Pluripotent stem cells (PSC) provide insight into development and may underpin new cell therapies, yet controlling PSC differentiation to generate functional cells remains a significant challenge. In this study we explored the concept that mimicking the local in vivo microenvironment during mesoderm specification could promote the emergence of hematopoietic progenitor cells from embryonic stem cells (ESCs). First, we assessed the expression of early phenotypic markers of mesoderm differentiation (E-cadherin, brachyury (T-GFP), PDGFRα, and Flk1: +/-ETPF) to reveal that E-T+P+F+ cells have the highest capacity for hematopoiesis. Second, we determined how initial aggregate size influences the emergence of mesodermal phenotypes (E-T+P+F+, E-T-P+/-F+, and E-T-P+F-) and discovered that colony forming cell (CFC) output was maximal with ~100 cells per PSC aggregate. Finally, we introduced these 100-cell PSC aggregates into a low oxygen environment (5%; to upregulate endogenous VEGF secretion) and delivered two potent blood-inductive molecules, BMP4 and TPO (bone morphogenetic protein-4 and thrombopoietin), locally from microparticles to obtain a more robust differentiation response than soluble delivery methods alone. Approximately 1.7-fold more CFCs were generated with localized delivery in comparison to exogenous delivery, while combined growth factor use was reduced ~14.2-fold. By systematically engineering the complex and dynamic environmental signals associated with the in vivo blood developmental niche we demonstrate a significant role for inductive endogenous signaling and introduce a tunable platform for enhancing PSC differentiation efficiency to specific lineages.
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