Abstract
Purpose :
Retinal Pigment Epithelium (RPE) has been implicated in pathogenesis of various vision-threatening retinal disorders, yet, in vitro diagnostic tools to evaluate RPE health in a patient-specific manner are not available. Urine represents an attractive, non-invasive source of induced pluripotent stem cells (iPSC), however, culture conditions for RPE induction of urine-derived iPSC have not been optimized yet. In this study, we sought to establish a protocol for rapid RPE induction from urine-derived, human iPSC.
Methods :
Human iPSCs were generated from urinary tract progenitors. For RPE induction, iPSCs were cultured on matrigel-precoated microplates (adherent culture, AC) or on low adherence U-bottom microplates (suspension culture, SC) for up to 2 weeks. Glasgow’s modified essential medium supplemented with either 5% fetal bovine serum or small molecule cocktail comprised of B27, N2, NEAA, glutamine, taurine, hydrocortisone, triiodothyronine, IGF1, Dkk1, Noggin and bFGF was used to test efficiency of RPE lineage commitment and phenotypic induction.
Results :
Expression of pluripotency marker OCT4 decreased by 63.6+1% in SC vs 20.6+2% in AC after 1 week of induction with serum. In parallel, genes for eye field lineage commitment, including PAX6, RAX, SIX3, LHX2 and OTX2, were found upregulated ranging from 10- to 100-fold in SC, while remaining unaltered in AC. In contrast, iPSC in small molecule-containing media failed to exit pluripotent state in AC and SC. Genes for eye field development were moderately upregulated by <10-fold.
After 2 weeks of serum SC, RAX, SIX3 and LHX2 expression were found reduced to baseline levels, while RPE phenotypic genes MITF and BEST1 were remarkably activated (15- and 6.38-fold, respectively). In contrast, small molecules-containing media failed to express RPE-specific genes.
Conclusions :
Suspension culture with serum-containing media efficiently induced iPSC towards RPE differentiation. Further protocol optimization will benefit urine-derived iPSC for clinical scale tissue engineering.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.