Expression Of Crx Research Articles

Pigment epithelium‐derived factor induced CRX alterations in the mouse retina

The gene Serpinf1 encodes for Pigment Epithelium‐Derived Factor (PEDF), which protects photoreceptors from cell death. The Serpinf1 null mouse exhibits normal retinal function; however, PEDF deficiency increases retinal degeneration susceptibility in rd10 mouse. CRX is a transcription factor that regulates expression of several photoreceptor specific genes, including opsins and phosphodiesterase. This study aims to explore whether regulation of CRX activation is one mechanism by which PEDF exerts photoreceptor survival.Serpinf1‐/‐, Serpinf1+/‐ and Serpinf1+/+ mice at 3 months of age were used. We prepared cultures of mouse retinal explants. They were treated with recombinant human PEDF. Zaprinast (a PDE inhibitor) was added to induce photoreceptor death. We determined cell death using PSVue‐550, a visible fluorescent probe for detection of phosphatidylserine on the surface of cells. Subcellular distribution of CRX, PDE6A and pan‐acetylation was detected by immunofluorescence. The transcriptional levels of Crx, Pde6a, Opn1mw and Opn1sw were assessed using qPCR.PEDF treatment induced the expression of Crx and its regulated genes Pde6a, Opn1mw and Opn1sw. PEDF also enhanced the CRX immunoreactivity in the nuclei of photoreceptors. In contrast, the nuclear CRX immunoreactivity was suppressed in photoreceptors from retinas of Serpinf1‐/‐ mice deficient of PEDF. PDE6A immunoreactivity also decreased in Serpinf1‐/‐ photoreceptors compared to those from Serpinf1+/+ mice. Histone acetylation was detected in discrete photoreceptors in Serpinf1+/+ but was undetectable in the Serpinf1‐/‐ retinas. Zaprinast‐induced photoreceptor death was more pronounced in Serpinf1‐/‐ retinal explants than in wild type controls. PEDF pre‐treatment prior to the zaprinast treatment improved photoreceptor survival and enhanced CRX immunoreactivity in both Serpinf1‐/‐ and Serpinf1+/+ retinal explants. Deletion in PEDF in the Serpinf1 null mice accelerated retinal degeneration induced by zaprinast, which was prevented by exogenous addition of PEDF in retinal explants.The findings imply that PEDF activated the CRX‐associated transcription factor network. They provide a novel insight linking extracellular PEDF to nuclear CRX during photoreceptor survival.

Interaction of human CRX and NRL in live HEK293T cells measured using fluorescence resonance energy transfer (FRET)

CRX and NRL are retina-specific transcription factors that control rod photoreceptor differentiation and synergistically activate rod phototransduction gene expression. Previous experiments showed they interact in vitro and in yeast two-hybrid assays. Here, we examined CRX-NRL interaction in live HEK293T cells using two fluorescence resonance energy transfer (FRET) approaches: confocal microscopy and flow cytometry (FC-FRET). FC-FRET can provide measurements from many cells having wide donor–acceptor expression ranges. FRET efficiencies were calibrated with a series of donor (EGFP)-acceptor (mCherry) fusion proteins separated with linkers between 6–45 amino acids. CRX and NRL were fused at either terminus with EGFP or mCherry to create fluorescent proteins, and all combinations were tested in transiently transfected cells. FRET signals between CRX or NRL homo-pairs were highest with both fluorophores fused to the DNA binding domains (DBD), lower with both fused to the activation domains (AD), and not significant when fused on opposite termini. NRL had stronger FRET signals than CRX. A significant FRET signal between CRX and NRL hetero-pairs was detected when donor was fused to the CRX DNA binding domain and the acceptor fused to the NRL activation domain. FRET signals increased with CRX or NRL expression levels at a rate much higher than expected for collisional FRET alone. Together, our results show the formation of CRX-NRL complexes in live HEK293T cells that are close enough for FRET.

