Proliferation Of Retinal Progenitor Cells Research Articles

Compound anisodine affects the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells and brain neural stem cells via the p-ERK1/2/HIF-1α/VEGF pathway.

As a Traditional Chinese Medicine, compound anisodine (CA) has previously been shown to regulate the vegetative nervous system, improve microcirculation and scavenge reactive oxygen species, and has been commonly utilized as a neuroprotective agent to treat ischemic optic neuropathy and choroidoretinopathy. The present study aimed to investigate the neuroprotective effects of CA on the proliferation and calcium overload of hypoxia-induced rat retinal progenitor cells (RPCs) and brain neural stem cells (BNSCs) harvested from neonatal Sprague-Dawley rats. Cells were treated with CA at 0.126, 0.252, 0.505 or 1.010 g/l for four hours prior to or after hypoxia (<1% oxygen) for four h, followed by re-oxygenation for four hours; a normal control group and a CA-untreated hypoxia model group were also included. An MTT assay demonstrated that the cell viability was markedly improved following treatment with 0.126–1.010 g/l CA, compared with that in the hypoxia model group (P<0.05). Bromodeoxyuridine (BrdU) immunocytochemical staining and flow cytometry indicated that after culture in hypoxia for 4 h, the number of BrdU+ RPCs and BNSCs was significant decreased, as well as the cell population in S+G2 phase of the cell cycle, which was significantly attenuated by treatment with 1.010 g/l CA for 4 h prior to hypoxia (P<0.05). Furthermore, laser scanning confocal microscopy showed that the intracellular calcium concentration in hypoxia-cultured RPCs and BNSCs was markedly increased, which was attenuated by 0.126–1.010 g/l CA in a concentration-dependent manner (P<0.05). Furthermore, western blot analysis demonstrated that after hypoxia, the protein levels of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were upregulated in RPCs and BNSCs, whereas phosphorylated extracellular signal-regulated kinase (phospho-ERK 1/2Thr202/Tyr204) and Cyclin D1 were downregulated; of note, treatment with 1.010 g/l CA significantly attenuated these changes (P<0.05). The results of the present study suggested that CA may improve the proliferation and inhibit calcium overload in hypoxia-induced RPCs and BNSCs by altering the protein levels of Cyclin D1 as well as signaling through the p-ERK1/2/HIF-1α/VEGF pathway.

MiR-29a regulates the proliferation and differentiation of retinal progenitors by targeting Rbm8a.

During development, tight regulation of the expansion of retinal progenitor cells (RPCs) and their differentiation into neuronal and glial cells is important for retinal formation and function. Our study demonstrated that microRNA (miR)-29a modulated the proliferation and differentiation of RPCs by suppressing RBM8A (one of the factors in the exon junction complex). Particularly, overexpression of miR-29a reduced RPC proliferation but accelerated RPC differentiation. By contrast, reduction of endogenous miR-29a elicited the opposite effects. Overexpression of miR-29a repressed the translation of Rbm8a, thus negatively regulating RPC proliferation and promoting the neuronal and glial differentiation of RPCs, and knockdown of endogenous Rbm8a phenocopied the observed effects of miR-29a overexpression. Furthermore, a luciferase reporter assay showed that miR-29a directly interacted with the Rbm8a mRNA 3′UTR, which indicated that Rbm8a is the direct target of miR-29a. To further verify the result, co-overexpression of the Rbm8a 3′ UTR-wt (plasmids into which the Rbm8a 3′ UTR sequence had been introduced) and miR-29a in RPCs rescued the phenotype associated with miR-29a overexpression, reversing the promotion of differentiation and inhibition of proliferation. These results show a novel mechanism by which miR-29a regulates the proliferation and differentiation of RPCs through Rbm8a.

Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina.

All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During this time frame, RPCs will differentiate, and the fate of the BrdU+ RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabe et al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuette et al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, and Pten as a modulator of the Tgfβ2 response (Ma et al., 2007; Tachibana et al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.

SUMOylation Regulation of Retina Development and Functions.

The structure and developmental mechanisms of vertebrate retina are highly conserved. One of the most distinctive events during retinogenesis is the temporally and spatially generation of seven types of retinal cells from the multipotent retinal progenitor cells. The importance and prevalence of SUMOylation in regulation of this process through modulation of gene expression and protein function diversity have been increasingly appreciated. Here, we review the biological significance of SUMOylation in retina development, examine how SUMOylation balances the proliferation and cell cycle exit of retinal progenitor cells, and finally discuss the molecular mechanisms mediating the specification of different retina neurons and photoreceptors through modulation of various transcription factors. The potential role of SUMOylation in normal retina function is illustrated by the abundant expression of key components of SUMOylation machinery in mouse retina, and is also exemplified by the highly conserved SUMOylation site on neurotransmission receptors in ganglion cells.

