Abstract
Purpose :
While it is possible to obtain photoreceptors by direct reprogramming from fibroblasts in low quantities, efficient 2D protocols to generate photoreceptors in vitro from human induced pluripotent stem cells (hiPSCs) needs to be established. Forward programming relies on a transcription factors’ (TF) abilities to activate distinct differentiation pathways in stem cells. Aiming at finding a TF combination that drives efficient differentiation of stem cells into photoreceptors, we performed a TF-library on library screen.
Methods :
A hiPSC reporter line was transduced with lentiviruses each carrying one of the 16 TFs known from in vivo photoreceptor development and with a comprehensive library consisting of 1748 human TFs. The hiPSC reporter line, expressing GFP under the cone arrestin and dsRed under the rhodopsin promoter, allowed for fluorescent cell sorting of photoreceptor-like cells. Sorted single cells were qPCR-tested for OTX, CRX, RCVRN, RHO, OPN1SW and OPN1LW and sequenced to identify the overexpressed TFs. Currently, we are validating TF combinations in the hiPSC reporter line using flow cytometry detecting the loss of a pluripotency marker (Tra1-60) and upregulation of neuronal markers (NCAM) and fluorescence from the reporter cassette.
Results :
87% of the sorted cells were qPCR-positive for at least one of the tested photoreceptor-specific genes indicating the cell-type-precision of our screen. Some of the tested TF combinations led to a significant loss of the Tra1-60 and upregulation of the NCAM marker (hiPSCs: 0.47 ± 0.07 %, hiPSCs-TFs: 75.23 ± 3.7 %; mean ± SEM, two-tailed t-test; p = 0.002) after 5 days of overexpression, indicating that cells are differentiating towards the neuronal lineage. Furthermore, fluorescence microscopy and flow cytometry detected GFP-positive cells after 10 days suggesting the presence of cone photoreceptors.
Conclusions :
We systematically screened TFs to find the combination that would help us reaching a final goal of engineering human photoreceptors in vitro. Our data suggest that the known factors were insufficient to drive photoreceptor differentiation, indicating that photoreceptor genesis from hiPSCs requires additional TFs. In vitro-engineered photoreceptors might serve as a donor material for cell transplantation to treat blindness as sufficient quantities can be generated within 10 days compared to hundreds of days if dissociated from 3D human retinal organoids.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.