Identification of a modular super-enhancer in murine retinal development

Super-enhancers are expansive regions of genomic DNA comprised of multiple putative enhancers that contribute to the dynamic gene expression patterns during development. This is particularly important in neurogenesis because many essential transcription factors have complex developmental stage– and cell–type specific expression patterns across the central nervous system. In the developing retina, Vsx2 is expressed in retinal progenitor cells and is maintained in differentiated bipolar neurons and Müller glia. A single super-enhancer controls this complex and dynamic pattern of expression. Here we show that deletion of one region disrupts retinal progenitor cell proliferation but does not affect cell fate specification. The deletion of another region has no effect on retinal progenitor cell proliferation but instead leads to a complete loss of bipolar neurons. This prototypical super-enhancer may serve as a model for dissecting the complex gene expression patterns for neurogenic transcription factors during development. Moreover, it provides a unique opportunity to alter expression of individual transcription factors in particular cell types at specific stages of development. This provides a deeper understanding of function that cannot be achieved with traditional knockout mouse approaches.

Mechanisms underlying microglial colonization of developing neural retina in zebrafish

Microglia are brain-resident macrophages that function as the first line of defense in brain. Embryonic microglial precursors originate in peripheral mesoderm and migrate into the brain during development. However, the mechanism by which they colonize the brain is incompletely understood. The retina is one of the first brain regions to accommodate microglia. In zebrafish, embryonic microglial precursors use intraocular hyaloid blood vessels as a pathway to migrate into the optic cup via the choroid fissure. Once retinal progenitor cells exit the cell cycle, microglial precursors associated with hyaloid blood vessels start to infiltrate the retina preferentially through neurogenic regions, suggesting that colonization of retinal tissue depends upon the neurogenic state. Along with blood vessels and retinal neurogenesis, IL34 also participates in microglial precursor colonization of the retina. Altogether, CSF receptor signaling, blood vessels, and neuronal differentiation function as cues to create an essential path for microglial migration into developing retina.

Chaperone-Mediated Autophagy Plays an Important Role in Regulating Retinal Progenitor Cell Homeostasis

Purpose: Autophagy is a key regulator of stem cell quiescence and self-renewal, especially in mesenchymal stem cells but related research on neural retinal stem cells is still limited. We are aimed to explore the function and mechanism of autophagy in the neural retinal stem cell.Methods: The published single cell sequencing data was involved to analysis the expression time course of IFITM3 in the mouse neural retinal progenitor cells (mNRPCs). The RNA interference was used to knock down the expression of IFITM3 in the mNRPCs. And the normal mNRPCs and mNRPCs with knockdown of IFITM3 were analysis with the CCK8 for the cell viability, RNA-seq for the mRNA expression, real-time quantitative PCR, immunofluorescence assay for the location of relative proteins, western blot for the levels of relative proteins and autophagy flux assay.Results: This study showed the mNRPCs in vivo and in vitro high expressed IFITM3 which are expressed in the mNRPCs. The proliferation of mNRPCs was greatly inhibited, and cell viability was greatly reduced after IFITM3 knockdown. Moreover, RNA-seq analysis showed that lysosomes were significant changed after IFITM3 knockdown. When cells were treated with rapamycin (RAMP), lysosome activation and agglomeration were evident in all groups. However, there was no significant difference between IFITM3 knockdown groups. The expression of LAMP1 was significantly increased, accompanied by increased lysosome agglomeration, in RAMP-treated cells and especially in IFITM3-knockdown cells. Further detection showed that SQSTM1/p62, HSC70 and LAMP-2A were upregulated, while there was no significant difference in LC3A/B expression, which demonstrated that the MA pathway was not activated but the CMA pathway was activated when knockdown of IFITM3. Conclusion: Our findings indicate that IFITM3 participates in regulating mNRPC viability and proliferation mainly through the CMA pathway, indicating that IFITM3 plays a significant role in maintaining the homeostasis of progenitor cell self-renewal by sustaining low-level activation of the CMA pathway to eliminate factors that are deleterious to cells and acts as a very important protector of RPCs.