Adenine Nucleotides Control Proliferation In Vivo of Rat Retinal Progenitors by P2Y1 Receptor.

Previous studies demonstrated that exogenous ATP is able to regulate proliferation of retinal progenitor cells (RPCs) in vitro possibly via P2Y1 receptor, a G protein-coupled receptor. Here, we evaluated the function of adenine nucleotides in vivo during retinal development of newborn rats. Intravitreal injection of apyrase, an enzyme that hydrolyzes nucleotides, reduced cell proliferation in retinas at postnatal day 2 (P2). This decrease was reversed when retinas were treated together with ATPγ-S or ADPβ-S, two hydrolysis-resistant analogs of ATP and ADP, respectively. During early postnatal days (P0 to P5), an increase in ectonucleotidase (E-NTPDase) activity was observed in the retina, suggesting a decrease in the availability of adenine nucleotides, coinciding with the end of proliferation. Interestingly, intravitreal injection of the E-NTPDase inhibitor ARL67156 increased proliferation by around 60% at P5 rats. Furthermore, immunolabeling against P2Y1 receptor was observed overall in retina layers from P2 rats, including proliferating Ki-67-positive cells in the neuroblastic layer (NBL), suggesting that this receptor could be responsible for the action of adenine nucleotides upon proliferation of RPCs. Accordingly, intravitreal injection of MRS2179, a selective antagonist of P2Y1 receptors, reduced cell proliferation by approximately 20% in P2 rats. Moreover, treatment with MRS 2179 caused an increase in p57KIP2 and cyclin D1 expression, a reduction in cyclin E and Rb phosphorylated expression and in BrdU-positive cell number. These data suggest that the adenine nucleotides modulate the proliferation of rat RPCs via activation of P2Y1 receptors regulating transition from G1 to S phase of the cell cycle.

Poly(1,3-propylene sebacate) and Poly(sebacoyl diglyceride): A Pair of Potential Polymers for the Proliferation and Differentiation of Retinal Progenitor Cells.

Using suitable polymers as a carrier for growing and delivering retinal progenitor cells (RPCs) is a promising therapeutic strategy in retinal cell-replacement therapy. Herein recently developed polymer, poly(sebacoyl diglyceride) (PSeD), is selected and its nonhydroxylized counterpart poly(1,3-propylene sebacate) (PPS) is designed to evaluate their potentials for RPC growth and future RPC application. The structures and mechanical properties of the polymers are characterized. The cytocompatibility and effects of these polymers on RPC proliferation, differentiation, and migration are systematically investigated in vitro. Our data show that PPS and PSeD display excellent cytocompatibility with low expression of inflammation and apoptosis factors, which benefit RPC growth. In proliferation assays reveal that RPCs expands well on the polymers, but PPS performs the best for RPC expansion, indicating that PPS can remarkably promote RPC proliferation. In differentiation conditions, RPCs grown on PSeD are more likely to differentiate toward retinal neurons, including photoreceptors, the most interesting type of cells for retinal cell-replacement therapy. Additionally, our results demonstrate that RPCs grown on PSeD display an outstanding ability to migrate. In conclusion, PPS can markedly promote RPC proliferation, whereas PSeD can enhance RPC differentiation toward retinal neurons, suggesting that PSeD and PPS have potential applications in future retinal cell-replacement therapies.

Low Intake of Choline During Pregnancy Leads to Aberrant Retinal Architecture and Poor Visual Function in the Offspring

Choline, an essential nutrient, regulates brain development through its role in maintaining the pool of neural progenitor cells. The role of choline in the development of the retina, however, is unknown. In this study, we modulated choline intake in mice during pregnancy and assessed the effects of low choline (LC) diet on the development and function of the retina in the offspring. We find that adequate dietary supply of choline between days 11 and 17 of mouse gestation is required to support the proliferation and differentiation of retinal progenitor cells. Furthermore, retinal structural integrity is disrupted in the embryos of dams receiving LC diet as early as embryonic day 17. As a result of aberrant retinal development, retinal function is abnormal in LC animals, as measured by electroretinography and assessment of visuomotor behavior. Together, our data demonstrate an essential role for choline in retinal health.Support or Funding InformationThis work was funded by grants from Nestlé S.A. and from the NIH (DK056350)

