Photoreceptor-like cells from reprogramming cultured mammalian RPE cells.

PURPOSE: Previous studies showed that chick retinal pigment epithelium (RPE) cells can be reprogrammed by a specific gene to take on the path of photoreceptor differentiation. In this study, we tested whether this reprogramming scheme could be applied to mammalian RPE cells. METHODS: Human RPE cell lines ARPE-19, a spontaneously transformed line of RPE cells derived from a 19-year-old person, and hTERT-RPE1, a telomerase-immortalized RPE cell line derived from a 1-year-old person, were commercially obtained and cultured as recommended. Primary RPE cell cultures were established using RPE isolated from 3- to 6-month-old pig and postnatal day 5 mouse. Cultured cells were transduced with a virus expressing neuroD, neurogenin1 (ngn1), or ngn3, basic helix-loop-helix (bHLH) genes previously identified as capable of inducing RPE-to-photoreceptor reprogramming in the chick system. Alternatively, cells in the culture were transfected chemically or physically through electroporation with vector DNA expressing one of the three genes. The cultures were then analyzed for RPE-to-photoreceptor reprogramming with in situ hybridization and/or immunostaining for photoreceptor gene expression. RESULTS: Both hTERT-RPE1 and ARPE-19 cultures gave rise to cells bearing markers of photoreceptors after transduction or transfection with vehicles expressing neuroD or ngn1. The new cells expressed genes encoding photoreceptor proteins, including interphotoreceptor retinoid-binding protein IRBP), recoverin, retinal cone arrestin 3, transducin α-subunit, Cone-rod homeobox protein (Crx), and red opsin. They displayed morphologies resembling differentiating photoreceptor cells. In primary porcine and mouse RPE cell cultures, transduction with lenti virus (Lvx-IRES-ZsGreen1) expressing ngn1 or ngn3 resulted in the emergence of ZsGreen1+ cells that exhibited morphologies reminiscent of differentiating photoreceptor cells. Immunochemistry showed that some ZsGreen1+ cells were positive for neural marker microtubule-associated protein 2 (Map2) and photoreceptor hallmark proteins red opsin and rhodopsin. CONCLUSIONS: The results suggest that cells in human RPE cell lines and in primary cultures of porcine and mouse RPE respond to gene-induced reprogramming by giving rise to photoreceptor-like cells. The responsiveness of primary RPE cells, especially those from porcine cells, enhances the biologic feasibility of exploring RPE-to-photoreceptor reprogramming for in situ mammalian photoreceptor replacement without cell transplantation.

Reprogramming retinal pigment epithelium to differentiate toward retinal neurons with Sox2.

Guiding non-neural, retinal pigment epithelium (RPE) to produce retinal neurons may offer a source of developing neurons for cell-replacement. Sox2 plays important roles in maintaining neural progenitor/stem cell properties and in converting fibroblasts into pluripotent stem cells. This study tests the possibility of using Sox2 to reprogram RPE to differentiate toward retinal neurons in vivo and in vitro. Expression of Sox2 in the chick retina was detected in progenitor cells, in cells at a discrete location in the layers of amacrine and ganglion cells, and in Muller glia. Overexpression of Sox2 in the developing eye resulted in hypopigmentation of the RPE. In the affected regions, expression of retinal ganglion cell markers became apparent in the RPE layer. In RPE cell culture, Sox2 promoted the expression of retinal ganglion and amacrine markers, and suppressed the expression of genes associated with RPE properties. Mechanistic investigation using the developing retina revealed a coexpression of Sox2 and basic fibroblast growth factor (bFGF), a growth factor commonly used in stem cell culture and capable of inducing RPE-to-retina transdifferentiation (or reprogramming) during early development. Similar patterns of changes in Sox2 expression and in bFGF expression were observed in atrophic retina and in injured retina. In RPE cell culture, Sox2 and bFGF mutually enhanced one another's expression. Upregulation of bFGF expression by Sox2 also occurred in the retina. These results suggest that Sox2 can initiate a reprogramming of RPE cells to differentiate toward retinal neurons and may engage bFGF during the process.

Neurogenin3 promotes early retinal neurogenesis.

