Epigenetic regulation in the commitment of progenitor cells during retinal development and regeneration.

Retinal development is initiated by multipotent retinal progenitor cells, which undergo several rounds of cell divisions and subsequently terminal differentiation. Retinal regeneration is usually considered as the recapitulation of retinal development, which share common mechanisms underlying the cell cycle re-entry of adult retinal stem cells and the differentiation of retinal neurons. However, how proliferative retinal progenitor cells perform a precise transition to postmitotic retinal cell types during the process of development and regeneration remains elusive. It is proposed that both the intrinsic and extrinsic programming are involved in the transcriptional regulation of the spatio-temporal fate commitment. Epigenetic modifications and the regulatory mechanisms at both DNA and chromatin levels are also postulated to play an important role in the timing of differentiation of specific retinal cells. In the present review, we have summarized recent knowledge of epigenetic regulation that underlies the commitment of retinal progenitor cells in the settings of retinal development and regeneration.

Unifying developmental programs for embryonic and postembryonic neurogenesis in the zebrafish retina.

The zebrafish retina grows for a lifetime. Whether embryonic and postembryonic retinogenesis conform to the same developmental program is an outstanding question that remains under debate. Using single-cell RNA sequencing of ∼20,000 cells of the developing zebrafish retina at four different stages, we identified seven distinct developmental states. Each state explicitly expresses a gene set. Disruption of individual state-specific marker genes results in various defects ranging from small eyes to the loss of distinct retinal cell types. Using a similar approach, we further characterized the developmental states of postembryonic retinal stem cells (RSCs) and their progeny in the ciliary marginal zone. Expression pattern analysis of state-specific marker genes showed that the developmental states of postembryonic RSCs largely recapitulated those of their embryonic counterparts, except for some differences in rod photoreceptor genesis. Thus, our findings reveal the unifying developmental program used by the embryonic and postembryonic retinogenesis in zebrafish.

miR-124-3p regulates the proliferation and differentiation of retinal progenitor cells through SEPT10.

Retinal degenerative diseases such as glaucoma, retinitis pigmentosa, and age-related macular degeneration pose serious threats to human visual health due to lack of effective therapeutic approaches. In recent years, the transplantation of retinal progenitor cells (RPCs) has shown increasing promise in the treatment of these diseases; however, the application of RPC transplantation is limited by both their poor proliferation and their differentiation capabilities. Previous studies have shown that microRNAs (miRNA) act as essential mediators in the fate determination of stem/progenitor cells. In this study, we hypothesized that miR-124-3p plays a regulatory role in the fate of RPC determination by targeting Septin10 (SEPT10) in vitro. We observed that the overexpression of miR124-3p downregulates SEPT10 expression in RPCs, leading to reduced RPC proliferation and increased differentiation, specifically towards both neurons and ganglion cells. Conversely, antisense knockdown of miR-124-3p was shown to boost SEPT10 expression, enhance RPC proliferation, and attenuate differentiation. Moreover, overexpression of SEPT10 rescued miR-124-3p-caused proliferation deficiency while weakening the enhancement of miR-124-3p-induced-RPC differentiation. Results from this study show that miR-124-3p regulates RPC proliferation and differentiation by targeting SEPT10. Furthermore, our findings enable a more comprehensive understanding into the mechanisms of proliferation and differentiation of RPC fate determination. Ultimately, this study may be useful for helping researchers and clinicians to develop more promising and effective approaches to optimize the use of RPCs in treating retinal degeneration diseases.

Absence of Connexin 43 Results in Smaller Retinas and Arrested, Depolarized Retinal Progenitor Cells in Human Retinal Organoids.