Activation of type 5 metabotropic glutamate receptor promotes the proliferation of rat retinal progenitor cell via activation of the PI-3-K and MAPK signaling pathways

The metabotropic glutamate receptor 5 (mGluR5) regulates neurogenesis in the brain, but the effect of mGluR5 on retinal progenitor cells (RPCs) remains unknown. In this study, we found that mGluR5 promoted the proliferation of rat RPCs with activation of the phosphatidylinositol-3-kinase (PI-3-K) and mitogen-activated protein kinase (MAPK) signaling pathways in vitro. The mGluR5 agonist (S)-3,5-dihydroxyphenylglycine hydrate (DHPG) increased the cellular viability in a concentration- and time-dependent manner, whereas the mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine hydrochloride (MTEP) had the opposite effect, as shown by 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTT) assay. Treatment with DHPG (100μM) also promoted the proliferation of RPCs, as indicated by 5-Bromo-2-deoxyUridine (BrdU) staining and flow cytometry, and likewise, MTEP (100μM) and mGluR5 knockdown abolished the action of mGluR5 activity. Western blot demonstrated that the activation of mGluR5 enhanced the expression of Cyclin D1 and the phosphorylation level of PKC however, MTEP or mGluR5 knockdown also abrogated the effect of DHPG on RPCs. Furthermore, we found that activation of the extracellular signal-regulated protein kinase (ERK) and protein kinase B (AKT) signaling pathways was involved in the proliferation of RPC. After DHPG treatment, the levels of both p-ERK1/2 and p-AKT increased in a time-dependent manner. Then we used MTEP, mGluR5 knockdown, the ERK1/2 inhibitor U0126 and the AKT inhibitor LY294002 to pretreat the cells, and all of them clearly eliminated the influence of DHPG. These results demonstrated that mGluR5 regulates neurogenesis in RPCs through the MAPK and PI-3-K signaling pathways, and these findings may motivate a pharmacological study investigating a potential mechanism for the treatment of retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD).

RONIN Is an Essential Transcriptional Regulator of Genes Required for Mitochondrial Function in the Developing Retina.

A fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell-cycle progression.

Coordinating progenitor cell cycle exit and differentiation in the developing vertebrate retina

ABSTRACTThe proper development of the vertebrate retina relies heavily on producing the correct number and type of differentiated retinal cell types. To achieve this, proliferating retinal progenitor cells (RPCs) must exit the cell cycle at an appropriate time and correctly express a subset of differentiation markers that help specify retinal cell fate. Homeobox genes, which encode a family of transcription factors, have been accredited to both these processes, implicated in the transcriptional regulation of important cell cycle components, such as cyclins and cyclin-dependent kinases, and proneural genes. This dual regulation of homeobox genes allows these factors to help co-ordinate the transition from the proliferating RPC to postmitotic, differentiated cell. However, understanding the exact molecular targets of these factors remains a challenging task. This commentary highlights the current knowledge we have about how these factors regulate cell cycle progression and differentiation, with particular emphasis on a recent discovery from our lab demonstrating an antagonistic relationship between Vsx2 and Dmbx1 to control RPC proliferation. Future studies should aim to further understand the direct transcriptional targets of these genes, additional co-factors/interacting proteins and the possible recruitment of epigenetic machinery by these homeobox genes.

Decellularized retinal matrix: Natural platforms for human retinal progenitor cell culture

