Elucidating the Roles of Physio-Chemical Cues in Determining Endothelial Fate and Function from Human Induced Pluripotent Stem Cells

Cardiovascular diseases (CVD) are the leading cause of death in the United States, claiming more lives annually than all cancers combined. A signature of these complex diseases involves the hardening and narrowing of blood vessels, which are comprised of endothelial (ECs) and supporting stromal cells. This remodeling of the vasculature leads to inadequate delivery of oxygen and nutrients to the surrounding tissue. One promising approach for CVD therapeutics is to introduce healthy human induced pluripotent stem cell (hiPSC) derivatives to compromised vasculature where they can act to replace or augment diseased tissue. hiPSCs are an attractive cell source, not only due to their immune-competence, and unlimited in vitro expansion, but also their ability to be guided to any lineage. Current methods to differentiate ECs from hiPSCs mainly rely on chemical signals, but the role of physical cues in EC differentiation have yet to be fully elucidated. In this work, we explore the roles of several physio-chemical cues, namely substrate stiffness, shear stress, and geometric confinement on the differentiation and functionality of hiPSC-EC derivatives. We first explored the role of substrate compliance on mesoderm differentiation kinetics and its impact on downstream EC commitment from hiPSCs. To this end, we compared the effects of physiologically stiff 1.7 megapascal (MPa) and soft 3 kilopascal (kPa) polydimethylsiloxane (PDMS) substrates, on mesodermal gene expression in serum and chemically defined media compared to traditionally used polystyrene plates. Subsequent to priming mesoderm induction on compliant substrates in serum containing conditions, we found that hiPSCs differentiated more efficiently towards EC phenotypes. When serum free media was used in tandem with PDMS substrates for mesoderm induction, differentiation efficiency was not only enhanced but also led to the rapid maturation of ECs. We next explored the functionality and shear responsiveness of hiPSC-EC derivatives. The promise of applying hiPSC-ECs to augment diseased vasculature and re-establishing blood flow, relies on their functional integration and response to hemodynamic shear forces. In vivo and in vitro evidence suggest the mechanosensing role of primary cilia is essential in EC maturation and shear responsiveness, however the role of cilia presentation in hiPSC-ECs has yet to be elucidated. Using a microfluidic platform and various hiPSC sources, we found that derived ECs lacking cilia presentation were unable to response to shear application, and propose that the degree of ciliogenesis is an important functional marker in characterization of hiPSC-ECs. Finally, using a micropatterning approach, we studied the role of geometric confinement in mimicking spatial organization found in human development. With circular patterns, we found that the loss of pluripotency occurs in a stochastic manner, where small domains show bimodality in pluripotent and differentiated populations, whereas larger micropatterns induce mixed populations of stem and non stem cells. Additionally, we show that confinement induces spatial organization of differentiated populations in a manner dependent on shape. Collectively, understanding the interplay between physical and chemical cues in hiPSC maturation kinetics, and functional outcome of their corresponding derivatives, is essential in the putative promise of stem cell technology for regenerative medicine applications