The transcriptional regulatory network governing the establishment of retinal neuron diversity is not well delineated. We report experimental results suggesting proneural gene neurogenin3 (ngn3) participating in this regulatory network. Retinal expression of chick ngn3 was confined to early neurogenesis. Overexpression of ngn3 in chick retina reduced cell proliferation and expanded the population of ganglion cells into the territory normally occupied by amacrine cells. Ngn3 overexpression altered the expression of a number of regulatory genes, including ash1, ath3, ath5, chx10, neuroD, ngn1, ngn2, and NSCL1. Early gene ngn1 was induced, but ash1, ngn2, ath3, and chx10, whose expressions persist through later phases of neurogenesis, were down-regulated. Expression of ath5 was up-regulated at the locale corresponding to young ganglion cells, but was down-regulated at the locale corresponding to progenitor cells. These results suggest that ngn3 regulates retinal neurogenesis by inducing regulatory genes for early-born neurons and repressing those for later-born cells.

Reprogramming chick RPE progeny cells to differentiate towards retinal neurons by ash1.

PURPOSE: Harnessing a cell culture of retinal pigment epithelium (RPE) to give rise to retinal neurons may offer a source of developing neurons for cell-replacement studies. This study explores the possibility of reprogramming RPE progeny cells to differentiate toward retinal neurons with achaete-scute homolog 1 (ash1), a proneural gene that is expressed in progenitor cells in the developing retina and promotes amacrine cell production when overexpressed in the chick retina. METHODS: Replication Competent Avian Splice (RCAS) retrovirus was used to drive the ectopic expression of ash1 in cell cultures of dissociated RPE isolated from day 6 chick embryos. RCAS expressing green fluorescent protein (RCAS-GFP) was used as control. The cultures were examined for de novo generation of neuron-like cells by molecular, cellular, and physiologic criteria. RESULTS: In control cultures infected with RCAS-GFP, RPE cells appeared cobblestone-like and often darkly pigmented. In cultures infected with RCAS-ash1, however, cells remained de-pigmented and frequently formed clusters. Further examination at the morphological and molecular levels showed the development of elaborate processes characteristic of neurons and the expression of genes/markers that identify different types of retinal neurons. The most prevalently expressed neural marker was calretinin, which in the chick retina identifies amacrine, ganglion, and horizontal cells. As an assay for functional maturation, the reprogrammed cells were analyzed for the presence of functional, ionotropic glutamate receptors that lead to a rise in the cytosolic free calcium (Ca(2+)) concentration. Calcium imaging showed that reprogrammed cells responded to glutamate and N-methyl-D-aspartate (NMDA) by increasing their Ca(2+) concentrations, which, after reaching a peak level, returned to the basal level. The response curves of reprogrammed cells resembled those of cultured retinal neurons. CONCLUSIONS: These results suggest that RPE progeny cells can be reprogrammed by ash1 to develop molecular, morphological, and physiologic properties that are characteristic of retinal neurons.

Exploring RPE as a source of photoreceptors: differentiation and integration of transdifferentiating cells grafted into embryonic chick eyes.

PURPOSE: To study the possibility of generating photoreceptors through programming RPE transdifferentiation by examining cell differentiation after transplantation into the developing chick eye. METHODS: RPE was isolated, and the cells were dissociated, cultured, and guided to transdifferentiate by infection with retrovirus expressing neuroD (RCAS-neuroD), using RCAS-green fluorescence protein (GFP) as a control. The cells were then harvested and microinjected into the developing eyes of day 5 to day 7 chick embryos, and their development and integration were analyzed. RESULTS: Cells from the control culture integrated into the host RPE. When grafted cells were present in large number, multilayered RPE-like tissues were formed, and the extra tissues consisted of grafted cells and host cells. None of the cells from the control culture expressed photoreceptor-specific genes. In contrast, most cells from RCAS-neuroD-infected culture remained depigmented. A large number of them expressed photoreceptor-specific genes, such as visinin and opsins. Antibodies against red opsin decorated the apical tips and the cell bodies of the grafted, transdifferentiating cells. In the subretinal space, visinin(+) cells aligned along the RPE or an RPE-like structure. When integrated into the host outer nuclear layer, grafted cells emanated elaborate, axonal arborization into the outer plexiform layer of the host retina. CONCLUSIONS: Cultured RPE cells retained their remarkable regenerative capabilities. Cells guided to transdifferentiate along the photoreceptor pathway by neuroD developed a highly ordered cellular structure and could integrate into the outer nuclear layer. These data suggest that, through genetic programming, RPE cells could be a potential source of photoreceptor cells.

