Transcriptomics of CD29+/CD44+ cells isolated from hPSC retinal organoids reveals a single cell population with retinal progenitor and Müller glia characteristics

Müller glia play very important and diverse roles in retinal homeostasis and disease. Although much is known of the physiological and morphological properties of mammalian Müller glia, there is still the need to further understand the profile of these cells during human retinal development. Using human embryonic stem cell-derived retinal organoids, we investigated the transcriptomic profiles of CD29+/CD44+ cells isolated from early and late stages of organoid development. Data showed that these cells express classic markers of retinal progenitors and Müller glia, including NFIX, RAX, PAX6, VSX2, HES1, WNT2B, SOX, NR2F1/2, ASCL1 and VIM, as early as days 10-20 after initiation of retinal differentiation. Expression of genes upregulated in CD29+/CD44+ cells isolated at later stages of organoid development (days 50-90), including NEUROG1, VSX2 and ASCL1 were gradually increased as retinal organoid maturation progressed. Based on the current observations that CD24+/CD44+ cells share the characteristics of early and late-stage retinal progenitors as well as of mature Müller glia, we propose that these cells constitute a single cell population that upon exposure to developmental cues regulates its gene expression to adapt to functions exerted by Müller glia in the postnatal and mature retina

Cell-Based Therapies for Glaucoma

Glaucomatous optic neuropathy (GON) is the major cause of irreversible visual loss worldwide and can result from a range of disease etiologies. The defining features of GON are retinal ganglion cell (RGC) degeneration and characteristic cupping of the optic nerve head (ONH) due to tissue remodeling, while intraocular pressure remains the only modifiable GON risk factor currently targeted by approved clinical treatment strategies. Efforts to understand the mechanisms that allow species such as the zebrafish to regenerate their retinal cells have greatly increased our understanding of regenerative signaling pathways. However, proper integration within the retina and projection to the brain by the newly regenerated neuronal cells remain major hurdles. Meanwhile, a range of methods for in vitro differentiation have been developed to derive retinal cells from a variety of cell sources, including embryonic and induced pluripotent stem cells. More recently, there has been growing interest in the implantation of glial cells as well as cell-derived products, including neurotrophins, microRNA, and extracellular vesicles, to provide functional support to vulnerable structures such as RGC axons and the ONH. These approaches offer the advantage of not relying upon the replacement of degenerated cells and potentially targeting earlier stages of disease pathogenesis. In order to translate these techniques into clinical practice, appropriate cell sourcing, robust differentiation protocols, and accurate implantation methods are crucial to the success of cell-based therapy in glaucoma. Translational Relevance: Cell-based therapies for glaucoma currently under active development include the induction of endogenous regeneration, implantation of exogenously derived retinal cells, and utilization of cell-derived products to provide functional support

MicroRNA profile of extracellular vesicles released by Müller glial cells

IntroductionAs with any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions, and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells.MethodsWe conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture and used bioinformatics tools to evaluate their potential role in the neuroprotective signalling attributed to these cells.ResultsSequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b, and the let-7 family. A total of 51 miRNAs were found to be differentially enriched in EV compared to the whole cells from which EV originated. Bioinformatics analyses also indicated that preferential enrichment of species was demonstrated to regulate genes involved in cell proliferation and survival, including PTEN, the master inhibitor of the PI3K/AKT pathway.DiscussionThe results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks is likely to represent a significant proportion of the neuroprotective effect observed after the transplantation of these cells into animal models of retinal ganglion cell (RGC) depletion. Future studies will seek to evaluate the modulation of putative genes as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons

Downregulation of the Canonical WNT Signaling Pathway by TGFβ1 Inhibits Photoreceptor Differentiation of Adult Human Müller Glia with Stem Cell Characteristics

Müller glia are responsible for the retina regeneration observed in zebrafish. Although the human retina harbors Müller glia with stem cell characteristics, there is no evidence that they regenerate the retina after disease or injury. Transforming growth factor-β (TGFβ) and Wnt signaling regulate retinal neurogenesis and inflammation, but their roles in the neural differentiation of human Müller stem cells (hMSC) are not known. We examined hMSC lines in vitro for the expression of various Wnt signaling components and for their modulation by TGFβ1, as well as the effect of this cytokine on the photoreceptor differentiation of these cells. Culture of hMSC with a combination of factors that induce photoreceptor differentiation of hMSC (FGF2, taurine, retinoic acid, and insulin-like growth factor type1; FTRI), markedly upregulated the expression of components of the canonical Wnt signaling pathway, including WNT2B, DKK1, and active β-CATENIN. Although FTRI did not modify mRNA expression of WNT5B, a component of the noncanonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFβ1 not only decreased WNT2B expression, but also inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, RHODOPSIN, and RECOVERIN. Inhibition of TGFβ1 signaling by an ALK5 inhibitor prevented TGFβ1-induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical WNT signaling by XAV-939 prevented FTRI-induced photoreceptor differentiation. These observations suggest that TGFβ may play a key role in preventing neural differentiation of hMSC and may constitute a potential target for induction of endogenous regeneration of the human retina

Table_1_MicroRNA profile of extracellular vesicles released by Müller glial cells.XLSX

IntroductionAs with any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions, and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells.MethodsWe conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture and used bioinformatics tools to evaluate their potential role in the neuroprotective signalling attributed to these cells.ResultsSequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b, and the let-7 family. A total of 51 miRNAs were found to be differentially enriched in EV compared to the whole cells from which EV originated. Bioinformatics analyses also indicated that preferential enrichment of species was demonstrated to regulate genes involved in cell proliferation and survival, including PTEN, the master inhibitor of the PI3K/AKT pathway.DiscussionThe results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks is likely to represent a significant proportion of the neuroprotective effect observed after the transplantation of these cells into animal models of retinal ganglion cell (RGC) depletion. Future studies will seek to evaluate the modulation of putative genes as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons.</p

Data_Sheet_1_MicroRNA profile of extracellular vesicles released by Müller glial cells.PDF

IntroductionAs with any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions, and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells.MethodsWe conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture and used bioinformatics tools to evaluate their potential role in the neuroprotective signalling attributed to these cells.ResultsSequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b, and the let-7 family. A total of 51 miRNAs were found to be differentially enriched in EV compared to the whole cells from which EV originated. Bioinformatics analyses also indicated that preferential enrichment of species was demonstrated to regulate genes involved in cell proliferation and survival, including PTEN, the master inhibitor of the PI3K/AKT pathway.DiscussionThe results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks is likely to represent a significant proportion of the neuroprotective effect observed after the transplantation of these cells into animal models of retinal ganglion cell (RGC) depletion. Future studies will seek to evaluate the modulation of putative genes as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons.</p