Downregulation of CRX, a Group 3-specific oncogenic transcription factor, inhibits TGF-β/activin signaling in medulloblastoma cells

Medulloblastoma, the most common malignant brain tumor in children, consists of four molecular subgroups WNT, SHH, Group 3, and Group 4. Group 3 has the worst survival rate among the four subgroups and is characterized by the expression of retina-specific genes. CRX, the master regulator of the photoreceptor differentiation, is aberrantly expressed in Group 3 medulloblastomas. CRX expression increased the proliferation, anchorage-independent growth, invasion potential, and tumorigenicity of medulloblastoma cells indicating the oncogenic role of CRX in medulloblastoma pathogenesis. CRX knockdown resulted in the downregulation of expression of several retina-specific genes like IMPG2, PDC, RCVRN. and Group 3 specific genes like GABRA5, MYC, PROM1. Thus, CRX plays a major role not only in the expression of retina-specific genes but also in defining Group 3 identity. Increased expression of several pro-apoptotic genes upon CRX knockdown suggests that CRX could protect Group 3 medulloblastoma cells from cell death. Several negative regulators of the TGF-β signaling pathway like SMAD7, PMEPA1, KLF2 were upregulated upon the CRX knockdown. Western blot analysis showed a decrease in the levels of (Phospho)-SMAD2, total levels of SMAD2, SMAD4, and an increase in the levels of SMAD7 indicating inhibition of the TGF-β signaling pathway upon CRX knockdown. Copy number variations in several genes involved in the TGF-β signaling pathway occur in a subset of Group 3 tumors. Autocrine TGF-β/activin signaling has recently been reported to be active in a subset of Group 3 medulloblastomas. CRX knockdown resulting in the inhibition of the TGF-β/activin signaling pathway demonstrates an interaction between the two Group 3 specific oncogenic pathways and suggests simultaneous targeting of both CRX and TGF-β signaling as a possible therapeutic strategy.

Change of transcription level of photoreceptor-specific CRX gene in the peripheral blood of the participants of an arctic world oceanic international flight

To reveal the dynamics of CRX expression level in humans after a prolonged temporary exposure to polar day conditions. The study included 6 pilots (males from 39 to 69 y.o.) who participated in the Arctic World Oceanic International Flight Sever Vash (West to East, from 62°N 74°E to 72°N 114°E). Samples of peripheral blood for RNA isolation were collected at the start and at the end of the flight. The level of mRNA in the samples was evaluated based on the data of quantitative real-time PCR of the CRX gene, as well as the b2M and TBP housekeeping genes (reference). Expression of the CRX gene in the studied group (p<0.01) was revealed; the total average level of mRNA was about 3 times higher prior to, and approximately 7 times higher after normalization to the b2M gene. Five pilots had increased expression of the CRX gene within the range of -1.53 to -3.07 (Z-score of <0 before the flight and >0 after the flight). In one pilot, the level of CRX expression was higher at the start, but it reduced by the end of the circumnavigation flight. The results confirm the hypothesis that the CRX gene is expressed after a prolonged temporary exposure to polar day conditions. It was also revealed that during rapid adaptation, equal changes in the illumination of retinal photoreceptors lead to different individual dynamics of CRX expression.

Crx Is Posttranscriptionally Regulated by Light Stimulation in Postnatal Rat Retina.