A role of ath5 in inducing neuroD and the photoreceptor pathway.

Photoreceptors in the vertebrate retina are light-sensitive neurons, and their degeneration results in irreversible visual loss. Understanding how photoreceptor fate is determined is a prerequisite for developing photoreceptor replacement therapies. Previous studies identified two basic helix-loop-helix genes, neurogenin2 (ngn2) and neuroD, participating in a genetic pathway leading to photoreceptor genesis. Here we present experimental data suggesting that ath5, which is known for its critical role in retinal ganglion cell development, may also lead to photoreceptor production. In the developing retina, ath5 expression was detected in two zones of cells, and coexpression with neuroD was observed in the zone adjacent to young photoreceptor cells accumulating on the retinal pigment epithelial side. Retroviral-driven misexpression of ath5 in retinal cells increased the population of photoreceptor cells, as well as ganglion cells, in a developmental stage-dependent manner that is consistent with ath5 being involved in the development of multiple types of retinal neurons. Ectopic ath5 expression in cultures of non-neural retinal pigment epithelial cells elicited transdifferentiation into cells that expressed photoreceptor-specific genes and displayed photoreceptor-like morphologies. Gene expression analysis showed that ngn2 did not induce ath5, and ath5 did not induce ngn2, but both induced neuroD and RaxL. These data suggest a pathway of "ath5 --> neuroD --> photoreceptor genes" separate from yet convergent with the ngn2 pathway.

bHLH genes and retinal cell fate specification.

The various cell types in the vertebrate retina arise from a pool of common progenitors. The way that the cell types are specified has been a long-standing issue. Decades of research have yielded a large body of information regarding the involvement of extrinsic factors, and only recently has the function of intrinsic factors begun to emerge. This article reviews recent studies addressing the role of basic helix-loop-helix (bHLH) factors in specifying retinal cell types, with an emphasis on bHLHhierarchies leading to photoreceptor production. Photoreceptor genesis appears to employ two transcriptional pathways: ngn2-->neuroD-->raxL and ath5-->neuroD-->raxL. ngn2 and ath5 function in progenitors, which can potentially develop into different cell types. neuroD represents one of the central steps in photoreceptor specification. Ath5 is also essential for ganglion cell development. It remains to be demonstrated whether a bHLH gene functions as a key player in specifying the other types of retinal cells. Genetic knockout studies have indicated intricate cross-regulation among bHLH genes. Future studies are expected to unveil the mechanism by which bHLH factors network with intrinsic factors and communicate with extrinsic factors to ensure a balanced production of the various types of retinal cells.

Chick homeobox gene cbx and its role in retinal development.

Homeobox genes play important roles in animal development. We isolated a chick homeobox gene, cbx, and studied its function during embryonic development. The deduced Cbx protein contained 376 amino acid residues. Its homeodomain was related (with 65-71% sequence identity) to that of human Crx, human Cart-1, and chick Alx-4. On searching the human genome sequence, a human homologue was found, which had 78% overall sequence identity and a 100% identical homeodomain. In the developing chick retina, cbx was expressed in a small fraction of post-mitotic cells residing at anatomical locations typical of bipolar cells. These cells were Goalpha(+) and protein kinase C(-), suggesting that they were probably cone bipolar cells. cbx mRNA was also detected outside the retina, particularly in the tectum and Rathke's pouch. Replication-competent retrovirus was used to drive misexpression of cbx and of an Engrailed repression construct. Engrailed-mediated repression of Cbx was embryonic lethal, while misexpression of cbx itself was tolerated. In the retina, misexpression of cbx resulted in fewer PKC(+) bipolar cells. Our data suggest that cbx is essential for embryonic survival and may participate in the development of bipolar, probably cone bipolar, cells in the retina.