The development of the vertebrate retina relies on complex regulatory mechanisms to achieve its characteristic layered morphology containing multiple neuronal cell types. While connexin 43 (CX43) is not expressed by mature retinal neurons, mutations in its gene GJA1 are associated with microphthalmia and low vision in patients. To delineate how lack of CX43 affects retinal development, GJA1 was disrupted in human induced pluripotent stem cells (hiPSCs) (GJA1-/-) using CRISPR/Cas9 editing, and these were subsequently differentiated into retinal organoids. GJA1-/- hiPSCs do not display defects in self-renewal and pluripotency, but the resulting organoids are smaller with a thinner neural retina and decreased abundance of many retinal cell types. CX43-deficient organoids express lower levels of the neural marker PAX6 and the retinal progenitor cell (RPC) markers PAX6, SIX3, and SIX6. Conversely, expression of the early neuroectoderm markers SOX1 and SOX2 remains high in GJA1-/- organoids throughout their development. The lack of CX43 results in an increased population of CHX10-positive RPCs that are smaller, disorganized, do not become polarized, and possess a limited ability to commit to retinal fate specification. Our data indicate that lack of CX43 causes a developmental arrest in RPCs that subsequently leads to pan-retinal defects and stunted ocular growth.

Foxn4 is a temporal identity factor conferring mid/late-early retinal competence and involved in retinal synaptogenesis.

During development, neural progenitors change their competence states over time to sequentially generate different types of neurons and glia. Several cascades of temporal transcription factors (tTFs) have been discovered in Drosophila to control the temporal identity of neuroblasts, but the temporal regulation mechanism is poorly understood in vertebrates. Mammalian retinal progenitor cells (RPCs) give rise to several types of neuronal and glial cells following a sequential yet overlapping temporal order. Here, by temporal cluster analysis, RNA-sequencing analysis, and loss-of-function and gain-of-function studies, we show that the Fox domain TF Foxn4 functions as a tTF during retinogenesis to confer RPCs with the competence to generate the mid/late-early cell types: amacrine, horizontal, cone, and rod cells, while suppressing the competence of generating the immediate-early cell type: retinal ganglion cells (RGCs). In early embryonic retinas, Foxn4 inactivation causes down-regulation of photoreceptor marker genes and decreased photoreceptor generation but increased RGC production, whereas its overexpression has the opposite effect. Just as in Drosophila, Foxn4 appears to positively regulate its downstream tTF Casz1 while negatively regulating its upstream tTF Ikzf1. Moreover, retina-specific ablation of Foxn4 reveals that it may be indirectly involved in the synaptogenesis, establishment of laminar structure, visual signal transmission, and long-term maintenance of the retina. Together, our data provide evidence that Foxn4 acts as a tTF to bias RPCs toward the mid/late-early cell fates and identify a missing member of the tTF cascade that controls RPC temporal identities to ensure the generation of proper neuronal diversity in the retina.

In vivo study to assess dosage of allogeneic pig retinal progenitor cells: Long-term survival, engraftment, differentiation and safety.

Despite notable efforts and significant therapeutical advances, age-related macular degeneration remains the single most common reason for vision loss. Retinal progenitor cells (RPCs) are considered promising candidates for cellular treatments that repair and restore vision. In this allogenic study, the phenotypic profile of pig and human RPCs derived using similar manufacturing processes is compared. The long-term (12-week) survival of green fluorescent protein-pig retinal progenitor cells GFP-pRPC after subretinal transplantation into normal miniature pig (mini-pig) retina is investigated. Human eyes are both anatomically and physiologically mimicked by pig eyes, so the pig is an ideal model to show an equivalent way of delivering cells, immunological response and dosage. The phenotypic equivalency of porcine and clinically intended human RPCs was established. Thirty-nine mini-pigs are used in this study, and vehicle-injected eyes and non-injected eyes serve as controls. Six groups are given different dosages of pRPCs, and the cells are found to survive well in all groups. At 12 weeks, strong evidence of integration is indicated by the location of the grafted cells within the neuro-retina, extension of processes to the plexiform layers and expression of key retinal markers such as recoverin, rhodopsin and synaptophysin. No immunosuppression is used, and no immune response is found in any of the groups. No pRPC-related histopathology findings are reported in the major organs investigated. An initial dose of 250 k cells in 100 µl of buffer is established as an appropriate initial dose for future human clinical trials.

A single-cell guide to retinal development: Cell fate decisions of multipotent retinal progenitors in scRNA-seq.