Cone rod homeobox (Crx) plays a key role at the center of a regulatory network that coordinates many pathways in the retina. Its abnormal expression induces retinal disorders. However, the underlying regulatory mechanism of Crx expression is not well defined. Here, we present data that show that the levels of Crx mRNA were inconsistent with that of Crx protein in primary retinal neurocytes cultured in light conditions. Crx protein levels were significantly higher (2.56-fold) in cells cultured in the dark than in cells cultured in light, whereas Crx mRNA was not changed in either type of cell. Moreover, the expression of Crx protein showed a significant light intensity-dependent decrease. Consistently, Crx downstream genes rhodopsin and arrestin also decreased in retinal neurocytes upon light exposure. Furthermore, Crx promoter activity assay performed in primary retinal neurocytes further indicated that light exposure and darkness did not affect its inducibility. In addition, the inconsistency between Crx mRNA and protein expression after light exposure was not observed in 661w cells transfected with plasmid pcDNA3.1-Crx, suggesting that the inconsistency between Crx mRNA and protein induced by light was specific to the endogenous Crx. More importantly, this observation was confirmed in vivo in postnatal day 15 (P15) retinas but not in adult retinas, further implicating that the posttranscriptional regulation mechanism may be involved in Crx expression in the developing retina. Therefore, our study sheds light on the mechanism of Crx expression in postnatal rat retina.

Overexpression of MiR-183/96/182 Triggers Retina-Like Fate in Human Bone Marrow-Derived Mesenchymal Stem Cells (hBMSCs) in Culture.

Retinal degeneration is considered as a condition ensued by different blinding disorders such as retinitis pigmentosa, age-related macular degeneration, and diabetic retinopathy, which can cause loss of photoreceptor cells and also lead to significant vision deficiencies. Although there is no efficient treatment in this domain, transplantation of stem cells has been regarded as a therapeutic approach for retinal degeneration. Thus, the purpose of this study was to analyze the potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) to differentiate into photoreceptor cells via transfection of microRNA (miRNA) in vitro for regenerative medicine purposes. To this end, miR-183/96/182 cluster was transfected into hBMSCs; then, qRT-PCR was performed to measure the expression levels of miR-183/96/182 cluster and some retina-specific neuronal genes such as OTX2, NRL, PKCα, and recoverin. CRX and rhodopsin (RHO) levels were also measured through qRT-PCR and immunocytochemistry, and subsequently, cellular change morphology was detected. The findings showed no changes in the morphology of the given cells, and the expression of the neuroretinal genes such as OTX2, NRL, and PKCα. Moreover, recoverin was upregulated upon miR-183/-96/-182 overexpression in cultured hBMSCs. Ectopic overexpression of the miR-183 cluster could further increase the expression of CRX and RHO at the messenger RNA (mRNA) and protein levels. Furthermore, the data indicated that the miR-183 cluster could serve as a crucial function in photoreceptor cell differentiation. In fact, miRNAs could be assumed as potential targets to exploit silent neuronal differentiation. Ultimately, it was suggested that in vitro overexpression of miR-183 cluster could trigger reprogramming of the hBMSCs to retinal neuron fate, especially photoreceptor cells.

Eye Degeneration and Loss of otx5b Expression in the Cavefish Sinocyclocheilus tileihornes

Cave animals possess remarkable phenotypes associated with existence in their dark environments. The Chinese cavefish Sinocyclocheilus tileihornes shows substantial eye degeneration, a trait shared by most cave species. The extent to which independent evolution of troglomorphic traits uses convergent molecular genetic mechanisms is as yet unknown. We performed transcriptome-wide gene expression profiling in S. tileihornes eyes and compared results with those from the closely related surface species S. angustiporus and an independently derived congeneric cavefish, S. anophthalmus. In total, 52.85 million 100 bp long paired-end clean reads were generated for S. tileihornes, and we identified differentially expressed genes between the three possible pairs of species. Functional analysis of genes differentially expressed between S. tileihornes and S. angustiporus revealed that phototransduction (KEGG id: dre04744) was the most significantly enriched pathway, indicating the obvious differences in response to captured photons between the cavefish S. tileihornes and the surface species S. angustiporus. Analysis of key genes regulating eye development showed complete absence of otx5b (orthodenticle homolog 5) expression in S. tileihornes eyes, probably related to degradation of rods, but normal expression of crx (cone-rod homeobox). The enriched pathways and Otx5 are involved in phototransduction, photoreceptor formation, and regulation of photoreceptor-related gene expression. Unlike the S. tileihornes reported here, S. anophthalmus has reduced crx and otx5 expression. These results show that different species of cavefish within the same genus that independently evolved troglodyte characteristics can have different genetic mechanisms of eye degeneration.

