Abstract
Purpose :
The pluripotent cells represent an excellent resource for examining the ex-vivo stem cell approach to glaucomatous neuropathy and/or for generating glaucoma patient-specific model to understand the underlying mechanism of retinal ganglion cell (RGC) degeneration. The success of both approaches depends upon derivation of RGCs with stable phenotype reflected at molecular, cellular and functional levels. This requires that the acquisition of the phenotype is least random and achieved primarily through the recruitment of the normal developmental mechanism. Here, we present defined conditions that promote induced pluripotent stem (iPS) cell-derived retinal progenitor cells (RPCs) to acquire functional RGC properties by temporally and contextually recruiting signaling pathways of normal RGC genesis.
Methods :
Defined conditions for RGC generation, first tested on native and ES cell-derived RPCs, were used to differentiate human iPS cells along the RGC lineage. The human RPCs were exposed to conditions consisting of small molecules and recombinant growth factors to temporally influence Shh, FGF8, TGF b, and Notch signaling pathways for 15 days in vitro, followed by examination of RGC properties at molecular, cellular and functional levels. In addition, we examined the influence of culture substratum on the generation and complexities of RGC neurites.
Results :
We determined the temporal expression patterns of genes that constitute the regulatory hierarchy for RGC specification and differentiation. We observed a temporal decrease in the expression of Rx and Chx10 with concomitant temporal increase in the expression of regulators (Atoh7, Brn3b, Islet1, and RPF1) and mature markers (Thy1) of RGCs. Double immunohistochemical analysis was used to identify RGCs. The efficiency of RGC differentiation was ~80 % based on Atho7 and Tuj1 co-expression. Cells displayed TTX-sensitive sodium currents in voltage-clamp recordings and generated action potentials in response to depolarizing current injections in current-clamp recordings. Longer and more prominent axons, extending radially, were observed on matrigel compared to PDL/Laminin.
Conclusions :
Induction conditions, defined by small molecule and recombinant growth factors on the matrigel substratum, generate RGCs from a variety of RPCs, including those derived from human iPS cells by recapitulating normal mechanism of RGC genesis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.