Recent advances in high throughput single-cell RNA sequencing (scRNA-seq) technology have enabled the simultaneous transcriptomic profiling of thousands of individual cells in a single experiment. To investigate the intrinsic process of retinal development, researchers have leveraged this technology to quantify gene expression in retinal cells across development, in multiple species, and from numerous important models of human disease. In this review, we summarize recent applications of scRNA-seq and discuss how these datasets have complemented and advanced our understanding of retinal progenitor cell competence, cell fate specification, and differentiation. Finally, we also highlight the outstanding questions in the field that advances in single-cell data generation and analysis will soon be able to answer.

PAX6D instructs neural retinal specification from human embryonic stem cell-derived neuroectoderm.

PAX6 is essential for neural retina (NR) and forebrain development but how PAX6 instructs NR versus forebrain specification remains unknown. We found that the paired-less PAX6, PAX6D, is expressed in NR cells during human eye development and along human embryonic stem cell (hESC) specification to retinal cells. hESCs deficient for PAX6D failed to enter NR specification. Induced expression of PAX6D but not PAX6A in a PAX6-null background restored the NR specification capacity. ChIP-Seq, confirmed by functional assays, revealed a set of retinal genes and non-retinal neural genes that are potential targets of PAX6D, including WNT8B. Inhibition of WNTs or knocking down of WNT8B restored the NR specification capacity of neuroepithelia with PAX6D knockout, whereas activation of WNTs blocked NR specification even when PAX6D was induced. Thus, PAX6D specifies neuroepithelia to NR cells via the regulation of WNT8B.

Zeb2 regulates the balance between retinal interneurons and Müller glia by inhibition of BMP-Smad signaling.

The interplay between signaling molecules and transcription factors during retinal development is key to controlling the correct number of retinal cell types. Zeb2 (Sip1) is a zinc-finger multidomain transcription factor that plays multiple roles in central and peripheral nervous system development. Haploinsufficiency of ZEB2 causes Mowat-Wilson syndrome, a congenital disease characterized by intellectual disability, epilepsy and Hirschsprung disease. In the developing retina, Zeb2 is required for generation of horizontal cells and the correct number of interneurons; however, its potential function in controlling gliogenic versus neurogenic decisions remains unresolved. Here we present cellular and molecular evidence of the inhibition of Müller glia cell fate by Zeb2 in late stages of retinogenesis. Unbiased transcriptomic profiling of control and Zeb2-deficient early-postnatal retina revealed that Zeb2 functions in inhibiting Id1/2/4 and Hes1 gene expression. These neural progenitor factors normally inhibit neural differentiation and promote Müller glia cell fate. Chromatin immunoprecipitation (ChIP) supported direct regulation of Id1 by Zeb2 in the postnatal retina. Reporter assays and ChIP analyses in differentiating neural progenitors provided further evidence that Zeb2 inhibits Id1 through inhibition of Smad-mediated activation of Id1 transcription. Together, the results suggest that Zeb2 promotes the timely differentiation of retinal interneurons at least in part by repressing BMP-Smad/Notch target genes that inhibit neurogenesis. These findings show that Zeb2 integrates extrinsic cues to regulate the balance between neuronal and glial cell types in the developing murine retina.

Targeted deletion of Crb1/Crb2 in the optic vesicle models key features of leber congenital amaurosis 8.

The Crb1 and 2 (Crumbs homolog 1 & 2) genes encode large, single-pass transmembrane proteins essential for the apicobasal polarity and adhesion of epithelial cells. Crb1 mutations cause degenerative retinal diseases in humans, including Leber congenital amaurosis type 8 (LCA8) and retinitis pigmentosa type 12 (RP12). In LCA8, impaired photoreceptor development and/or survival is thought to cause blindness during early infancy, whereas, in RP12, progressive photoreceptor degeneration damages peripheral vision later in life. There are multiple animal models of RP12 pathology, but no experimental model of LCA8 recapitulates the full spectrum of its pathological features. To generate a mouse model of LCA8 and identify the functions of Crb1/2 in developing ocular tissues, we used an mRx-Cre driver to generate allelic combinations that enabled conditional gene ablation from the optic vesicle stage. In this series only Crb1/2 double knockout (dKO) mice exhibited characteristics of human LCA8 disease: locally thickened retina with spots devoid of cells, aberrant positioning of retinal cells, severely disrupted lamination, and depigmented retinal-pigmented epithelium. Retinal defects antedated E12.5, which is far earlier than the stage at which photoreceptor cells mainly differentiate. Most remarkably, Crb1/Crb2 dKO showed a severely attenuated electroretinogram at the eye opening stage. These results suggest that human LCA8 can be modeled in the mouse by simultaneously ablating Crb1/2 from the beginning of eye development. Importantly, they also indicate that LCA8 is caused by malfunction of retinal progenitor cells during early ocular development rather than by defective photoreceptor-Muller glial interaction, a mechanism proposed for RP12.

