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![Expression of the homeobox gene Cr x in 21-day differentiated PCM sphere colonies. (A) RT-PCR analysis of total RNA isolated from adult mouse retina control samples (lane 1) and differentiated adult PCM colonies (lane 2). Nested Cr x RT-PCR resulted in a 207– base pair product (arrow). No product was seen in an adult liver control sample (lane 3) or in adult retinal RNA with RT omitted (lane 4). Data are representative of 75 ng of adult retinal or liver RNA (controls). We used one differentiated PCM colony (13,000 cells) isolated from two separate cultures; we estimate that less RNA (more than one order of magnitude) was used from the PCM colony in comparison with the adult retinal and liver controls. (B and C) In situ hybridization analysis of Cr x on differentiated PCM colonies exposed to antisense Cr x probes. Cells expressing Cr x mRNA [arrowheads , top panel in (B)] and cells with no Cr x mRNA expression [arrowheads, top panel in (C)] could be identified in the same culture well exposed to antisense Cr x probes using phase (Ph3) contrast and focusing specifically on the silver grains in the emulsion layer. Bottom panels in (B) and (C) show the position of the same cells indicated by arrowheads under phase (Ph2) contrast focusing specifically on cell morphology . Culture wells exposed to sense Cr x probes were identical in appearance to (C). Data are representative of four adult PCM colonies exposed to antisense Cr x probes and three adult PCM colonies exposed to sense probes from two separate cultures. The same probes detected Cr x mRNA in sections of the adult mouse eye. Scale bar, 20 m.](profile/Brenda-Coles/publication/12596248/figure/fig3/AS:394427060244501@1471050147273/Expression-of-the-homeobox-gene-Cr-x-in-21-day-differentiated-PCM-sphere-colonies-A.png)
Expression of the homeobox gene Cr x in 21-day differentiated PCM sphere colonies. (A) RT-PCR analysis of total RNA isolated from adult mouse retina control samples (lane 1) and differentiated adult PCM colonies (lane 2). Nested Cr x RT-PCR resulted in a 207– base pair product (arrow). No product was seen in an adult liver control sample (lane 3) or in adult retinal RNA with RT omitted (lane 4). Data are representative of 75 ng of adult retinal or liver RNA (controls). We used one differentiated PCM colony (13,000 cells) isolated from two separate cultures; we estimate that less RNA (more than one order of magnitude) was used from the PCM colony in comparison with the adult retinal and liver controls. (B and C) In situ hybridization analysis of Cr x on differentiated PCM colonies exposed to antisense Cr x probes. Cells expressing Cr x mRNA [arrowheads , top panel in (B)] and cells with no Cr x mRNA expression [arrowheads, top panel in (C)] could be identified in the same culture well exposed to antisense Cr x probes using phase (Ph3) contrast and focusing specifically on the silver grains in the emulsion layer. Bottom panels in (B) and (C) show the position of the same cells indicated by arrowheads under phase (Ph2) contrast focusing specifically on cell morphology . Culture wells exposed to sense Cr x probes were identical in appearance to (C). Data are representative of four adult PCM colonies exposed to antisense Cr x probes and three adult PCM colonies exposed to sense probes from two separate cultures. The same probes detected Cr x mRNA in sections of the adult mouse eye. Scale bar, 20 m.
Source publication
The mature mammalian retina is thought to lack regenerative capacity. Here, we report the identification of a stem cell in the adult mouse eye, which represents a possible substrate for retinal regeneration. Single pigmented ciliary margin cells clonally proliferate in vitro to form sphere colonies of cells that can differentiate into retinal-speci...
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Citations
... Among them, the fetal retina has been proven to be a potential source of isolated RPCs/RSCs. Tropepe et al. first demonstrated the existence of RSCs in the ciliary margin of adult and embryonic mice using a neural stem cell colony-forming assay [37]. These cells, when transplanted into the eye, can directly differentiate into retina-specific cells within the microenvironment of the body, replacing damaged retinal cells and thereby restoring partial vision. ...
Retinal degeneration (RD) is a leading cause of blindness worldwide and includes conditions such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt’s disease (STGD). These diseases result in the permanent loss of vision due to the progressive and irreversible degeneration of retinal cells, including photoreceptors (PR) and the retinal pigment epithelium (RPE). The adult human retina has limited abilities to regenerate and repair itself, making it challenging to achieve complete self-replenishment and functional repair of retinal cells. Currently, there is no effective clinical treatment for RD. Stem cell therapy, which involves transplanting exogenous stem cells such as retinal progenitor cells (RPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs), or activating endogenous stem cells like Müller Glia (MG) cells, holds great promise for regenerating and repairing retinal cells in the treatment of RD. Several preclinical and clinical studies have shown the potential of stem cell-based therapies for RD. However, the clinical translation of these therapies for the reconstruction of substantial vision still faces significant challenges. This review provides a comprehensive overview of stem/progenitor cell-based therapy strategies for RD, summarizes recent advances in preclinical studies and clinical trials, and highlights the major challenges in using stem/progenitor cell-based therapies for RD.