Simvastatin protects photoreceptors from oxidative stress induced by all-trans-retinal, through the up-regulation of interphotoreceptor retinoid binding protein.

Simvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor with multiple targets and effects. It protects neurons in the brain, but its protective effects on photoreceptors are unclear. In this study, we evaluated the neuroprotective effect of simvastatin on photoreceptors exposed to stress induced by all-trans-retinal (atRAL). AlamarBlue and LDH assays were used to evaluate the viability and metabolic activity of Y79 cells (a retinoblastoma cell line) exposed to atRAL-induced stress with or without simvastatin pretreatment. Changes in cellular ROS were evaluated using flow cytometry and mitochondrial stress markers JC-1 and HSP60. Changes in levels of two photoreceptor-specific markers, cone-rod homeobox protein (CRX) and interphotoreceptor retinoid binding protein (IRBP), were evaluated with western blot. The results were validated in ex vivo human retinal explants and a mouse model of photoreceptor degeneration. Simvastatin improved mitochondrial function, alleviated oxidative stress and up-regulated the photoreceptor-specific markers IRBP and its upstream regulator CRX in Y79 cells and ex vivo human retinal explants under atRAL-induced stress. Simvastatin attenuated photoreceptor degeneration in association with up-regulation of IRBP and CRX expression after knockdown of IRBP in a murine model. Our findings suggest that simvastatin has a novel role in protecting photoreceptors from atRAL-induced stress. Simvastatin treatment resulted in up-regulation of IRBP and its upstream transcription factor CRX in Y79 cells, ex vivo human retinal explants, and murine retinas in vivo. Further studies of simvastatin to treat photoreceptor degeneration are warranted.

CRX Expression in Pluripotent Stem Cell-Derived Photoreceptors Marks a Transplantable Subpopulation of Early Cones.

Death of photoreceptors is a common cause of age‐related and inherited retinal dystrophies, and thus their replenishment from renewable stem cell sources is a highly desirable therapeutic goal. Human pluripotent stem cells provide a useful cell source in view of their limitless self‐renewal capacity and potential to not only differentiate into cells of the retina but also self‐organize into tissue with structure akin to the human retina as part of three‐dimensional retinal organoids. Photoreceptor precursors have been isolated from differentiating human pluripotent stem cells through application of cell surface markers or fluorescent reporter approaches and shown to have a similar transcriptome to fetal photoreceptors. In this study, we investigated the transcriptional profile of CRX‐expressing photoreceptor precursors derived from human pluripotent stem cells and their engraftment capacity in an animal model of retinitis pigmentosa (Pde6brd1), which is characterized by rapid photoreceptor degeneration. Single cell RNA‐Seq analysis revealed the presence of a dominant cell cluster comprising 72% of the cells, which displayed the hallmarks of early cone photoreceptor expression. When transplanted subretinally into the Pde6brd1 mice, the CRX+ cells settled next to the inner nuclear layer and made connections with the inner neurons of the host retina, and approximately one‐third of them expressed the pan cone marker, Arrestin 3, indicating further maturation upon integration into the host retina. Together, our data provide valuable molecular insights into the transcriptional profile of human pluripotent stem cells‐derived CRX+ photoreceptor precursors and indicate their usefulness as a source of transplantable cone photoreceptors. Stem Cells 2019;37:609–622

Exogenous CRX gene induces Müller cell-derived progenitors to differentiate into photoreceptors