Tissue decellularization strategies have enabled engineering of scaffolds that preserve native extracellular matrix (ECM) structure and composition. In this study, we developed decellularized retina (decell-retina) thin films. We hypothesized that these films, mimicking the retina niche, would promote human retinal progenitor cell (hRPC) attachment, proliferation and differentiation. Retinas isolated from bovine eyes were decellularized using 1% w/v sodium dodecyl sulfate (SDS) and pepsin digested. The resulting decell-retina was biochemically assayed for composition and cast dried to develop thin films. Attachment, viability, morphology, proliferation and gene expression of hRPC cultured on the films were studied in vitro. Biochemical analyses of decell-retina compared to native retina indicated the bulk of DNA (94%) was removed, while the majority of sulfated GAGs (55%), collagen (83%), hyaluronic acid (87%), and key growth factors were retained. The decell-retina films supported hRPC attachment and growth, with cell number increasing 1.5-fold over a week. RT-PCR analysis revealed hRPC expression of rhodopsin, rod outer membrane, neural retina-specific leucine zipper neural and cone-rod homeobox gene on decell-retina films, indicating photoreceptor development. In conclusion, novel decell-retina films show promise as potential substrates for culture and/or transplantation of retinal progenitor cells to treat retinal degenerative disorders. Statement of significanceIn this study, we report the development of a novel biomaterial, based on decellularized retina (decell-retina) that mimics the retina niche and promotes human retinal progenitor cell (hRPC) attachment, proliferation and differentiation. We estimated, for the first time, the amounts of collagen I, GAGs and HA present in native retina, as well as the decell-retina. We demonstrated that retinas can be decellularized using ionic detergents and can be processed into mechanically stable thin films, which can act as substrates for culturing hRPCs. Rhodopsin, ROM1, NRL and CRX gene expression on the decell-retina films indicated photoreceptor development from RPCs. These results support the potential of decell-retina as a cell delivery platform to treat and manage retinal degenerative disease like AMD.

Electrospun SF/PLCL nanofibrous membrane: a potential scaffold for retinal progenitor cell proliferation and differentiation.

Biocompatible polymer scaffolds are promising as potential carriers for the delivery of retinal progenitor cells (RPCs) in cell replacement therapy for the repair of damaged or diseased retinas. The primary goal of the present study was to investigate the effects of blended electrospun nanofibrous membranes of silk fibroin (SF) and poly(L-lactic acid-co-ε-caprolactone) (PLCL), a novel scaffold, on the biological behaviour of RPCs in vitro. To assess the cell-scaffold interaction, RPCs were cultured on SF/PLCL scaffolds for indicated durations. Our data revealed that all the SF/PLCL scaffolds were thoroughly cytocompatible, and the SF:PLCL (1:1) scaffolds yielded the best RPC growth. The in vitro proliferation assays showed that RPCs proliferated more quickly on the SF:PLCL (1:1) than on the other scaffolds and the control. Quantitative polymerase chain reaction (qPCR) and immunocytochemistry analyses demonstrated that RPCs grown on the SF:PLCL (1:1) scaffolds preferentially differentiated toward retinal neurons, including, most interestingly, photoreceptors. In summary, we demonstrated that the SF:PLCL (1:1) scaffolds can not only markedly promote RPC proliferation with cytocompatibility for RPC growth but also robustly enhance RPCs’ differentiation toward specific retinal neurons of interest in vitro, suggesting that SF:PLCL (1:1) scaffolds may have potential applications in retinal cell replacement therapy in the future.

MicroRNA-7a regulates Müller glia differentiation by attenuating Notch3 expression

miRNA-7a plays critical roles in various biological aspects in health and disease. We aimed to reveal roles of miR-7a in mouse retinal development by loss- and gain-of-function analyses of miR-7a. Plasmids encoding miR-7a or miR-7a-decoy (anti-sense miR-7a) were introduced into mouse retina at P0, and the retina was cultured as explant. Then, proliferation of retinal progenitors and differentiation of retinal subtypes were examined by immunostaining. miR-7a had no apparent effect on the proliferation of retinal progenitor cells. However, the expression of Müller glia marker, cyclin D3, was reduced by miR-7a overexpression and up-regulated by miR-7a decoy, suggesting that miR-7a negatively regulates differentiation of Müller glia. Targets of miR-7a, which were predicted by using a public program miRNA.org, and Notch3 was suggested to be one of candidate genes of miR-7a target. Notch3 3′ UTR appeared to contain complementary sequence to the seed sequence of miR-7a. A reporter assay in NIH3T3 cells using a plasmid containing multiple repeats of potential target sequence of 3′ Notch UTR showed that miR-7a suppress expression of reporter EGFP through 3′UTR region. Expression of sh-Notch3 and over-expression of NICD3 in retina suggested that miR-7a regulates Müller glia differentiation through attenuation of Notch3 expression. Taken together, we revealed that the miR-7a regulates the differentiation of Müller glia through the suppression of Notch3 expression.