Graphical Abstract
... which arises from the CMZ was postulated to contain a population of pigmented cells that were able to form clonogenic spheres and could be differentiated to express marker genes found in photoreceptors, bipolar and Muller glia cells 14 . However subsequent studies demonstrated that these pigmented ciliary epithelial cells fail to differentiate into retinal neurones in vitro or in vivo 15 . ...
The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein, we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors, which transit to late, and further to transient neurogenic progenitors, that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples, we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells, we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs, which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids.
... 329 We discuss the mechanisms previously put forward, and review the evidence for this novel third possible modality of action which proposes the involvement of PET. Netarsudil has been used to promote corneal clearance following DWEK in FECD, iridocorneal endothelial syndrome, and graft failure following penetrating keratoplasty; 288 [373][374][375] retinal SCs at the pigmented ciliary margin, 376,377 conjunctival epithelium SCs in the fornices, 378 at the eyelid margin, 379 in lacrimal 380 and meibomian glands, 381 and more recently, SCs in the transition zone bordering the CE and trabecular meshwork (TM). 141,143,258,382 ( ...
Currently, disease of the corneal endothelium – of which Fuchs’ corneal endothelial dystrophy is the most prevalent (estimated to affect 4% of the US population)1 – are largely managed with corneal transplantation which is highly invasive, costly, prone to rejection and limited by availability of donor tissue. A large proportion of patients with visually significant early-stage disease – which left untreated leads to progressive visual deterioration – do not qualify for transplantation based on risk/benefit considerations and are left without treatment options. Therefore, research exploring minimally invasive modalities for treating corneal endothelial disease is of high priority. The advent of Descemet’s Stripping Only (DSO) – a surgical technique for treating corneal endothelial disease (facilitated by pharmacological stimulation of corneal endothelial progenitors)2 – circumvents limitations inherent in transplantation, yet remains invasive, carries surgical risks including infective complications, and is relatively costly. This project aims to address these needs by exploring the feasibility of treating corneal endothelial disease in an ex vivo model using licensed ophthalmic lasers – which are widely available, affordable, and can be rapidly repurposed – with a view towards clinical application of this minimally invasive laser version of DSO (laser-DSO) procedure. Using electron microscopy to evaluate the corneal endothelium of 17 human donor corneas following laser treatment using either a long-pulsed or short-pulsed frequency-doubled Nd:YAG laser, we have demonstrated (1) proof of concept that by applying the long-pulsed 532 nm laser directly to the corneal endothelium these cells can be removed, and (2) successful corneal endothelial removal using a clinically relevant model wherein the long-pulsed 532 nm laser was applied trans-corneally when the endothelium was photosensitised using trypan blue dye prior to laser treatment. These results support the feasibility of a laser-DSO procedure where a photosensitising agent is employed and warrant further research directed towards clinical application.
... Unfortunately, the adult mammalian retina does not have a proliferative CMZ or an RSC cell source available, resulting in an ineffective capacity to regenerate after tissue damage. However, the mammalian ciliary epithelium (CE) is a structure topologically analogous to the adult CMZ of lower vertebrates and presents a small subset of cells that exhibit promising stem-cell-like properties that could be targeted for future regenerative therapy development [2,3]. ...
... At the transcriptional level, adult PE cells exposed to GF upregulated the expression of proliferation markers (Ki67 and Cyclins) and neural progenitor genes specific to the RSC phenotype (Nestin and Pax6), while at the same time downregulating the expression of terminally differentiated cell markers, such as the epithelial gene Rab27b [7,8]. PE-derived neurospheres were capable of differentiating into different retinal neuron phenotypes and presented electrophysiological responses [2,3,9]. These results proved the existence of a subpopulation of PE cells with the ability to undergo progenitor/stem cell reprogramming, self-renewal, and generation of functional neurons upon non-cell-autonomous influences [9]. ...