Objective: To investigate whether exogenous CRX gene would be able to induce Müller cells-derived progenitors to differentiate into photoreceptors. Methods: Experimental study. Müller cells-derived progenitors resulted from primary Müller cells isolated from KunMing mice(5-7 days old) and cultured in free-serum media. Markers of Müller cells(glutamine synthetase, GS and Vimentin) and stem cells (Nestin and Sox2) were analysed by immnocytochemical assays. The secondary passage progenitors were divided into three groups: (1)the control group; (2)the empty vector group was transfected with lentivirus GFP; (3)the treated group was transfected with lentivirus GFP-CRX. After differentiation for 7 days, 7 days after differentiation, the expression of markers of photoreceptors were analyzed by q-PCR and Western blot assay. Results: There were 96.03%±1.21% of Müllerz cells cultured in vitro were immunoreactive to both GS and Vimentin. The dedifferentiation cells expressed Nestin and Sox2. After 7 days of induction, Exogenous CRX induced Müller cell-derived progenitors to differentiate into rod-like cells showed appearance like neuron morphology. q-PCR demonstrated that mRNAs of CRX and Rhodopsin were upregulated greatly. CRX mRNA were 9 times (P<0.05) and Rhodopsin mRNA were 20 times (P<0.05). The difference between the control group and the empty vector group was not statistically significant. Western blot showed that the expression of CRX was upregulated significantly, and was 2.7 times(P<0.05). But expression of Rhodopsin was weak and was nearly not detected in the control group and empty vector group. The expression of S-opsin was not detected. Conclusion: CRX gene could induce the differentiation of Müller cell-derived progenitor into rod photoreceptors, indicating a new avenue to study müller cells as endogenous seed cells for retinal photoreceptor. (Chin J Ophthalmol, 2018, 54: 923-928).

Loss of zebrafish Ataxin-7, a SAGA subunit responsible for SCA7 retinopathy, causes ocular coloboma and malformation of photoreceptors.

Polyglutamine (polyQ) expansion in Ataxin-7 (ATXN7) results in spinocerebellar ataxia type 7 (SCA7) and causes visual impairment. SCA7 photoreceptors progressively lose their outer segments (OSs), a structure essential for their visual function. ATXN7 is a subunit of the transcriptional coactivator Spt-Ada-Gcn5 Acetyltransferase complex, implicated in the development of the visual system in flies. To determine the function of ATXN7 in the vertebrate eye, we have inactivated ATXN7 in zebrafish. While ATXN7 depletion in flies led to gross retinal degeneration, in zebrafish, it primarily results in ocular coloboma, a structural malformation responsible for pediatric visual impairment in humans. ATXN7 inactivation leads to elevated Hedgehog signaling in the forebrain, causing an alteration of proximo-distal patterning of the optic vesicle during early eye development and coloboma. At later developmental stages, malformations of photoreceptors due to incomplete formation of their OSs are observed and correlate with altered expression of crx, a key transcription factor involved in the formation of photoreceptor OS. Therefore, we propose that a primary toxic effect of polyQ expansion is the alteration of ATXN7 function in the daily renewal of OS in SCA7. Together, our data indicate that ATXN7 plays an essential role in vertebrate eye morphogenesis and photoreceptor differentiation, and its loss of function may contribute to the development of human coloboma.

Bicistronic 2A-peptide-based co-expression reporter knock-in strategy by CRISPR/Cas9 system: application to the labeling of specific cell lineages and gene expression monitoring

