Abstract
Purpose :
To study whether the retinal progenitor cells derived from normal human corneal induced pluripotent stem cell (iPSC) could be used as model to study human retinal diseases.
Methods :
Normal human corneal stromal fibroblasts from three male donors ages (45, 50 and 55 years) were reprogramed directly to iPSC by delivering reprogramming factors in a single virus using 2A “self-cleaving” peptides, using a single polycistronic lentiviral vector co-expressing four transcription factors (Oct 4, Sox2, Klf4 and Myc). These iPS cells were immunofluorescentally characterized for stem cell markers (SSEA4, Oct4 and Sox2), and were differentiated using a retinal differentiation medium [DMEM F-12 medium containing 10% knockout serum replacement, 2% B27 supplement, 1% N2 supplement, 1% L-glutamine, 1% 100X NEAA, 1% penicillin/streptomycin, 1 ng/ml noggin, 1 ng/ml DKK-1, 1 ng/ml IGF-1 and 0.5 ng/ml bFGF]. The differentiated cells were immunocytochemically characterized and were transfected with CCR1 plasmid (Chemokine receptors, belong to the class of rhodopsin-like family of GPCRs). The 661W photoreceptor cell line was used as a control.
Results :
iPSC colonies were positive for all the stem cell markers (SSEA4, Oct4 and Sox2). The iPS cells were differentiated for 33 days; and the immunocytochemical analysis showed the expression of two retinal photoreceptor markers, recoverin and rhodopsin. CCR1 transfection resulted in an increased cell death in differentiated photoreceptor cells after 24 h and also in 661W cell line. This was in concordance with a previous report showing that expression of CCR1 in the photoreceptor cells led to an increased progression of retinal degeneration in rd mice cells.
Conclusions :
Normal human corneal fibroblasts were reprogramed to iPSC using single polycistronic virus co-expressing four transcription factors and the iPSC-derived retinal progenitor cells expressed photoreceptor markers (rhodopsin and recoverin). The retinal precursor cells could be used as model to study human retinal diseases, and also as a source for transplantation in retinal degenerative diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.