Derivation of Human Differential Photoreceptor-like Cells from the Iris by Defined Combinations of CRX, RX and NEUROD

Abstract

Examples of direct differentiation by defined transcription factors have been provided for beta-cells, cardiomyocytes and neurons. In the human visual system, there are four kinds of photoreceptors in the retina. Neural retina and iris-pigmented epithelium (IPE) share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons. We here define the transcription factor combinations that can determine human photoreceptor cell fate. Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Phototransduction-related genes as well as opsin genes were up-regulated in those cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that generated photoreceptor cells, induced by CRX, RX and NEUROD, responded to light. The response was an inward current instead of the typical outward current. These data suggest that photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX generate immature photoreceptors: and additional NEUROD promotes maturation. These findings contribute substantially to a major advance toward eventual cell-based therapy for retinal degenerative diseases.

Derivation of human differential photoreceptor cells from adult human dermal fibroblasts by defined combinations of CRX, RAX,OTX2 and NEUROD

“…Expression of the CRX, RAX and NEUROD and genes in the transgene‐induced cells was analyzed by RT‐PCR, using the exogenous and endogenous gene‐specific primers (Seko et al . ). Equal amounts of RNAs were examined as determined by normalization by expression of the G3PDH gene.…”

“…CRX, RAX and NEUROD are essential factors that induce photoreceptor cells from human iris cells (Seko et al . ) and POU1f1, Sox2 and Otx2 bind to the Rx promoter (Martinez‐de Luna et al . ).…”

“…We recently employed the strategy of ‘direct reprogramming’ to generate retinal photoreceptor cells from human somatic cells, defining a combination of transcription factors, CRX, RAX and NEUROD , that induce light responsive photoreceptor cells (Seko et al . ). In that study, we induced ‘iris cells’ into photoreceptor cells.…”

Regenerative Medicine: Solution in Sight

“…By forcing expression of the photoreceptor specific homeobox gene Crx, primary cells derived from adult rat iris tissue could produce photoreceptor-like cells that expressed rhodopsin and recoverin (Haruta et al 2001). Combinations of (1) Crx and Otx2, (2) Crx, Nrl and NeuroD or (3) Crx, Rx and NeuroD produced similar results, and generated photoreceptor-like cells that express photoreceptor-specific markers and exhibited rod photoreceptor-specific electrophysiological responses to light stimuli (Akagi et al 2004(Akagi et al , 2005Seko et al 2012). A related strategy has been applied to generate RPE-like cells from human fibroblast cells.…”

Ocular stem cells: a status update!

“…These cells can be transdifferentiated into retinal neuronal cells expressing retinal-specific markers [30]. Further studies are needed before iris pigment epithelial cells can be used clinically.…”