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The molecular pathways and microenvironmental cues that choreograph the conversion of endothelial cells (ECs) into long-term repopulating hematopoietic stem cells (HSCs) remain poorly defined. This is due to lack of models that recreate the ephemeral transition of an endothelial cell to a hemogenic state to the emergence of HSCs.

To provide a platform to deconvolute the process by which endothelial-to-hematopoietic transition is possible.

Here, we have developed a modular in vitro model in which—by precise, conditional expression of transcription factors: FosB, Gfi1, Runx1, and Spi1 (FGRS), and reintroduction of a proper inductive niche—adult mouse ECs were reprogrammed into HSCs (rEC-HSCs) with multi-lineage engraftment potential (rEC-MPPs). Adult, non-lymphatic ECs isolated from various organs of Runx1-IRES-GFP reporter mice were transduced with FGRS and co-cultured in direct contact with vascular niche.

Within 14 days, ECs initiated a hematopoietic program, turning on the endogenous expression of Runx1 and transitioning into hematopoietic cells. Expansion of these cells for another 14 days resulted in generation of rEC-HSCs and rEC-MPPs. Transplantation of rEC-HSCs and rEC-MPPs (CD45.2⁺) into lethally irradiated mice (CD45.1⁺) reconstituted both short-term (rEC-MPPs) and long-term hematopoiesis, with secondary engraftment potential (rEC-HSCs). rEC-HSCs gave rise to both functional myeloid and lymphoid cells with full complement of polarized T cell subsets. rEC-HSC-derived T cells undergo T-cell receptor (TCR) rearrangement and restore adaptive immune function in Rag1^-/- mice.

This multi-phasic, step-wise approach provided an interrogable model to decipher pathways involved in EC transition into hematopoietic cells. This will provide cues to devise strategies to convert autologous ECs into large numbers of HSCs for genetic modification and subsequent treatment of both genetic and acquired hematological disorders.