(502e) Mechanical Influences on Endoderm Differentiation

Introduction: Stem cell differentiation has been a well-studied and important aspect of cellular biology over the last several decades; however, while significant advancements have been made in the study of soluble cues, not all effects of the mechanical environment in and outside of the cell have been fully investigated at this point. Specifically, the mechanical dependencies of endoderm specification have not been fully investigated. The mechanical context of tissue development and homeostasis in vivo vastly differs from in vitro approaches to derive endoderm derivatives. To this end, this work will explore how mechanical cues impact endoderm differentiation efficiency, which can lead to improvements in differentiated cell models, enhancement of differentiation protocols, and improvements to mimic adult cell tissues better than currently generated products.

Approach: We intend to modulate how differentiating cells perceive their microenvironments by culturing cells in substrates of various stiffness, using functionalized gelatin gels, and controlling cytoskeletal tension inside the cells. For cytoskeletal modulation, we first ran a toxicity screen on iPSCs with the cytoskeletal modulating drugs Latrunculin A, Nocodazole, Cytochalasin D, Jasplakinolid, Blebbistatin, and Y-27632, to determine both their effect on endoderm differentiation and maximum allowable concentrations. To investigate the temporal effect of cellular microenvironment conditions, we softened or stiffened the cytoskeleton of cells for a portion of the differentiation before monitoring protein expression via IF and transcriptional changes through mRNA expression.

Result: We have observed that a stiffer microenvironment caused by the depolymerization of microtubules through Nocodazole increased NANOG expression compared to a control differentiation by 1.8-fold. In addition, disruption of actin polymerization by Cytochalasin D led to a 3.1-fold increase of GATA4 expression compared to a control differentiation. Overall, we find that disrupting F-actin polymerization enhances endoderm differentiation while enhanced polymerization limits proper differentiation.