Induction of ectopic retina-like tissue by transgenic expression of neurogenin.

Degeneration of retinal neurons is an underlying cause of several major types of blinding diseases, and effective therapies remain to be developed. The suppositive strategy of repopulating a degenerative retina with new cells generated onsite faces serious challenges, because the mammalian retina seems to lack the ability to regenerate itself or replace its lost neurons. We investigated the possibility of using a transcriptional factor with proneural activities to reprogram ocular tissue with regenerative capability to give rise to retinal cells. Transgenic mice were generated with DNA constructs that targeted the expression in the retinal pigment epithelium of proneural gene neurogenin1 from the promoter of Bestrophin1, or neurogenin3 from RPE65 promoter. Here we report the presence of ectopic retina-like tissue in some of the transgenic mice, young and aged. The ectopic retina-like tissue contained cells positive for photoreceptor proteins Crx, recoverin, red opsin, and rhodopsin, and cells positive for proteins that label other types of retinal neurons, including AP2α and Pax6 for amacrine cells, Otx2 for bipolar cells, and Brn3A for ganglion cells. The retina-like tissue often co-existed with darkly pigmented tissue positive for RPE proteins: cytokeratin 18, Otx2, and RPE65. The ectopic retina-like tissue was detected in the subretinal space, including two retinae co-existing in the same eye, and/or in the optic nerve or in the vicinity of the optic nerve head. On rare occasions, it was detected in the choroid and in the vicinity of the ciliary body. The presence of ectopic retina-like tissue in the transgenic mouse supports the possibility of inducing retinal regeneration in the mammalian eyes through gene-directed reprograming.

Requirement of neuroD for photoreceptor formation in the chick retina.

PURPOSE: The genetic control of photoreceptor cell fate in the vertebrate retina is poorly understood. Published studies suggest that the genetic program underlying photoreceptor production involves neuroD, a proneural basic helix-loop-helix (bHLH) gene. The present study investigates whether neuroD is necessary for photoreceptor cell development, by using loss-of-function analyses. METHOD: Engrailed-mediated active repression, antisense oligonucleotides, and small interfering RNA (siRNA) were used to attenuate neuroD expression and function in embryonic chick retina. The development of the retina was subsequently analyzed to determine whether these experimental manipulations would yield photoreceptor deficits in otherwise normal retina. RESULTS: Chick embryos infected with retroviruses expressing an active repression construct, En-NeuroDDeltaC, exhibited severe photoreceptor deficits. The outer nuclear layer (ONL) of the retina was no longer a contiguous structure, but became fragmented with regions that contained fewer or no photoreceptor cells. Photoreceptor deficiency was evident even before the retina became laminated, suggesting that active repression of NeuroD may have affected photoreceptor genesis. No deficiency was observed in other types of retinal cells. Culturing retinal cells in the presence of siRNA against neuroD resulted in a more than 50% reduction in the number of photoreceptor cells and an increase in the number of chx10+ cells. Subjecting the developing retina to antisense oligonucleotides against neuroD yielded fewer photoreceptor cells both in vivo and in vitro. Consistent with these observations, anti-NeuroD antibody specifically labeled the nuclei of the ONL. CONCLUSIONS: The data suggest a specific and an essential role of neuroD in photoreceptor formation in the chick retina.

Enhanced retinal ganglion cell differentiation by ath5 and NSCL1 coexpression.