Fluorescent cell labeling is used to identify the specific cell lineages in a tissue or a whole organism. Transgenic organisms with fluorescent reporter genes have been created to visualize specific cell lineages and to investigate cell specific morphologies, motilities, gene expressions, neural activities, intracellular signaling, etc. However, in human cells, transgenes are often silenced during cell differentiation, and so knock-in technology was adopted to label the specific human cell lineages, although the establishment of knock-in human pluripotent stem cells (hPSCs) required considerable efforts. Genome editing technology paved the way to more efficient and useful knock-in methods. Also, we applied a bicistronic 2A-peptide-based co-expression (B2AC) system to the knock-in strategy for the fluorescent cell labeling. By using these technologies, knock-in hPSC lines were established, and the expression of Crx, a specific photoreceptor marker, was revealed during three-dimensional retinal organoid culture. The Crx expression and fluorescent intensity in the cells were positively correlated, suggesting that the B2AC reporter system functioned during human retinal development. The immunohistochemistry of Crx and the maturation of fluorescent reporter cells after long-term differentiation culture indicated that knock-in of the reporter gene did not affect the function of the target Crx gene. B2AC reporter cells successfully represented Crx upregulation by DAPT, a Notch signal inhibitor, during retinal differentiation from hPSC. These results indicated that the B2AC reporter knock-in system could be used to investigate cell transplantation, developmental mechanisms, disease signaling, drug screening, and intracellular signaling.

Morphological and genetical changes of endothelial progenitor cells after in-vitro conversion into photoreceptors

BackgroundRetinal degeneration is a condition ensued by various ocular disorders such as artery occlusion, diabetic retinopathy, retrolental fibroplasia and retinitis pigmentosa which cause abnormal loss of photoreceptor cells and lead to eventual vision impairment. No efficient treatment has yet been found, however, the use of stem cell therapy such as bone marrow and embryonic stem cells has opened a new treatment modality for retinal degenerative diseases. The major goal of this study is to analyze the potential of endothelial progenitor cells derived from bone marrow to differentiate into retinal neural cells for regenerative medicine purposes. MethodsIn this study, endothelial progenitor cells were induced in-vitro with photoreceptor growth factor (taurine) for 21 days. Subsequently, the morphology and gene expression of CRX and RHO of the photoreceptors-induced EPCs were examined through immunostaining assay. FindingsThe results indicated that the induced endothelial progenitor cells demonstrated positive gene expression of CRX and RHO. Our findings suggested that EPC cells may have a high advantage in cell replacement therapy for treating eye disease, in addition to other neural diseases, and may be a suitable cell source in regenerative medicine for eye disorders.

Adult human periodontal ligament-derived stem cells delay retinal degeneration and maintain retinal function in RCS rats

BackgroundRetinal degeneration (RD) is a leading cause of irreversible blindness, affecting millions of people worldwide. Stem cell transplantation has been considered a promising therapy for retinal degenerative diseases. This study aimed to investigate the therapeutic potential of human periodontal ligament-derived stem cells (hPDLSCs) for intervention in the progress of this degeneration in the Royal College Surgeons (RCS) rat.MethodshPDLSCs were injected into the subretinal space of 3-week-old RCS rats. Control animals received a phosphate-buffered saline injection or were untreated. Retinal function was assessed by electroretinography recording. Eyes were collected afterward for histology and molecular studies.ResultsRetinal function maintenance was observed at 2 weeks and persisted for up to 8 weeks following hPDLSC transplantation. Retinal structure preservation was demonstrated in hPDLSC-transplanted eyes at 4 and 8 weeks following transplantation, as reflected in the preservation of outer nuclear layer thickness and gene expression of Rho, Crx, and Opsin. The percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic photoreceptors was significantly lower in the hPDLSC-injected retinas than in those of the control groups. hPDLSCs were also found to express multiple neurotrophic factors, including vascular endothelial growth factor, bioactive basic fibroblast growth factor, brain-derived neurotrophic factor, neurotrophin-3, insulin-like growth factor 1, nerve growth factor, and glial cell line-derived neurotrophic factor.ConclusionsOur findings suggest that hPDLSC transplantation is effective in delaying photoreceptor loss and provides significant preservation of retinal function in RCS rats. This study supports further exploration of hPDLSCs for treating RD.

Assessment of retinoblastoma RNA reflux after intravitreal injection of melphalan

BackgroundIntravitreal injection of chemotherapy in retinoblastoma eyes with vitreous seeds may lead to a risk of extraocular tumour dissemination that has not been assessed so far.AimsTo develop a sensitive and...