The protein tyrosine phosphatase interacting protein 51 (PTPIP51) is required for the differentiation of photoreceptors

Proliferation and differentiation of retinal progenitor cells (RPCs) are tightly controlled by extrinsic cues and distinct combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we investigated the role of the protein tyrosine phosphatase interacting protein 51 (PTPIP51) in the differentiation of RPCs. The expression pattern of PTPIP51 was analyzed by immunostaining during post-natal retinal development in the rat. Ex vivo electroporation has been used to silence or misexpress PTPIP51 in post-natal retinal explants, and the retinal phenotype was investigated after 3–7days in vitro (div). PTPIP51 expression in the retina started postnatally and was maintained throughout adulthood, especially in retinal ganglion cells and in the inner segment of photoreceptor cells. Silencing of Ptpip51 expression in postnatal retina failed to modify the commitment of late RPCs in the different lineages but severely impaired the final differentiation of photoreceptors, observed by a decrease in the fraction of Rhodopsin-positive cells after 7div. By contrast, misexpression of PTPIP51 in early or late RPCs failed to modify the differentiation of the RPCs. Our data demonstrate that PTPIP51 is implicated in the differentiation process of immature photoreceptors. Because PTPIP51 is specifically localized in the inner segment, PTPIP51 may contribute to the complex stage of maturation of the apical segment of these cells.

Genetic chimeras reveal the autonomy requirements for Vsx2 in embryonic retinal progenitor cells.

BackgroundVertebrate retinal development is a complex process, requiring the specification and maintenance of retinal identity, proliferative expansion of retinal progenitor cells (RPCs), and their differentiation into retinal neurons and glia. The homeobox gene Vsx2 is expressed in RPCs and required for the proper execution of this retinal program. However, our understanding of the mechanisms by which Vsx2 does this is still rudimentary. To define the autonomy requirements for Vsx2 in the regulation of RPC properties, we generated chimeric mouse embryos comprised of wild-type and Vsx2-deficient cells.ResultsWe show that Vsx2 maintains retinal identity in part through the cell-autonomous repression of the retinal pigment epithelium determinant Mitf, and that Lhx2 is required cell autonomously for the ectopic Mitf expression in Vsx2-deficient cells. We also found significant cell-nonautonomous contributions to Vsx2-mediated regulation of RPC proliferation, pointing to an important role for Vsx2 in establishing a growth-promoting extracellular environment. Additionally, we report a cell-autonomous requirement for Vsx2 in controlling when neurogenesis is initiated, indicating that Vsx2 is an important mediator of neurogenic competence. Finally, the distribution of wild-type cells shifted away from RPCs and toward retinal ganglion cell precursors in patches of high Vsx2-deficient cell density to potentially compensate for the lack of fated precursors in these areas.ConclusionsThrough the generation and analysis of genetic chimeras, we demonstrate that Vsx2 utilizes both cell-autonomous and cell-nonautonomous mechanisms to regulate progenitor properties in the embryonic retina. Importantly, Vsx2’s role in regulating Mitf is in part separable from its role in promoting proliferation, and proliferation is excluded as the intrinsic timer that determines when neurogenesis is initiated. These findings highlight the complexity of Vsx2 function during retinal development and provide a framework for identifying the molecular mechanisms mediating these functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-015-0039-5) contains supplementary material, which is available to authorized users.

Neuropeptide Y system in the retina: From localization to function.

The retina is a highly complex structure where several types of cells communicate through countless different molecules to codify visual information. Each type of cells plays unique roles in the retina, presenting a singular expression of neurotransmitters. Some neurotransmitter systems in the retina are well understood, while others need to be better explored to unravel the intricate signaling system involved. Neuropeptide Y (NPY), a 36 amino acid peptide, is one of the most common peptide neurotransmitter in the CNS and a highly conserved peptide among species. We review the localization of NPY and NPY receptors (mainly NPY Y1, Y2, Y4 and Y5) in retinal cells. Common features of the expression of NPY and NPY receptors in mammalian and non-mammalian species indicate universal roles of this system in the retina. In the present review, we highlight the putative roles of NPY receptor activation in the retina, discussing, in particular, their involvement in retinal development, neurotransmitter release modulation, neuroprotection, microglia and Muller cells function, retinal pigmented epithelium changes, retinal endothelial physiology and proliferation of retinal progenitor cells. Further studies are needed to confirm that targeting the NPY system might be a potential therapeutic strategy for retinal degenerative diseases.

Activated adult microglia influence retinal progenitor cell proliferation and differentiation toward recoverin-expressing neuron-like cells in a co-culture model.