PURPOSE: The molecular mechanism underlying retinal ganglion cell (RGC) differentiation is not fully understood. In this study, the role of the basic helix-loop-helix (bHLH) genes ath5 and NSCL1 in RGC differentiation was examined, by testing whether their coexpression would promote RGC differentiation to a greater extent than either gene alone. METHODS: The replication-competent avian RCAS retrovirus was used to coexpress ath5 and NSCL1 through an internal ribosomal entry site. The effect of the coexpression on RGC differentiation was assayed in vivo in the developing chick retina and in vitro in RPE cell cultures derived from day 6 chick embryos. RESULTS: Coexpression of ath5 and NSCL1 in RPE cells cultured in the presence of bFGF promoted RPE transdifferentiation toward RGCs, and the degree of transdifferentiation was much higher than with either gene alone. Cells expressing RGC markers, including RA4, calretinin, and two neurofilament-associated proteins, displayed processes that were remarkably long and thin and often had numerous branches, characteristics of long-projecting RGCs. In the developing chick retina, retroviral expression of NSCL1 resulted in a moderate increase in the number of RGCs, results similar to retroviral expression of ath5. Coexpression of ath5 and NSCL1 yielded increases in RGCs greater than the sum of their increases when expressed separately. CONCLUSIONS: Both in vitro and in vivo data indicate that the combination of ath5 and NSCL1 promotes RGC differentiation to a greater degree than either gene alone, suggesting a synergism between ath5 and NSCL1 in advancing RGC development.

Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii ("Clostridium butylicum").

Clostridium beijerinckii ("Clostridium butylicum") NRRL B592 and NRRL B593 were grown in batch cultures without pH control. The use of more sensitive and accurate procedures for the determination of solvents in cultures led to the recognition of the onset of solvent production about 2 h earlier than the previously assigned point and at a higher culture pH for both strains. Reliable assays for solvent-forming enzyme activities in cell extracts have also been developed. The results showed that activities of solvent-forming enzymes in strain NRRL B592 started to increase about 1 h before the measured onset of solvent production and that the increase in activities of solvent-forming enzymes was not simultaneous. The degree of increase of these enzyme activities for both strains ranged from 2- to 165-fold, with acetoacetate decarboxylase and butanol-isopropanol dehydrogenase showing the largest activity increases. However, the pattern of increase of enzyme activities differed significantly in the two strains of C. beijerinckii. When an increase in solvent-forming enzyme activities was first detected in strain NRRL B592, the culture pH was at 5.7 and the concentrations of total acetic and butyric acids were 5.2 and 3.6 mM, respectively. For strain NRRL B593, the corresponding pH was 5.5. Thus, the culture conditions immediately preceding the expression of solvent-forming enzyme activities differed significantly from those that have been correlated with the production of solvents at later stages of growth.

The Retinal Pigment Epithelium: a Convenient Source of New Photoreceptor cells?

Recent success in restoring visual function through photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to generate or regenerate photoreceptor cells for the ultimate goal of using cell replacement therapy for blindness caused by photoreceptor degeneration. Current research on deriving new photoreceptors for replacement, as regenerative medicine in general, focuses on the use of embryonic stem cells and induced pluripotent stem (iPS) cells to generate transplantable cells. Nonetheless, naturally occurring regeneration, such as wound healing, involves awakening cells at or near a wound site to produce new cells needed to heal the wound. Here we discuss the possibility of tweaking an ocular tissue, the retinal pigment epithelium (RPE), to produce photoreceptor cells in situ in the eye. Unlike the neural retina, the RPE in adult mammals maintains cell proliferation capability. Furthermore, progeny cells from RPE proliferation may differentiate into cells other than RPE. The combination of proliferation and plasticity opens a question of whether they could be channeled by a regulatory gene with pro-photoreceptor activity towards photoreceptor production. Studies using embryonic chick and transgenic mouse showed that indeed photoreceptor-like cells were produced in culture and in vivo in the eye using gene-directed reprogramming of RPE cells, supporting the feasibility of using the RPE as a convenient source of new photoreceptor cells for in situ retinal repair without involving cell transplantation.

Photoreceptor-like cells in transgenic mouse eye.