An enigmatic translocation of the vertebrate primordial eye field.

The primordial eye field of the vertebrate embryo is a single entity of retinal progenitor cells spanning the anterior neural plate before bifurcating to form bilateral optic vesicles. Here we review fate mapping data from zebrafish suggesting that prior to evagination of the optic vesicles the eye field may undergo a Maypole-plait migration of progenitor cells through the midline influenced by the anteriorly subducting diencephalon. Such an enigmatic translocation of scaffolding progenitors could have evolutionary significance if pointing, by way of homology, to an ancient mechanism for transition of the single eye field in chordates to contralateral eye fields in vertebrates.

ARS2 is required for retinal progenitor cell S-phase progression and Müller glial cell fate specification.

During a developmental period that extends postnatally in the mouse, proliferating multipotent retinal progenitor cells produce one of 7 major cell types (rod, cone, bipolar, horizontal, amacrine, ganglion, and Müller glial cells) as they exit the cell cycle in consecutive waves. Cell production in the retina is tightly regulated by intrinsic, extrinsic, spatial, and temporal cues, and is coupled to the timing of cell cycle exit. Arsenic-resistance protein 2 (ARS2, also known as SRRT) is a component of the nuclear cap-binding complex involved in RNA Polymerase II transcription, and is required for cell cycle progression. We show that postnatal retinal progenitor cells (RPCs) require ARS2 for proper progression through S phase, and ARS2 disruption leads to early exit from the cell cycle. Furthermore, we observe an increase in the proportion of cells expressing a rod photoreceptor marker, and a loss of Müller glia marker expression, indicating a role for ARS2 in regulating cell fate specification or differentiation. Knockdown of Flice Associated Huge protein (FLASH), which interacts with ARS2 and is required for cell cycle progression and 3'-end processing of replication-dependent histone transcripts, phenocopies ARS2 knockdown. These data implicate ARS2-FLASH-mediated histone mRNA processing in regulating RPC cell cycle kinetics and neuroglial cell fate specification during postnatal retinal development.

miR-17 regulates the proliferation and differentiation of retinal progenitor cells by targeting CHMP1A.

MicroRNAs have a vital effect on the differentiation of many types of progenitor cells. Recent studies have suggested that miR-17 plays an important role in the differentiation process of brain neural progenitor cells (NPC). Nevertheless, its detailed functions in regulating retinal progenitor cells (RPC) remain unclear. In our study, overexpression and knockdown of miR-17 were performed by transfecting RPC with mimics and inhibitors, respectively. Next, we investigated the role of miR-17 in RPC proliferation and differentiation by the following experiments: qPCR, CCK8, Edu staining, immunostaining and Western blot. The results revealed that miR-17 inhibited RPC proliferation but enhanced differentiation. Furthermore, according to a web-based database analysis, we identified charged multivesicular body protein 1A (CHMP1A) as a target gene. A dual luciferase reporter system showed that miR-17 specifically binds to the CHMP1A 3' untranslated region (UTR). Next, our data showed upregulation of miR-17 decreased CHMP1A protein level, causing reduced proliferation and enhanced differentiation of RPC. Downregulation of miR-17 led to enhanced CHMP1A protein expression, increased RPC proliferation and decreased differentiation. Taken together, our data provide a proven pathway by which miR-17 regulates RPC proliferation and differentiation by targeting CHMP1A.

Low-oxygen and knock-out serum maintain stemness in human retinal progenitor cells.

