Sticking It to Conventional Models; Micropatterned hiPSC Culture as a Novel Approach for Studying Complex Molecular and Cellular Systems

Walsh, Sabrina.

Undergraduate Honors Thesis

Sticking It to Conventional Models; Micropatterned hiPSC Culture as a Novel Approach for Studying Complex Molecular and Cellular Systems Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/tt44pp507

Alternative Title

hiPSC Micropatterned Adhesive Islands

Abstract

Studying complex molecular and cellular systems in humans presents a crucial challenge that demands specialized models for visualization and experimentation. While human induced pluripotent stem cells (hiPSCs) offer a valuable tool due to their ability to maintain a pluripotent state and differentiate upon direction, they can yield irreproducible results. To overcome this limitation, better-controlled and more sophisticated hiPSC differentiation models are needed. One promising approach to the study of tissue-scale cell fate patterning involves pre-patterned geometrical confinement (Warmflash et al., 2014; Etoc et al, 2016). In this study, we aimed to test a modified methodology for producing hiPSC micropatterned adhesive islands to investigate complex molecular systems, specifically intermediate filament protein vimentin and the effects of molecular chaperones. We were able to construct two CRISPR-Cas9 plasmids that targeted the human vimentin gene and showed that upon transfection into HeLa cells, they induced a vimentin-null phenotype. We then successfully adapted the Warmflash et al. protocol to produce adhesive islands in HeLa and hiPSC which constrained the cells using pre-patterned geometric constraints. This is a crucial step in studying complex molecular systems and genetic manipulations in differentiating human cells. Our modifications also enhanced the cost-effectiveness of our model potentially making it more applicable to a teaching setting (CU MCDB Skills Center) . In the long term, this approach has the potential to shed light on the role(s) of vimentin and the effects of molecular chaperones on hiPSC differentiation and development.

Keywords: human induced pluripotent stem cells; adhesive islands; micropatterning; intermediate filament proteins; vimentin; differentiation; chaperones.

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