PURPOSE: Recent success of rescuing vision by photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to identify approaches to generate photoreceptors cells for future replacement therapies. We explored the possibility of whether in the mouse eye photoreceptor-like cells could arise from the RPE experimentally manipulated to express a regulatory gene participating in transcriptional networks leading to photoreceptor genesis during retinal development. METHODS: Transgenic mice were generated with a DNA construct that would express neurogenin1 from RPE bestrophin-1 promoter or neurogenin3 from RPE65 promoter. Transgenic mice were examined with histology and immunohistology for the presence of photoreceptor-like cells and for the presence of cells that might represent transitional stages in RPE-to-photoreceptor reprogramming. Explant culture of "sclera+choroid+RPE" eyecup was used to examine whether cells with photoreceptor traits could arise from the eyecup derived from transgenic mice. RESULTS: Transgenic animals showed varied degrees of phenotype manifestation. Approximately 60% of offspring from ∼50% of founders contained photoreceptor-like cells in the subretinal space. These cells expressed photoreceptor proteins recoverin, red opsin, and rhodopsin, and displayed morphologic similarities to photoreceptors. In these eyes, the RPE was maintained. Cells seemingly amid RPE-to-photoreceptor transformation were observed in young and aged mice, suggesting old animals were responsive to the reprogramming scheme. De novo generation of photoreceptor-like cells was detected in "sclera+choroid+RPE" eyecup explants derived from adult animals. CONCLUSIONS: Our results point to a potential way to generate photoreceptor cells in situ in adult mammalian eyes.

Production of high-titer RCAS retrovirus.

RCAS (B/P) is a replication-competent avian retrovirus engineered by Hughes et al. (J Virol 61:3004-3012, 1987) and is referred to in this chapter as RCAS for simplicity. The RCAS retrovirus has been widely used as a vehicle for stable transduction of a gene into cells both in the developing chick embryo and tissue/cell culture. It can be used for both gain- and loss-function experiments. The ability of this virus to spread among proliferating cells makes it possible to achieve widespread gene transduction in the developing retina. The transduction efficiency of RCAS is highly depending on the titer of the viral stock, particularly for experiments involving solid tissues such as the developing retina. Here, we describe the procedure that we have used for 15 years to generate RCAS viral stocks with a titer of 1-5 × 10(8) pfu/ml.

Chick retinal pigment epithelium transdifferentiation assay for proneural activities.

We describe a cell culture system for assaying proneural activities of genes hypothesized to play instrumental roles in neuronal fate specification during vertebrate retinal development. The retinal pigment epithelium (RPE) is collected from embryonic day 6 (E6) chick to establish a primary RPE cell culture. The culture is then infected with a replication competent retrovirus RCAS expressing the gene of interest. The presence of retinal neurons in the otherwise nonneural, RPE cell culture is examined between 4 and 10 days after the administration of the virus. Taking advantage of the plasticity and the relative simplicity of RPE cells, this method offers an informative assay for proneural activities prior to planning for large-scale in vivo experiments.

Using neurogenin to reprogram chick RPE to produce photoreceptor-like neurons.

PURPOSE: One potential therapy for vision loss from photoreceptor degeneration is cell replacement, but this approach presents a need for photoreceptor cells. This study explores whether the retinal pigment epithelium (RPE) could be a convenient source of developing photoreceptors. METHODS: The RPE of chick embryos was subjected to reprogramming by proneural genes neurogenin (ngn)1 and ngn3. The genes were introduced into the RPE through retrovirus RCAS-mediated transduction, with the virus microinjected into the eye or added to retinal pigment epithelial explant culture. The retinal pigment epithelia were then analyzed for photoreceptor traits. RESULTS: In chick embryos infected with retrovirus RCAS-expressing ngn3 (RCAS-ngn3), the photoreceptor gene visinin (the equivalent of mammalian recoverin) was expressed in cells of the retinal pigment epithelial layer. When isolated and cultured as explants, retinal pigment epithelial tissues from embryos infected with RCAS-ngn3 or RCAS-ngn1 gave rise to layers of visinin-positive cells. These reprogrammed cells expressed genes of phototransduction and synapses, such as red opsin, the alpha-subunit of cone transducin, SNAP-25, and PSD-95. Reprogramming occurred with retinal pigment epithelial explants derived from virally infected embryos and with retinal pigment epithelial explants derived from normal embryos, with the recombinant viruses added at the onset of the explant culture. In addition, reprogramming took place in retinal pigment epithelial explants from both young and old embryos, from embryonic day (E)6 to E18, when the visual system becomes functional in the chick. CONCLUSIONS: The results support the prospect of exploring the RPE as a convenient source of developing photoreceptors for in situ cell replacement.