Using stem and progenitor cells to treat retinal disorders holds great promise. Using defined culture conditions to maintain the desires phenotype is of utmost clinical importance. We cultured human retinal progenitor cells (hRPCs) in different conditions: such as normoxia (20% oxygen), and hypoxia (5% oxygen) with and without knock-out serum replacement (KOSR) to evaluate its effect on these cells. KOSR is known nutrient supplement often used to replace bovine serum for culturing embryonic or pluripotent stem cells, especially those destined for clinical applications. The purpose of this study was to identify the impact of different environmental and chemical cues to determine if this alters the fate of these cells. Our results indicate that cells cultured with or without KOSR do not show significant differences in viability, but that the oxygen tension can significantly change their viability (higher in hypoxia than normoxia). However, cells with KOSR in hypoxia condition expressed significantly higher stemness markers such as C-myc and Oct4 (31.20% and 13.44% respectively) in comparison to hRPCs cultured in KOSR at normoxia (12.07% and 4.05%). Furthermore, levels of markers for retinal commitment such as rhodopsin were significantly lower in the KOSR supplemented cells in hypoxia culture compared to normoxia. KOSR is known to improve proliferation and maintain stemness of embryonic cells and our experiments suggest that hRPCs maintain their proliferation and stemness characteristics in hypoxia with KOSR supplement. Normoxia, however, results in mature cell marker expression, suggesting a profound effect of oxygen tension on these cells.

Neurogenesis and Specification of Retinal Ganglion Cells.

Across all species, retinal ganglion cells (RGCs) are the first retinal neurons generated during development, followed by the other retinal cell types. How are retinal progenitor cells (RPCs) able to produce these cell types in a specific and timely order? Here, we will review the different models of retinal neurogenesis proposed over the last decades as well as the extrinsic and intrinsic factors controlling it. We will then focus on the molecular mechanisms, especially the cascade of transcription factors that regulate, more specifically, RGC fate. We will also comment on the recent discovery that the ciliary marginal zone is a new stem cell niche in mice contributing to retinal neurogenesis, especially to the generation of ipsilateral RGCs. Furthermore, RGCs are composed of many different subtypes that are anatomically, physiologically, functionally, and molecularly defined. We will summarize the different classifications of RGC subtypes and will recapitulate the specification of some of them and describe how a genetic disease such as albinism affects neurogenesis, resulting in profound visual deficits.

A novel electro-chemotactic approach to impact the directional migration of transplantable retinal progenitor cells.

Photoreceptor degeneration is a significant cause of visual impairment in the United States and globally. Cell replacement therapy shows great promise in restoring vision by transplanting stem-like cells into the sub-retinal space as substitutes for damaged photoreceptors. However, vision repair via transplantation has been limited, in large part, by low numbers of replacement cells able to migrate into damaged retinal tissue and integrate with native photoreceptors. Projects have used external chemical fields and applied electric fields to induce the chemotaxis and electrotaxis of replacement cells, respectively, with limited success. However, the application of combined electro-chemotactic fields in directing cells within biomaterials and host tissue has been surprisingly understudied. The current work examined the ability of combined electro-chemotactic fields to direct the migration of transplantable retinal progenitor cells (RPCs) in controlled microenvironments. Experiments used our established galvano-microfluidic system (Gal-MµS) to generate tunable chemotactic concentration fields with and without superimposed electric fields. Result illustrate that combination fields increased the distance migrated by RPCs by over three times that seen in either field, individually, and with greater directionality towards increasing gradients. Interestingly, immunofluorescence assays showed no significant differences in the distribution of the total and/or activated cognate receptor of interest, indicating that changes in ligand binding alone were not responsible for the measured increases in migration. Bioinformatics analysis was then performed to identity potential, synergistic mechanistic pathways involved in the electro-chemotaxis measured. Results indicate that increased RPC migration in electro-chemotactic fields may arise from down-regulation of cell adhesion proteins in tandem with up-regulation of cytoskeletal regulation proteins. These comprehensive results point towards a novel migration-targeted treatment that may dramatically improve transplantation outcomes as well as elucidate unreported synergy across biological mechanisms in response to electro-chemotactic fields.

S1P transporter SPNS2 regulates proper postnatal retinal morphogenesis.