Pax6 is essential for the generation of late-born retinal neurons and for inhibition of photoreceptor-fate during late stages of retinogenesis

In the developing retina, as in other regions of the CNS, neural progenitors give rise to individual cell types during discrete temporal windows. Pax6 is expressed in retinal progenitor cells (RPCs) throughout the course of retinogenesis, and has been shown to be required during early retinogenesis for generation of most early-born cell types. In this study, we examined the function of Pax6 in postnatal mouse retinal development. We found that Pax6 is essential for the generation of late-born interneurons, while inhibiting photoreceptor differentiation. Generation of bipolar interneurons requires Pax6 expression in RPCs, while Pax6 is required for the generation of glycinergic, but not for GABAergic or non-GABAergic-non-glycinergic (nGnG) amacrine cell subtypes. In contrast, overexpression of either full-length Pax6 or its 5a isoform in RPCs induces formation of cells with nGnG amacrine features, and suppresses generation of other inner retinal cell types. Moreover, overexpression of both Pax6 variants prevents photoreceptor differentiation, most likely by inhibiting Crx expression. Taken together, these data show that Pax6 acts in RPCs to control differentiation of multiple late-born neuronal cell types.

Nuclear CRX and FOXJ1 Expression Differentiates Non-Germ Cell Pineal Region Tumors and Supports the Ependymal Differentiation of Papillary Tumor of the Pineal Region.

Papillary tumor of the pineal region (PTPR) is a neuroepithelial neoplasm first described in 2003. Despite the anatomic association of PTPR with the pineal gland, the features of these tumors resemble those of the ependymal circumventricular subcommissural organ (SCO) of the posterior third ventricle. Given the presumed distinct derivation of PTPR and pineal parenchymal tumors, we hypothesized that expression of lineage-specific transcription factors could distinguish these tumors and provide additional insight into the differentiation of PTPR. A broad series of pineal region samples was reviewed, including 7 benign pineal glands, 4 pineal cysts, 13 pineocytomas, 28 pineal parenchymal tumors of intermediate differentiation, 11 pineoblastomas, and 18 PTPR. All samples were evaluated by immunohistochemistry for expression of CRX, a master transcriptional regulator of photoreceptor differentiation expressed in pineal gland and retina and/or FOXJ1, a master transcriptional regulator of ciliogenesis expressed in normal ependymal cells and ependymal neoplasms. Diffuse nuclear CRX expression is present in 100% of pineal samples. FOXJ1 is negative in all pineal samples. CRX staining is present in 53% of PTPR, though expression is nearly always limited to rare cells. Diffuse nuclear FOXJ1 expression is present in 100% of PTPR. Fetal human SCO diffusely expressed FOXJ1 but was negative for CRX. Immunohistochemistry for FOXJ1 and CRX differentiates non-germ cell pineal region tumors with high sensitivity and specificity, including pineal parenchymal tumors and PTPR. Our findings support the hypothesis that PTPR have ependymal differentiation and are phenotypically more similar to SCO than pineal gland.

MicroRNA-28 potentially regulates the photoreceptor lineage commitment of M\xfcller glia-derived progenitors