Microglia contribute to immune homeostasis of the retina, and thus act as a potential regulator determining successful repair or retinal stem cell transplantation. We investigated the interaction between human microglia and retinal progenitor cells in cell co-culture to further our exploration on developing a new therapeutic strategy for retinal degeneration. Microglia and retinal progenitor cultures were developed using CD11b(+) and CD133(+), respectively, from adult donor retina. Microglia activation was developed using interferon-gamma and lipopolysaccharide. Retinal progenitor differentiation was analysed in co-culture with or without microglial activation. Retinal progenitor proliferation was analysed in presence of conditioned medium from activated microglia. Phenotype and function of adult human retinal cell cultures were examined using cell morphology, immunohistochemistry and real-time PCR. By morphology, neuron-like cells generated in co-culture expressed photoreceptor marker recoverin. Neurospheres derived from retinal progenitor cells showed reduced growth in the presence of conditioned medium from activated microglia. Delayed retinal progenitor cell migration and reduced cellular differentiation was observed in co-cultures with activated microglia. In independent experiments, activated microglia showed enhanced mRNA expression of CXCL10, IL-27, IL-6, and TNF-alpha compared to controls. Adult human retina retains retinal progenitors or potential to reprogram cells to then proliferate and differentiate into neuron-like cells in vitro. Human microglia support retinal progenitor differentiation into neuron-like cells, but such capacity is altered following microglial activation. Modulating microglia activity is a potential approach to promote retinal repair and facilitate success of stem-cell transplantation.

Lycium barbarum polysaccharides promotes in vivo proliferation of adult rat retinal progenitor cells.

Lycium barbarum is a widely used Chinese herbal medicine prescription for protection of optic nerve. However, it remains unclear regarding the effects of Lycium barbarum polysaccharides, the main component of Lycium barbarum, on in vivo proliferation of adult ciliary body cells. In this study, adult rats were intragastrically administered low- and high-dose Lycium barbarum polysaccharides (1 and 10 mg/kg) for 35 days and those intragastrically administered phosphate buffered saline served as controls. The number of Ki-67-positive cells in rat ciliary body in the Lycium barbarum polysaccharides groups, in particular low-dose Lycium barbarum polysaccharides group, was significantly greater than that in the phosphate buffered saline group. Ki-67-positive rat ciliary body cells expressed nestin but they did not express glial fibrillary acidic protein. These findings suggest that Lycium barbarum polysaccharides can promote the proliferation of adult rat retinal progenitor cells and the proliferated cells present with neuronal phenotype.

The guanine nucleotide exchange factor Vav3 regulates differentiation of progenitor cells in the developing mouse retina.

The seven main cell types in the mammalian retina arise from multipotent retinal progenitor cells, a process that is tightly regulated by intrinsic and extrinsic signals. However, the molecular mechanisms that control proliferation, differentiation and cell-fate decisions of retinal progenitor cells are not fully understood yet. Here, we report that the guanine nucleotide exchange factor Vav3, a regulator of Rho-GTPases, is involved in retinal development. We demonstrate that Vav3 is expressed in the mouse retina during the embryonic period. In order to study the role of Vav3 in the developing retina, we generate Vav3-deficient mice. The loss of Vav3 results in an accelerated differentiation of retinal ganglion cells and cone photoreceptors during early and late embryonic development. We provide evidence that more retinal progenitor cells express the late progenitor marker Sox9 in Vav3-deficient mice than in wild-types. This premature differentiation is compensated during the postnatal period and late-born cell types such as bipolar cells and Müller glia display normal numbers. Taken together, our data imply that Vav3 is a regulator of retinal progenitor cell differentiation, thus highlighting a novel role for guanine nucleotide exchange factors in retinogenesis.

Effects of let-7b and TLX on the proliferation and differentiation of retinal progenitor cells in vitro.

MicroRNAs manifest significant functions in brain neural stem cell (NSC) self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. Let-7b is expressed in the mammalian brain and regulates NSC proliferation and differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal. Whether let-7b and TLX act as important regulators in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. Here, our data show that let-7b and TLX play important roles in controlling RPC fate determination in vitro. Let-7b suppresses TLX expression to negatively regulate RPC proliferation and accelerate the neuronal and glial differentiation of RPCs. The overexpression of let-7b downregulates TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, whereas antisense knockdown of let-7b produces robust TLX expression,enhanced RPC proliferation and decreased differentiation. Moreover, the inhibition of endogenous TLX by small interfering RNA suppresses RPC proliferation and promotes RPC differentiation. Furthermore, overexpression of TLX rescues let-7b-induced proliferation deficiency and weakens the RPC differentiation enhancement caused by let-7b alone. These results suggest that let-7b, by forming a negative feedback loop with TLX, provides a novel model to regulate the proliferation and differentiation of retinal progenitors in vitro.