Modulation of Mesodermal Patterning Combined with High VEGFA Concentrations Promote Robust Arterial Hemogenic Endothelium Differentiation from Human iPSCs

One of the most promising objectives of clinical hematology is to derive transplantable autologous hematopoietic stem/progenitor cells (HSPCs) from human iPSCs ex vivo, but efficient and clinically relevant methodologies remain unavailable. Observations in the developing embryo indicate that definitive HSPCs arise in the dorsal aorta from hemogenic endothelium (HE) in close association with an arterial vascular endothelial niche. The Notch pathway plays a key role in arterial and HSC differentiation; in the embryo, only HE populations found in arterial regions with active Notch signaling through Delta-like ligand 4 (Dll4) and Jagged 1 (Jag1) lead to HSPCs with repopulating potential. Recent studies have also shown that modulation of mesodermal patterning through repression and activation of Activin/Nodal and Wnt/β-catenin pathways, respectively, promotes arterial programs and definitive hematopoiesis. To facilitate the development of functional HSPCs from human iPSCs, we previously 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, mesodermal specification is induced using morphogens (bFGF, BMP4, VEGF 10ng/mL) and, for the subsequent 18 days, cells are further differentiated into HSPCs with the addition of hematopoietic cytokines (SCF, Flt3L and TPO). As previously presented by our group, this differentiation system recapitulates the successive waves of hematopoiesis during development and leads to robust production of immunophenotypic HSC-like cells (CD34⁺CD38^-CD90⁺CD45RA^-CD49f⁺). However, these cells do not result in efficient, long-term engraftment in immunodeficient (NSG) mouse models. Characterization of the supportive monolayer from which HSPCs arise during vitro differentiation revealed limited percentages of arterial HE (CD43-CD45-CD34^hiCD144+CD73-Dll4+) and arterial endothelium (CD43-CD45-CD34^hiCD144+CD73^midCD184+), and overabundance of stromal cells (CD43-CD45-CD34-CD144-) which upregulate MSC markers CD105, CD73, and CD90 at later stages in culture. This provides a possible explanation for the lack of engraftment potential of iPSC-derived HSPCs in this system (Panel A). To restrict stromal development and further promote differentiation and maintenance of a supportive arterial endothelial niche, we modified the standard differentiation protocol by addition of CHIR99021 (CHIR) and SB431542 (SB) during the mesodermal stage of differentiation (days 2-3) to activate Wnt/β-catenin and block of Activin/Nodal signaling, respectively. Given that VEGF acts upstream of the Notch pathway during arterial endothelial differentiation, we also increased the concentration of VEGFA 20-fold throughout differentiation (200ng/mL). Our results showed that mesodermal patterning alone (CHIR/SB) activated critical HoxA cluster genes in both early endothelial and late HSPC populations but was insufficient to repress stromal production and maintain an endothelial niche beyond early culture days. However, increased VEGF concentrations, alone or in combination with CHIR/SB, markedly reduced stromal differentiation (Panel B) and enhanced arterial endothelium formation (Panel C) compared to the standard system (control). Importantly, combination treatments also led to significantly higher percentages of arterial HE at days 5 and 7 (panel D). Current assessment of these treatments on the hematopoietic potential of the system is ongoing, and include NSG mouse transplantations. Overall, our data indicate that commercially available technologies can be further modified and improved to move closer to chemically-defined and scalable HSPC differentiation protocols.

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