Retinal degenerative diseases ultimately result into irreversible photoreceptor death or loss. At present, the most promising treatment for these diseases is cell replacement therapy. Müller glia are the major glia in the retina, displaying cardinal features of retinal progenitor cells, and can be candidate of seed cells for retinal degenerative diseases. Here, mouse retinal Müller glia dissociated and cultured in vitro amplified and were dedifferentiated into Müller glia-derived progenitors (MGDPs), demonstrating expression of stem/progenitor cell markers Nestin, Sox2 and self-renewal capacity. MicroRNAs (miRNAs) play unique roles in the retinogenesis, so we hypothesized miRNAs would contribute to photoreceptor lineage commitment of MGDPs. By TargetScan, Miranda, and Pictar bioinformatics, gain/loss-of-function models, dual luciferase assay, we identified and validated that miR-28 targeted the photoreceptor-specific CRX transcription factor. Anti-miR-28 could induce MGDPs to differentiate into neurons strongly expressing CRX and Rhodopsin, while miR-28 mimic suppressed CRX and Rhodopsin expression. Knockdown of CRX by siRNA blocked the expression of CRX and Rhodospin upregulated by anti-miR-28, indicating that anti-miR-28 potentially induced photoreceptor commitment of MGDPs by targeting CRX, but more experiments are necessary to confirm their role in differentiation.

Activin Signals through SMAD2/3 to Increase Photoreceptor Precursor Yield during Embryonic Stem Cell Differentiation.

SummaryIn vitro differentiation of mouse embryonic stem cells (ESCs) into retinal fates can be used to study the roles of exogenous factors acting through multiple signaling pathways during retina development. Application of activin A during a specific time frame that corresponds to early embryonic retinogenesis caused increased generation of CRX+ photoreceptor precursors and decreased PAX6+ retinal progenitor cells (RPCs). Following activin A treatment, SMAD2/3 was activated in RPCs and bound to promoter regions of key RPC and photoreceptor genes. The effect of activin on CRX expression was repressed by pharmacological inhibition of SMAD2/3 phosphorylation. Activin signaling through SMAD2/3 in RPCs regulates expression of transcription factors involved in cell type determination and promotes photoreceptor lineage specification. Our findings can contribute to the production of photoreceptors for cell replacement therapy.

Fibrin gel as a scaffold for photoreceptor cells differentiation from conjunctiva mesenchymal stem cells in retina tissue engineering

Stem cell-based therapies are attraction approaches for regenerative medicine for treating retinal diseases. One of the limitations in cell therapy is cell death following post-injection whit preventing functional integration with retinal tissue. Fibrin gel, a bio-polymeric material with excellent biocompatibility, provides numerous advantages as a tissue engineering scaffold and a stem cell carrier. Therefore, current research is focusing on developing fibrin hydrogel scaffolds to protect stem cells during delivery and to stimulate endogenous regeneration through interactions of transplanted stem cells and retinal tissue. In this study fibrin gel was used as hydrogel scaffold for immobilization of cells. The structural characteristics of fibrin gel scaffold were examined with SEM. Rheological properties of fibrin gel were measured by rheometer and biodegradation rate of fibrin were assayed for 2 weeks. After isolation of stem cells CJMSCs, the cells were differentiated into photoreceptor-like cells by exposing with taurin for 14 days in tissue culture plate (TCP group) and fibrin hydrogel (3 D group). The attachment of cells was analyzed with SEM and MTT. The expression of rhodopsin, PKC, CRX, recoverin, peripherin, nestin and RPE65 as photoreceptor-like cell markers was evaluated by immunocytochemistry and quantitative real-time PCR (RT-PCR) in TCP and 3 D groups. The results of SEM analysis showed CJMSCs were well attached in fibrin gels and there were good integrity between cells and scaffold. The elastic modulus and constant degradation of the gel contributes to the growth and proliferation of cells. There was no toxicity effect of fibrin hydrogel on cells and the viability of cultured cells was higher in 3 D fibrin gels in comparison with TCP groups. After 2 weeks, the expression of rhodopsin, PKC, CRX, peripherin, recoverin, nestin and RPE65 as special markers of photoreceptor cells were detected by Real time PCR and immunofluorescence that these expressions in 3 D groups were higher than TCP groups. In conclusion, our findings showed that application of readily available sources of adult stem cells like human conjunctiva stem cells encapsulated in fibrin gel could be interesting strategy to enhance photoreceptor progenitor cell numbers for repair and regeneration of retina disease such as photoreceptor injury.