Human iPS-derived retinal Müller glia cells to model diabetic retinopathy

Abstract

Purpose : Given the central role of Müller glial cells (MGCs) in diabetic retinopathy (DR) pathogenesis, the use of human induced pluripotent stem cell-derived MGCs (hiMGCs) for DR disease modeling seems of high interest. Palmitic acid (PA) is a strong DR-relevant stimulus for primary human MGCs (pMGCs) activation. In this work, we aimed to present an innovative process to produce hiMGCs and to show that their reaction to PA is similar to that of pMGCs.

Methods : - hiMGCs generation
hiMGC were isolated from retinal organoids at D150 and cultured in serial passages. To ensure the reproducibility in the generation of hiMGCs, RNA seq analysis was done on 3 distinct pMGCs and on 3 hiMGCs from different origins.
- Disease modeling
To validate the cability ofhiMGCs to behave as pMGCs, we study their reaction to PA exposure. After 24h, RNA were collected to evaluate the expression of 3 key genes, ATF3, CXL1-2, and ANGPTL4 that were shown to be up-regulated in PA-treated pMGCs. We also investigate hiMGC abilities to remodel vessels, using an angiogenic blot assay and an ex-vivo aortic rings test in the presence of conditioned medium (CM) of hiMGCs.

Results : - hiMGCs generation
The thawed and fresh hiMGCs were VIM+/GFAP- and, GS+/SOX9+ consistent with in vivo retinal Muller glia phenotype. hiMGCs and pMGCs expressed a majority of transcripts in common (5922 transcripts) and only a limited subset of transcripts were restricted to pMGCs (676) or hiMGCs (564).
- Disease modeling
PA exposure resulted in a strong induction of the relative expression of ATF3, CXL1-2, and ANGPTL4, with a mean 28.4-, 3.7- and 5.0-fold increase respectively in pMGCs and 52.7-, 19.9-, 3.3-fold respectively in hiMGCs. The 24 hours-secretome of hiMGCs treated with PA contained detectable amount of 41 angiogenic-related proteins (out of the 55 blotted proteins). Among these proteins, 17 pro-angiogenic factors were found up-regulated. Conditioned media (CM) from hiMGCs treated with PA significantly affected vascular outgrowth in aortic rings while CM from non-treated hiMGCs failed at modifying endothelial cell growth.

Conclusions : For the first time,we described a methods to generate, isolate, amplified and bank human iPS-derived MGCs. This new iPS-based model represent an extremely valuable tool to better understand mechanisms of complex diseases as DR and for the development of new therapies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.