Assistant Professor University of Iowa, United States
Introduction:: Retinal degenerative diseases are a major cause of blindness involving the dysfunction of photoreceptors, retinal pigmented epithelium (RPE), or both. A promising treatment approach involves replacing these cells via surgical transplantation, and previous work has shown that cell delivery scaffolds are vital to ensure sufficient cell survival. Characterizing scaffold properties that lend themselves to effective cell scaffolds (such as suitable material and mechanical properties) is critical to ensuring a successful treatment approach. However, immortalized RPE cell lines commonly used for in vitro experiments, such as ARPE19, may not be representative of in vivo cells and tissues. For example, RPE cells are vulnerable to de-specialization via the epithelial-to-mesenchymal transition, which is suspected to be driven in part by mechanical stiffness. Yet, immortalized cell lines may not respond to substrate stiffness in the same way stem cell-derived and/or in vivo RPE cells would. In this project, our objective is to establish clearer RPE scaffold design guidelines by determining the effect of scaffold stiffness on human RPE attachment, survival, and differentiation, comparing immortalized and stem cell derived RPE cell lines.
Materials and Methods:: A well-characterized, spontaneously immortalized RPE cell line (ARPE-19) as well as human iPSC-derived RPE cells were used to study the effect of substrate stiffness on RPE behavior. In all studies, poly(dimethylsiloxane) (PDMS) was used as the substrate. To achieve varying stiffness, the crosslinker-to-base ratio was varied to alter compressive modulus (~15 kPa to ~800 kPa), and all samples were coated with cell basement membrane proteins. Post-attachment changes in gene and protein expression were assessed for up to two weeks using qPCR and immunocytochemistry. For both RPE lines, genes and proteins of interest included markers of proliferation, tight junction and membrane channel formation, and specialized RPE function (e.g., visual cycle).
Results, Conclusions, and Discussions:: Compared to tissue culture plastic, ARPE-19 cells at the two-week timepoint cultured on any stiffness had lower expression of genes encoding ZO-1 (tight junctions), but higher expression of genes encoding PEDF (a retinal protective glycoprotein). On the other hand, under the same conditions, iPSC derived RPE cells did not have any significant changes in gene expression compared to the tissue plastic control group. While these results highlight the differences in expression between immortalized and iPSC-derived RPE cells, they also suggest that stiffnesses in this range (~15-800 kPa) may not result in differences in RPE growth and maturation, an important consideration in scaffold design. Our data support the hypothesis that immortalized and iPSC derived RPE cells show different responses to substrate stiffness. Furthermore, substrate stiffness may play an important role in the health and function of RPE cells. However, the details of this relationship would be made clearer via future studies that include a greater number of timepoints and an in vitro system that enables dynamic changes in substrate stiffness, such that the dynamics of RPE cell behavior are better established.
Acknowledgements (Optional): : The authors would like to acknowledge the NIH (NEI RO1 EY033331), the Roy J. Carver Charitable Trust, and the University of Iowa Office of the Vice President for Research for funding this project.
