An optimized differentiation protocol produces robust endothelial cells essential for a physiologically relevant 3D in vitro angiogenesis model

An optimized differentiation protocol produces robust endothelial cells essential for a physiologically relevant 3D in vitro angiogenesis model/nBy/nSylvia Lucia Natividad/nDoctor of Philosophy in Bioengineering/nUniversity of California, Berkeley/nProfessor Kevin Healy, Chair/nHuman induced pluripotent stem cell (hiPSC) derived angiogenesis models present a unique opportunity for patient-specific platforms to study the complex process of angiogenesis and the endothelial cell response to biochemical and biophysical changes in a defined microenvironment. This dissertation presents a robust method for differentiating hiPSCs into a CD31+ endothelial cell population (hiPSC-ECs) using a chemically defined basal medium from the pluripotency stage to the final stage of differentiation and the development of a physiologically relevant in vitro angiogenesis model. The endothelial cell differentiation was validated through phenotype characterization, gene expression studies, and lineage-specific function assays. This protocol produced robust, functional endothelial cells in a shorter period of time relative to current reports since a maturation period was not required. Subsequently, the hiPSC-ECs were incorporated into a tunable, growth factor sequestering hyaluronic acid (HyA) matrix that formed stable, capillary-like networks that responded to environmental stimuli in a physiologically relevant manner. An in vitro angiogenesis model containing the HyA matrix and hiPSC-ECs was then developed within a tri-chamber microfluidic device that demonstrated perfusion of the capillary networks. Finally, an in vitro cardiovascular tissue model was developed by culturing hiPSC derived cardiomyocytes (CMs) in the HyA hydrogel with the hiPSC-ECs. The hiPSC-CMs demonstrated a limited ability to survive, function, and support angiogenesis within the hydrogel./nChapter 3 presents a novel endothelial cell differentiation method from human pluripotent stem cells that was developed and validated in this study. The phenotype and gene expression of the total differentiated population and the purified CD31+ population were characterized for endothelial lineage with phase contrast microscopy, flow cytometry, fluorescence microscopy, and RT-qPCR analysis. The purified endothelial cell population was further validated with functional assays including Ac-LDL uptake and network formation within a Matrigel angiogenesis assay./nChapter 4 presents a study to assess the behavior of the hiPSC-ECs (from chapter 3) in a growth factor sequestering hyaluronic acid (HyA) matrix, which promoted cell survival and maintenance of lineage-specific function. The system’s ability to respond to biochemical and biophysical cues was demonstrated through characterization of changes in tube formation with confocal microscopy and measurement of nitric oxide production using a fluorescence-based Griess assay. The dependence of network formation on proangiogenic signaling was further demonstrated by treatment with a small-molecule VEGFR2/FGFR inhibitor, which eliminated network formation and permitted the calculation of an IC50 value. An in vitro angiogenesis model containing the HyA matrix and hiPSC-ECs was then developed within a tri-chamber microfluidic device that demonstrated perfusion of the hollow capillary tube networks. A fluid dynamics analysis of the HyA flow through the microfluidic device’s capillary burst valve (CBV) system during sample loading is also discussed./nChapter 5 demonstrates the study of an in vitro cardiovascular tissue model that is developed by culturing hiPSC-CMs in the HyA hydrogel alone and with the hiPSC-ECs. The hiPSC-CMs ability to survive, function, and support angiogenesis within the hydrogel was analyzed through live/dead fluorescence staining, beat rate analysis with motion tracking software, and confocal microscopy characterization of the cellular morphology, organization, and sarcomere structure.

Advisor: Healy, Kevin E.