The retina is a central nervous tissue essential to visual perception and highly susceptible to environmental damage. Lower vertebrate retinas activate intrinsic regeneration mechanisms in response to retinal injury regulated by a specialized population of progenitor cells. The mammalian retina does not have populations of progenitor/stem cells available to activate regeneration, but contains a subpopulation of differentiated cells that can be reprogrammed into retinal stem cells, the ciliary epithelium (CE) cells. Despite the regenerative potential, stem cells derived from CE exhibit limited reprogramming capacity probably associated with the expression of intrinsic regulatory mechanisms. Platelet-activating factor (PAF) is a lipid mediator widely expressed in many cells and plays an important role in stem cell proliferation and differentiation. During mammalian development, PAF receptor signaling showed important effects on retinal progenitors’ cell cycle regulation and neuronal differentiation that need to be further investigated. In this study, our findings suggested a dynamic role for PAF receptor signaling in CE cells, impacting stem cell characteristics and neurosphere formation. We showed that PAF receptors and PAF-related enzymes are downregulated in retinal progenitor/stem cells derived from PE cells. Blocking PAFR activity using antagonists increased the expression of specific progenitor markers, revealing potential implications for retinal tissue development and maintenance.
... This indicates that these cells could serve as a viable substrate for retinal regeneration. 102 ...
Background
Degenerate eye disorders, such as glaucoma, cataracts and age-related macular degeneration (AMD), are prevalent causes of blindness and visual impairment worldwide. Other eye disorders, including limbal stem cell deficiency (LSCD), dry eye diseases (DED), and retinitis pigmentosa (RP), result in symptoms such as ocular discomfort and impaired visual function, significantly impacting quality of life. Traditional therapies are limited, primarily focus on delaying disease progression, while emerging stem cell therapy directly targets ocular tissues, aiming to restore ocular function by reconstructing ocular tissue.
Main text
The utilization of stem cells for the treatment of diverse degenerative ocular diseases is becoming increasingly significant, owing to the regenerative and malleable properties of stem cells and their functional cells. Currently, stem cell therapy for ophthalmopathy involves various cell types, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and retinal progenitor cells (RPCs). In the current article, we will review the current progress regarding the utilization of stem cells for the regeneration of ocular tissue covering key eye tissues from the cornea to the retina. These therapies aim to address the loss of functional cells, restore damaged ocular tissue and or in a paracrine-mediated manner. We also provide an overview of the ocular disorders that stem cell therapy is targeting, as well as the difficulties and opportunities in this field.
Conclusions
Stem cells can not only promote tissue regeneration but also release exosomes to mitigate inflammation and provide neuroprotection, making stem cell therapy emerge as a promising approach for treating a wide range of eye disorders through multiple mechanisms.
... After seven days in culture, the resulting neurospheres were collected and dissociated enzymatically and mechanically to form a stem/progenitor single-cell suspension, which was then cultured for seven days with the test item or control (secondary neurospheres). Increased proliferation was indicated by significantly increased object counts in the secondary neurosphere cultures [28,29]. In the described assay, after incubation with CompA, object counts were significantly increased compared with controls ( Figure 1C), without affecting nucleus size (Supplementary Figure S1B), whereas the vehicle control (DMSO) did not affect proliferation (Supplementary Figure S1C). ...
Citation: Mokady, D.; Charish, J.; Barretto-Burns, P.; Grisé, K.N.; Coles, B.L.K.; Raab, S.; Ortin-Martinez, A.; Müller, A.; Fasching, B.; Jain, P.; et al. Small-Molecule-Directed Endogenous Regeneration of Visual Function in a Mammalian Retinal Degeneration Model. Int. J. Mol. Sci. 2024, 25, 1521. Abstract: Degenerative retinal diseases associated with photoreceptor loss are a leading cause of visual impairment worldwide, with limited treatment options. Phenotypic profiling coupled with medicinal chemistry were used to develop a small molecule with proliferative effects on retinal stem/progenitor cells, as assessed in vitro in a neurosphere assay and in vivo by measuring Msx1-positive ciliary body cell proliferation. The compound was identified as having kinase inhibitory activity and was subjected to cellular pathway analysis in non-retinal human primary cell systems. When tested in a disease-relevant murine model of adult retinal degeneration (MNU-induced retinal degeneration), we observed that four repeat intravitreal injections of the compound improved the thickness of the outer nuclear layer along with the regeneration of the visual function, as measured with ERG, visual acuity, and contrast sensitivity tests. This serves as a proof of concept for the use of a small molecule to promote endogenous regeneration in the eye.
... The presence of RSCs in the retina of higher vertebrates is still uncertain 4,16,18,19 . Coles et al. suggested that RSCs could be found within the pigmented ciliary epithelium of both mouse and human CMZs [20][21][22] . These pigment cells were identi ed to be capable of forming clonal spheres through proliferation and differentiating into speci c retinal cell types in vitro, implying the presence of RSCs with regenerative potential in the CMZ of higher vertebrates. ...
... These cells are characterized by their distinctive spatial distributions, molecular pro les, and remarkable capacities in terms of self-renewal and differentiation. Earlier studies indicated that a group of pigmented RSCs were detected to be capable of forming cell spheres in vitro in the pigmented CMZ of mammals 20,21 . However, accumulating evidence suggests that these pigmented RSCs might only differentiate into RPE rather than neural retina 23,24 . ...
