Global Transcriptional and Epigenetic Reconfiguration during Chemical Reprogramming of Human Retinal Pigment Epithelial Cells into Photoreceptor-like Cells

Abstract

Retinal degenerative diseases are frequently caused by the loss of retinal neural cells such as photoreceptors. Cell replacement is regarded as one of the most promising therapies. Multiple types of stem and somatic cells have been tested for photoreceptor conversion. However, current induction efficiencies are still low and the molecular mechanisms underlying reprogramming remain to be clarified. In this work, by combining treatment with small molecules, we directly reprogrammed human fetal retinal pigment epithelial (RPE) cells into chemically induced photoreceptor-like cells (CiPCs) in vitro. Bulk and single-cell RNA sequencing, as well as methylation sequencing, were performed to understand the transcriptional and epigenetic changes during CiPCs conversion. A multi-omics analysis showed that the direct reprogramming process partly resembled events of early retina development. We also found that the efficiency of CiPCs conversion from RPE is much better than that from human dermal fibroblasts (HDF). The small molecules effectively induced RPE cells into CiPCs via suppression of the epithelial-to-mesenchymal transition (EMT). Among the signaling pathways involved in CiPCs conversion, glutamate receptor activation is prominent. In summary, RPE cells can be efficiently reprogrammed into photoreceptor-like cells through defined pharmacological modulations, providing a useful cell source for photoreceptor generation in cell replacement therapy for retinal degenerative diseases.

Recent Achievements in the Heterogeneity of Mammalian and Human Retinal Pigment Epithelium: In Search of a Stem Cell

“…Gene ontology analysis of RNA sequencing results from these cells revealed the upregulation of genes involved in neuronal generation, neurotransmitter uptake, and photoreceptor cell differentiation, such as SOX8, IGFN1, ASCL1, RXRG, THRB, and RORB. On day 10 of reprogramming, the transcriptome profile showed a stable activation of genes specific to rod photoreceptors, but not to cones [14]. Mouse and human RPE stem cells are capable of redifferentiating to the original phenotype.…”

“…Finally, at the terminal stage of RPESC differentiation, rod photoreceptor cells expressing REC, RHO, ARRESTIN, and GNAT1 were obtained [27]. Another study managed to differentiate fetal RPE cells into rod photoreceptors by chemical reprogram-ming [14]. Gene ontology analysis of RNA sequencing results from these cells revealed the upregulation of genes involved in neuronal generation, neurotransmitter uptake, and photoreceptor cell differentiation, such as SOX8, IGFN1, ASCL1, RXRG, THRB, and RORB.…”

“…The RPE in the human eye undergoes morphofunctional restructuring throughout ontogenesis [9][10][11]. Recently, many studies have been carried out to characterize the gene profile of the RPE and retina of mammals and humans, which have made it possible to create a transcriptomic atlas of these tissues [2,[12][13][14][15][16][17][18][19][20][21][22][23][24]. RPE cells demonstrate different susceptibilities to monogenic and polygenic retinal diseases, as well as to the effects of various drugs, depending on the topographic location of the cells in the RPE layer and the microenvironment [1,25,26].…”

“…Recent research confirms the high plasticity of RPE cells and shows that fetal RPE cells or the mature human RPE can be reprogrammed into a neuronal phenotype. For example, it is possible to reprogram fetal RPE cells into photoreceptors using small molecular factors, which opens up prospects for regenerative medicine [14,27]. In this regard, understanding the role of the entire genetic and functional diversity of cell subpopulations in the RPE, among which a special place is given to stem cells (SCs), in health and in retinal pathology determines the success of regenerative medicine.…”