Generating retinal neurons by reprogramming retinal pigment epithelial cells.

IMPORTANCE OF THE FIELD: Retinal degenerations cause blindness. One potential therapy is cell replacement. Because the human retina lacks regeneration capacity, much attention has been directed towards searching for cells that can differentiate into retinal neurons. AREAS COVERED IN THIS REVIEW: We discuss the possibility of using transcription factor genes to channel retinal pigment epithelial (RPE) cells' capabilities of proliferation and plasticity towards the production of retinal neurons. WHAT THE READER WILL GAIN: Experiments with chick embryos show that RPE cells - in the eye, in explant, or in a dissociated cell culture - can give rise to cells resembling retinal neurons when reprogrammed with regulatory genes involved in retinal neurogenesis. Depending on the regulatory gene used, reprogramming generates cells exhibiting traits of photoreceptor cells, amacrine cells and/or young ganglion neurons. TAKE HOME MESSAGE: Gene-directed reprogramming of chick RPE can efficiently generate cells that exhibit traits of retinal neurons. Remaining to be addressed is the question of whether the results from chicks apply to mammals. Since the RPE is located adjacent to the neural retina, RPE reprogramming, if successful in mammals, may offer an approach to repopulate the neural retina without involving cell transplantation.

Neurogenin1 effectively reprograms cultured chick retinal pigment epithelial cells to differentiate toward photoreceptors.

Photoreceptors are highly specialized sensory neurons in the retina, and their degeneration results in blindness. Replacement with developing photoreceptor cells promises to be an effective therapy, but it requires a supply of new photoreceptors, because the neural retina in human eyes lacks regeneration capability. We report efficient generation of differentiating, photoreceptor-like neurons from chick retinal pigment epithelial (RPE) cells propagated in culture through reprogramming with neurogenin1 (ngn1). In reprogrammed culture, a large number of the cells (85.0% +/- 5.9%) began to differentiate toward photoreceptors. Reprogrammed cells expressed transcription factors that set in motion photoreceptor differentiation, including Crx, Nr2E3, NeuroD, and RXRgamma, and phototransduction pathway components, including transducin, cGMP-gated channel, and red opsin of cone photoreceptors (equivalent to rhodopsin of rod photoreceptors). They developed inner segments rich in mitochondria. Furthermore, they responded to light by decreasing their cellular free calcium (Ca(2+)) levels and responded to 9-cis-retinal by increasing their Ca(2+) levels after photobleaching, hallmarks of photoreceptor physiology. The high efficiency and the advanced photoreceptor differentiation indicate ngn1 as a gene of choice to reprogram RPE progeny cells to differentiate into photoreceptor neurons in future cell replacement studies.

Proneural gene ash1 promotes amacrine cell production in the chick retina.

The diverse types of neurons and Müller glia in the vertebrate retina are believed to arise from common progenitor cells. To better understand how neural diversity is achieved during retinal neurogenesis, we examined the function of ash1, a proneural bHLH gene expressed in progenitor cells throughout retinal neurogenesis. Published studies using retinal explant culture derived from knockout mice concluded that ash1 is required for the production of late-born neurons, including bipolar cells. In this study, gain-of-function experiments were carried out in ovo in embryonic chick retina. In the developing chick retina, expression of ash1 temporally overlapped with, but spatially differed from, the expression of ngn2, also a proneural gene expressed in progenitor cells throughout retinal neurogenesis. Retrovirus-driven overexpression of ash1 in the developing chick retina decreased the progenitor population (BrdU+ or expressing ngn2), expanded the amacrine population (AP2alpha+ or Pax6+), and reduced bipolar (chx10 mRNA+) and Müller glial (vimentin+) populations. Photoreceptor deficiency occurred after the completion of neurogenesis. The number of ganglion cells, which are born first during retinal neurogenesis, remained unchanged. Similar overexpression of ngn2 did not produce discernible changes in retinal neurogenesis, nor in ash1 expression. These results suggest that ash1 promotes the production of amacrine cells and thus may participate in a regulatory network governing neural diversity in the chick retina.