Human retinal stem cells hold great promise in regenerative medicine, yet their existence and characteristics remain elusive. Here, we preformed single-cell multi-omics and spatial transcriptomics of human fetal retinas and uncovered a novel cell subpopulation, human neural retinal stem-like cells (hNRSCs), distinct from RPE stem-like cell and traditional retinal progenitor cells. These hNRSCs reside in the peripheral retina within the ciliary marginal zone, exhibiting substantial self-renewal and differentiation potential. We conducted single-cell and spatial transcriptomic analysis of human retinal organoids (hROs), and revealed hROs have remarkable similar hNRSCs consistent with fetal retina, capable of regenerating all retinal cells. Furthermore, we identified crucial transcription factors, notably MECOM , governing hNRSC commitment to neural retinogenesis and regulating hROs regeneration. Transplanting hRO-derived hNRSCs into the rd10 mouse of rapid retinal degeneration significantly repairs the degenerated retina and restores visual function. Together, our work identifies and characterizes a unique category of retinal stem cells from human retinas, underscoring their regenerative potential and promise for transplantation therapy.
... This area generates new neurons that are incorporated into the retina as it enlarges continually throughout the lifetime of an animal (Harris & Perron, 1998;Reh & Levine, 1998). This peripheral area of the adult eye may also contain retinal stem cell pools (Ahmad et al, 2000;Tropepe et al, 2000;Coles et al, 2006). These findings further confirm a key role of TMEM107 in differentiation of neural retina and also suggest that SOX2 regulation specifically requires TMEM107. ...
Primary cilia are cellular surface projections enriched in receptors and signaling molecules, acting as signaling hubs that respond to stimuli. Malfunctions in primary cilia have been linked to human diseases, including retinopathies and ocular defects. Here, we focus on TMEM107, a protein localized to the transition zone of primary cilia. TMEM107 mutations were found in patients with Joubert and Meckel–Gruber syndromes. A mouse model lacking Tmem107 exhibited eye defects such as anophthalmia and microphthalmia, affecting retina differentiation. Tmem107 expression during prenatal mouse development correlated with phenotype occurrence, with enhanced expression in differentiating retina and optic stalk. TMEM107 deficiency in retinal organoids resulted in the loss of primary cilia, down-regulation of retina-specific genes, and cyst formation. Knocking out TMEM107 in human ARPE-19 cells prevented primary cilia formation and impaired response to Smoothened agonist treatment because of ectopic activation of the SHH pathway. Our data suggest TMEM107 plays a crucial role in early vertebrate eye development and ciliogenesis in the differentiating retina.
... Immuno uorescence staining of frozen sections of eyeballs was performed as reported (22,23). Eyeball tissue sections were sealed at room temperature for 1 h in PBS containing 3% BSA and 0.3% TritonX-100. ...
Müller differentiated RGCs have potential therapeutic value for glaucoma. However, axonal regeneration of differentiated RGCs has been a difficult problem. Retinal stem cells were differenticated from rat retinal Müller cells. The stem cells were randomly divided into five groups (control group, AAV-STAT3 group, shSTAT3 group, Y27632 group and AAV-STAT3 + Y27632 group). Stem cells in different groups were injected into rat model of glaucoma. The length of axon regeneration in STAT3 combined with Y27632 group was significantly longer than that in other experimental groups. The AAV-STAT3 transfected RGCs treated with Y27632 significantly increased the mRNA levels of Esrrb, Prdm14, Sox2, and Rex1, while decreasing the mRNA levels of Nestin, Eomes, Mixl1, and Gata4. Meanwhile, Socs3, Pten, Klf9, and Mdm4 were significantly lowered, while Dclk2, Armcx1, C-MYC, and Nrn1 were elevated. After injecting differentiated RGCs into the glaucoma model rat eyes, the axon length, RGC layer thickness and the electrophysiology were superior to the glaucoma model group. These findings suggested that STAT3 combined with Y27632 can significantly improve the axonal growth level of Müller differentiated RGCs, and reveal the potential mechanism to induce pluripotency of RGCs.
... 3. Sourced from adult tissues where a few stem cells exist in almost all the organs [8] 4. From cord blood and cord lining mesenchymal and epithelial stem cells. [9] Techniques of delivery of RPE cells: 1. Sub-retinal injection of cell suspension 2. Implant of sub-retinal sheet of RPE cells without any substrate 3. Implant of monolayer RPE cells grown on a synthetic (such as polyethylene terephthalate, polyester, parylene-C membrane, and polylactic-co-glycolic acid) or biological substrate (amniotic membrane). ...
