate HSCs from pluripotent stem cells (PSC), providing novel insight into pathways that regulate HSC potential from hemogenic endothelial precursors. Future studies in our lab will focus on applying our findings to human PSC models in order to engineer HSCs in vitro for translational purposes in disease modelling and cellular therapies,” commented the authors. 

This work was funded by the American Society of Hematology Scholar Award, the National Heart, Lung, and Blood Institute of the National Institutes of Health and by the National Institute of Diabetes and Digestive and Kidney Diseases of the NIH.

UW/Fred Hutch Cancer Consortium members Brandon Hadland, Cole Trapnell and Irwin D Bernstein contributed to this work.

Dignum T, Varnum-Finney B, Srivatsan SR, Dozono S, Waltner O, Heck AM, Ishida T, Nourigat-McKay C, Jackson DL, Rafii S, Trapnell C, Bernstein ID, Hadland B. Multipotent progenitors and hematopoietic stem cells arise independently from hemogenic endothelium in the mouse embryo. Cell Rep. 2021 Sep 14;36(11):109675. doi: 10.1016/j.celrep.2021.109675. PMID: 34525376; PMCID: PMC8478150.