Retinal differentiation propensity is linked to pluripotency factor localization and donor cell epigenetic memory.

Abstract

Purpose : While we have previously shown that significant differences in retinal differentiation propensity exist between patient-derived iPSC lines, to our surprise undifferentiated iPSCs had few transcriptional differences. Interestingly, at just 7 days post-differentiation robust transcriptional profiles associated with retinal differentiation capacity were revealed. The purpose of this study was to determine if there were differences in pluripotency factor expression/subcellular localization, and if poor retinal differentiation capacity was linked to residual donor cell epigenetic memory.

Methods : Human cells (dermal fibroblasts and ectoderm progenitors) were reprogrammed into induced pluripotent stem cells (hiPSCs) via transduction with Sendai viral vectors expression OCT4, SOX2, KLF4 and C-MYC (CytoTune 2). Immunostaining (ICC), Western blot and FACs analysis were used to evaluate pluripotency marker expression. Retinal differentiation capacity was evaluated microscopically and validated using immunocytochemistry.

Results : Western Blot analysis revealed differences in pluripotency factor localization between undifferentiated hiPSCs determined to be good retinal producers vs. those determined to have poor retinal differentiation capacity (Figure 1). Compared to poor retinal producers, OCT4, SOX2, KLF5, and NANOG were predominantly expressed in the nucleus of good retinal producers. As repeat reprogramming of dermal fibroblasts from patients deemed poor retinal producers did not alter differentiation capacity, we next asked if the lack of retinal differentiation potential was linked to epigenetic memory. As such, a poor retinal producing line was differentiated into cells of ectodermal lineage and reprogrammed back into iPSCs. Unlike the founding mesoderm (fibroblast) derived iPSC line, resulting ectoderm derived iPSCs were found to be excellent retinal producers (Figure 1).

Conclusions : Striking differences in pluripotency factor localization were discovered in good vs poor retinal producers. Poor retinal producers appear to retain an epigenetic memory of their former self. By rederiving iPSC lines using cells of ectodermal lineage we were able to convert poor retinal producers into good retinal producers.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure 1. Cytoplasmic localization of pluripotency factors and residual mesodermal epigenetic memory are associated with poor retinal differentiation capacity.