Abstract
Purpose :
In clinical trials, retinal progenitor cells (RPCs) are anticipated to rescue vision in late-stage photoreceptor degeneration, but the availability of primary human RPCs is limited to fetal tissue. We developed a method to directly reprogram human fibroblasts (HFs) into RPCs with small molecules, and tested its safety and therapeutic efficacy in a rodent model of photoreceptor loss.
Methods :
HFs were transduced with Vsx2–eGFP reporter, to determine the conversion of induced retinal progenitor cells (iRPCs). By fluorescence activated cell sorting (FACS), we sorted Vsx2–eGFP+ cells, and checked the gene expression profile utilizing quantitative reverse transcription PCR (qRT-PCR), and bulk RNA sequencing. To test in vitro potency, we used fura-2-based imaging to study glutamate receptor-mediated [Ca2+] i changes in fibroblasts and iRPCs. To test in vivo safety and therapeutic efficacy, one eye of Royal College of Surgeons (RCS) rat at P21 were subretinally transplanted with 5 μL of 2*105 cell suspension containing PBS (N=4), HFs (N=4), or iRPCs (N=4). The rats were then analyzed with electroretinogram (ERG) and light aversion tests monthly. At 4 month age, the eyes were cryo-fixed and sectioned for immunohistochemistry.
Results :
After chemical reprogramming, there were 42.8% Vsx2–eGFP+ cells on FACS. In qRT-PCR, statistically significant increase (>1000 folds) of Vsx2 expression was found in iRPCs. In bulk RNA sequencing, iRPCs demonstrated upregulated expression of transcripts related to the extracellular matrix component, axon, dendrite, synaptic and postsynaptic membrane, and transport vesicle formation (p<0.003). In fura-2-based imaging, intracellular calcium concentrations were found to elevate in iRPCs upon glutamate stimulation (1mM), whereas HFs showed no response. In RCS rats, there was an improvement of ERG in the eyes transplanted with iRPCs at P28 and P56 (p<0.05). In light aversion test at P112, RCS rats transplanted with iRPCs were found to spend significantly more time in the dark space, indicating vision rescue in late-stage photoreceptor loss. In immunohistochemistry, we found survival and integration of iRPCs in layer of rat photoreceptors, with layer-by-layer retinal morphology well preserved.
Conclusions :
By chemical reprogramming of human fibroblasts into induced retinal progenitor cells, we demonstrated its therapeutic potential in photoreceptor degeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.