Spinster homolog 2 (SPNS2) is the membrane transporter of sphingosine-1-phosphate (S1P), and it participates in several physiologic processes by activating different S1P receptors (S1PRs). However, its functions in the nervous system remain largely unclear. We explored the important role of SPNS2 in the process of retinal morphogenesis using a spns2-deficient rat model. In the absence of the functional SPNS2 transporter, we observed progressively aggravating laminar disorganization of the epithelium at the postnatal stage of retinal development. Disrupted cell polarity, delayed cell-cycle exit of retinal progenitor cells, and insufficient migration of newborn neurons were proposed in this study as potential mechanisms accounting for this structural disorder. In addition, we analyzed the expression profiles of spns2 and s1prs, and proposed that SPNS2 regulated retinal morphogenesis by establishing the S1P level in the eye and activating S1PR3 signaling. These data indicate that SPNS2 is indispensable for normal retinal morphogenesis and provide new insights on the role of S1P in the developing retina using an established in vivo model.-Fang, C., Bian, G., Ren, P., Xiang, J., Song, J., Yu, C., Zhang, Q., Liu, L., Chen, K., Liu, F., Zhang, K., Wu, C., Sun, R., Hu, D., Ju, G., Wang, J. S1P transporter SPNS2 regulates proper postnatal retinal morphogenesis.

Stem cells from apical papilla promote differentiation of human pluripotent stem cells towards retinal cells.

Recently, we have found that human stem cells from apical papilla (SCAP) show a stromal cell-derived inducing activity (SDIA). To examine SDIA competence for retinal cells differentiation, we co-cultured SCAP with human pluripotent stem cells (hPSCs). In comparison with Matrigel-cultured hPSCs, SCAP significantly induces hPSCs to differentiate into rostral neural cells as demonstrated by upregulation of OTX2 and PAX6 and down-regulation of EN1, HOXB4 and HOXC8. Furthermore, the differentiated cells on SCAP significantly expressed eye-field markers, RAX, PAX6, LHX2 and SIX3 and showed five folds pigmented colonies. The generated hPSC-retinal pigmented epithelium (RPE) was hexagonal and highly expressed related markers, ZO-1, RPE65, BEST, CRALBP and MITF. They were able to phagocytose latex beads. Moreover, the assessment of the isolated neural tube-like structures on SCAP showed the expression of retinal progenitor cells (RPCs) - SIX3, RAX, and PAX6. SCAP highly expressed DKK3 and SFRP2, Wnt inhibitor factors and their target genes, Cyclin D1 and c-Myc were down-regulated significantly on SCAP. These results showed SCAP promoted the differentiation of hPSCs into retinal cells (RPE and RPCs) possibly through inhibition of Wnt signaling pathway. This simple and efficient approach provides human RPE generation for developing therapies for diseases such as age-related macular degeneration.

Identification of retinal homeobox (rax) gene-dependent genes by a microarray approach: The DNA endoglycosylase neil3 is a major downstream component of the rax genetic pathway.

BACKGROUND: The retinal homeobox (rx/rax) gene is a transcription factor expressed in the developing eye field that is necessary for normal eye development. rax is necessary for retinal specification and stem cell development. The genetic program of early retinal development, including rax expression, can be induced in naïve ectoderm by activation of insulin-like growth factor (IGF) signaling. We have undertaken a microarray-based approach to identify rax-dependent IGF-induced genes. RESULTS: We identified 21 IGF-induced genes that exhibit at least a two-fold decrease in expression when rax expression is knocked down. Ten of these genes were expressed in the developing eye, eight were expressed in the ciliary marginal zone of the mature tadpole retina, and four could significantly rescue the rax knockdown phenotype. One of these, the nei endonuclease VIII-like 3 (neil3) gene, rescued the rax knockdown phenotype to a remarkable degree. We found that neil3 is necessary for normal retinal lamination and retinal neuron differentiation. CONCLUSIONS: We have identified neil3 as a component of the rax genetic pathway necessary for normal retinal progenitor cell development. neil3 is involved in the base excision DNA repair pathway, suggesting that this pathway is essential for normal rax-dependent progenitor cell development in the mature retina. Developmental Dynamics 247:1199-1210, 2018. © 2018 Wiley Periodicals, Inc.

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro.

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs' ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2-Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4-Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC-pre-treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF-pre-treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.