Concurrent Ex Vivo Differentiation of a Vascular Niche and Hematopoietic Stem/Progenitor Cells from Human iPSCs

One of the most promising objectives of clinical hematology is to derive transplantable autologous hematopoietic stem/progenitor cells (HSPCs) ex vivo. Recent reports have described the generation of engraftable HSPCs via forced expression of key transcription factors in either endothelial cells, or hemogenic endothelium (HE) derived from induced pluripotent stem cells (iPSCs). While protocols for the generation of transgene-free engraftable HSPCs suitable for therapy remain unavailable, these studies, along with observations in the developing embryo, have shown the close association of HE populations with arterial vascular endothelium is of critical importance to HSPC production and maturation. To facilitate the development of a vascular niche supporting the emergence of transgene-free, functional HSPCs from human iPSCs, we developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiff^TM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, a mesodermal lineage specification was induced with morphogens (BMP4, bFGF and VEGF). For the subsequent 18 days, cells were further differentiated into HSPCs with the addition of hematopoietic cytokines (SCF, Flt3-L and TPO). Starting at day 5, an adherent monolayer composed in part of CD34+CD43- endothelial cells rapidly formed, followed by the initiation of endothelial-to-hematopoietic transition (EHT), as demonstrated by the emergence of round cells directly budding off the endothelial monolayer in time-lapse imaging studies (Panel A). Importantly, these cells were released within the culture supernatant and could be easily harvested by simple pipetting. Flow cytometric analysis of the supernatant cells indicated that the emerging cells initially had a predominant CD45-CD235a+ erythroid phenotype (maximum 69.9±12.4% at day 7), consistent with a primitive yolk-sac wave of hematopoiesis (PanelB). From day 7, CD45+CD34+ HSPCs increased gradually, with a maximum number (average 1x10⁵ cells/cm², n=10) observed at day 12, and a peak percentage at day 12 (47.4±12.4%) and 14 (48.8±9.9%) (Panel B). These cells could form colonies in clonogenic progenitor assays, albeit at reduced capacity compared to bona fide CD45+CD34+ cells. Strikingly, cells with a phenotype that enables the highest reported purity of human HSCs (CD34+ CD38- CD45RA- CD90+ CD49f+ Rho^lo) were highly enriched in the culture supernatant at day 10 through 14 (Panel C). However, these cells did not result in efficient, long-term engraftment in immuno-deficient (NSG) mice. To identify possible causes for the lack of durable repopulating potential of iPSC-derived HSPCs in this system, we further characterized the endothelial monolayer between day 5 and 12 of differentiation. We focused on recently described endothelial subsets within the CD34^hiCD43^- population, including the in vitro equivalent of definitive HE (CD34^hi CD43^- CD73^- CD184^- DLL4^-), arterial vascular endothelium (CD34^hi CD43^- CD73^mid CD184⁺), and venous vascular endothelium (CD34^hi CD43^- CD73^hi CD184^-) (Ditadi et al., Nat. Cell Biol 2015). At each timepoint analyzed, definitive HE comprised <1% of the total cell population (Panel D). Arterial vascular endothelium was also scarce, rising to a maximum of 6.6% at day 7 before declining to <1% by day 9 of culture (Panel D). Venous vascular endothelium accounted for approximately 2% of total cells (Panel D). The remaining cells of the monolayer expressed varying levels of CD34 and CD43, consistent with the recently demonstrated heterogeneity of cells undergoing EHT (Guibentif et al., Cell Rep 2017). Thus, our data suggest that the absence of a definitive HE population within a developing arterial niche, as occurs in the dorsal aorta (DA) during development, may explain in part the lack of engraftment potential of iPSC-derived HSPCs. The unique adaptable and scalable nature of this monolayer-based differentiation protocol will facilitate precise temporal activation or downregulation of signaling pathways essential for the generation of engraftable human HSPCs. Quantitative and temporal modulations of Wnt/Nodal pathways for mesodermal specification as well as VEGF/Notch pathways for arterial vascular endothelium are underway.

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