Human Retinal Progenitor Cells Derived Small Extracellular Vesicles Delay Retinal Degeneration: A Paradigm for Cell-free Therapy

Retinal degeneration is a leading cause of irreversible vision impairment and blindness worldwide. Previous studies indicate that subretinal injection of human retinal progenitor cells (hRPCs) can delay the progression of retinal degeneration, preserve retinal function, and protect photoreceptor cells from death, albeit the mechanism is not well understood. In this study, small extracellular vesicles derived from hRPCs (hRPC-sEVs) were injected into the subretinal space of retinal dystrophic RCS rats. We find that hRPC-sEVs significantly preserve the function of retina and thickness of the outer nuclear layer (ONL), reduce the apoptosis of photoreceptors in the ONL, and suppress the inflammatory response in the retina of RCS rats. In vitro, we have shown that hRPC-sEV treatment could significantly reserve the low-glucose preconditioned apoptosis of photoreceptors and reduce the expression of pro-inflammatory cytokines in microglia. Pathway analysis predicted the target genes of hRPC-sEV microRNAs involved in inflammation related biological processes and significantly enriched in processes autophagy, signal release, regulation of neuron death, and cell cycle. Collectively, our study suggests that hRPC-sEVs might be a favorable agent to delay retinal degeneration and highlights as a new paradigm for cell-free therapy.

Hyaluronan-CD44 Interaction Regulates Mouse Retinal Progenitor Cells Migration, Proliferation and Neuronal Differentiation

Background: Therapeutic applications of retinal progenitor cells (RPCs) are hindered by their limited proliferation and differentiation capacity and poor ability to migrate into damaged retinal tissue. Our study aimed to explore the effects of HA-CD44 interactions on the regulation of RPCs migration, proliferation and differentiation, and to investigate the underlying regulation mechanisms.Methods: Mouse RPCs were isolated and amplified. Western blot and flow cytometry analyses were used to investigate the expression of CD44 in RPCs. The effects of HA-CD44 interactions on the RPCs behaviors, including migration, proliferation and differentiation, were investigated by MTT assay, CCK8 assay, vertical collagen gel invasion assay, time-lapse imaging, immunocytochemistry, RT-PCR and western blot assay. Furthermore, the downstream signals of HA-CD44 interactions were investigated.Results: CD44 was expressed in RPCs, and HA-CD44 interaction markedly improved RPCs adhesion and migration. The stimulation of miR-21 expression by HA-CD44 interaction was PKC/Nanog-dependent in RPCs. Treatment of RPCs with PKC- or Nanog-specific ASODN or miR-21 antagomir effectively blocked HA-mediated RPCs adhesion and migration. Moreover, ROK/Gab-1 associated PI3K/AKT signaling activation was required in the HA-CD44 interaction mediated RPCs proliferation and neuronal differentiation.Conclusions: Our findings demonstrated new roles for HA-CD44 interaction in regulating both migration, proliferation and neuronal differentiation of RPCs. HA-CD44 signaling could comprise a novel approach to control RPC fates, which may be instructive for the application of RPCs for future therapeutic application.

mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia

Retinal progenitor cells (RPCs) divide in limited numbers to generate the cells comprising vertebrate retina. The molecular mechanism that leads RPC to the division limit, however, remains elusive. Here, we find that the hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) in an RPC subset by deletion of tuberous sclerosis complex 1 (Tsc1) makes the RPCs arrive at the division limit precociously and produce Müller glia (MG) that degenerate from senescence-associated cell death. We further show the hyperproliferation of Tsc1-deficient RPCs and the degeneration of MG in the mouse retina disappear by concomitant deletion of hypoxia-induced factor 1-a (Hif1a), which induces glycolytic gene expression to support mTORC1-induced RPC proliferation. Collectively, our results suggest that, by having mTORC1 constitutively active, an RPC divides and exhausts mitotic capacity faster than neighboring RPCs, and thus produces retinal cells that degenerate with aging-related changes.

Human primitive mesenchymal stem cell-derived retinal progenitor cells promoted neuroprotection and neurogenesis in rd12 mice

Retinal degenerative diseases (RDD) such as retinitis pigmentosa (RP) have no treatment. Stem cell-based therapies could provide promising opportunities to repair the damaged retina and restore vision. We investigated a novel approach in which human retinal progenitor cells (RPCs) derived from primitive mesenchymal stem cells (pMSCs) were examined to treat retinal degeneration in an rd12 mouse model of RP. Intravitreally transplanted cells improved retinal function and significantly increased retinal thickness. Transplanted cells homed, survived, and integrated to various retinal layers. They also induced anti-inflammatory and neuroprotective responses and upregulated neurogenesis genes. We found that RPCs were more efficacious than pMSCs in improving the retinal structure and function. RNA analyses suggest that RPCs promote neuroprotection and neuronal differentiation by activating JAK/STAT and MAPK, and inhibiting BMP signaling pathways. These promising results provide the basis for clinical studies to treat RDD using RPCs derived from pMSCs.

TLX, an orphan nuclear receptor with emerging roles in physiology and disease

TLX (NR2E1), an orphan member of the nuclear receptor superfamily, is a transcription factor that has been described to be generally repressive in nature. It has been implicated in several aspects of physiology and disease. TLX is best known for its ability to regulate the proliferation of neural stem cells and retinal progenitor cells. Dysregulation, overexpression, or loss of TLX expression has been characterized in numerous studies focused on a diverse range of pathological conditions, including: abnormal brain development, psychiatric disorders, retinopathies, metabolic disease, and malignant neoplasm. Despite the lack of an identified endogenous ligand, several studies have described putative synthetic and natural TLX ligands, suggesting that this receptor may serve as a therapeutic target. Therefore, this article aims to briefly review what is known about TLX structure and function in normal physiology, and provide an overview of TLX in regard to pathological conditions. Particular emphasis is placed on TLX and cancer, and the potential utility of this receptor as a therapeutic target.

Stimulation of c-Kit+ Retinal Progenitor Cells by Stem Cell Factor Confers Protection Against Retinal Degeneration

Background c-Kit/CD117, expressed in a series of tissue-specific progenitor cells, plays an important role in tissue regeneration and tissue homeostasis. We previously demonstrated that organoid-derived c-Kit+ retinal progenitor cells can facilitate the restoration of degenerated retina. Meanwhile, we have identified a population of endogenous c-Kit+ cells in retinas of adult mouse. However, the exact role of these cells in retinal degeneration remains unclear. Methods Retinal degeneration was induced by intravitreal injection of N-methyl-D-aspartate (NMDA). Two days post NMDA challenge, intravitreal injection of stem cell factor (SCF) was performed. Distribution and abundance of c-Kit+ cells and other retinal cells were evaluated by immunochemistry. Retinal function of treated mice was tested via flash electroretinogram (fERG) and the light/dark transition test. Possible regulatory pathways were evaluated by RNA sequencing. Results NMDA challenge increased the total number of c-Kit+ cells in the retinal ganglion cell layer (GCL), while slightly deregulated the protein level of SCF, which is mainly expressed in Müller cells. Both fERG and light/dark transition tests showed that intravitreal injection of SCF effectively improve the visual function of NMDA-treated mice. Consistently, the activation of microglia in injured retina has also been inhibited after SCF treatment. Mechanistically, SCF administration not only prevent the loss of retinal ganglion cells (RGCs), but also maintained the function of RGCs as quantified by fERG. Further, we performed transcriptome sequencing analysis of the retinal cells isolated from SCF-treated mice and the parallel control. Gene Ontology analysis showed that SCF-induced transcriptome changes were closely correlated with eye development-related pathways. Crystallins and several protective factors such as Pitx3 were significantly upregulated by SCF treatment. Conclusions Our results revealed the role of c-Kit+ cells in the protection of RGCs in NMDA-treated mice, via inhibiting the loss of RGCs. Administration of SCF can act as a potent strategy for treating retinal degeneration-related diseases.