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Transplantation of human bone marrow mesenchymal stem cells as a thin subretinal layer ameliorates retinal degeneration in a Rat model of retinal dystrophy

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Abstract

Vision incapacitation and blindness associated with retinal degeneration affect millions of people worldwide. Cell based therapy and specifically transplantation of human adult bone marrow-derived stem cells (hBM-MSCs) present possible treatment strategy. Subretinal transplantation of human or rat BM-MSCs was shown previously to improve retinal function in Royal College Surgeons (RCS) rats. In those studies cells were transplanted via a transscleral-transchoroidal approach, creating a localized subretinal bleb. Limited number cells could be injected and photoreceptor rescue was restricted to areas in proximity to the injection site. Here we describe a new surgical method for subretinal transplantation that facilitates uniform distribution of transplanted cells as a thin layer along most of the subretinal space. We assessed the therapeutic effect of hBM-MSCs on RCS rats when transplanted either subretinally or intravitreally. We also examined whether a second transplantation can prolong the therapeutic effect. A cell suspension of 2.5 × 10(6) cells in 5 μl was injected subretinally or intravitreally in RCS rats at 28 days postnatal. In the subretinal group, hBM-MSCs were transplanted posterior to the limbus in the superotemporal part of the eye through a longitudinal triangular scleral tunnel reaching the choroid. In the intravitreal group, the cells were injected into the superotemporal part of the vitreous cavity. In cross sections of subretinally transplanted eyes, removed 2 h following transplantation, hBM-MSCs were distributed as a near-homogenous thin layer along most of the subretinal space. In some animals the cells were also detected in the choroid. In the intravitreal injection group, hBM-MSCs were clustered in the vitreous cavity. Transplanted cells could be detected up to 2 weeks after transplantation but not at later time points. Retinal function and structure were assessed by electroretinogram (ERG) and histology analysis, respectively. Six weeks post transplantation, the mean maximal scotopic ERG b-wave amplitude response recorded in RCS control eyes was 1.2 μV. By contrast, in transplanted eyes mean responses of 56.4 μV and 66.2 μV were recorded in the intravitreally and subretinally transplanted eyes, respectively. In the subretinal group, retinal function was significantly higher in transplanted compared with control eyes up to 20 weeks following transplantation. By contrast, in the intravitreal group, rescue of retinal function persisted only up to 12 weeks following transplantation. Histological analysis revealed that 8 weeks following subretinal transplantation, the retinas of control eyes were dystrophic, with outer nuclear layer (ONL) containing a single cell layer. An extensive photoreceptor rescue was demonstrated in transplanted eyes at this time point, with 3-4 cell layers in the ONL along the entire retina. A second subretinal transplantation at 70 days postnatal did not enhance or prolong the therapeutic effect of hBM-MSCs. No immunosuppressants were used and long-term safety analysis demonstrated no gross or microscopic adverse effects. Taken together our findings suggest that transplantation of hBM-MSCs as a thin subretinal layer enhances the therapeutic effect and the safety of cell transplantation.

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... These cells can be obtained from several tissues and injected in the retina or close to it where they could in theory slow photoreceptor loss and, at the same time, replace the lost photoreceptors (Dias et al. 2018;Gagliardi et al. 2019;Singh et al. 2020;Holan et al. 2021). Particularly, SC harvested from the bone marrow have attracted interest as a therapy for retinal degenerations, because in studies using animal models of retinal degeneration it has been documented that these cells increase neuronal survival and regeneration (Li et al. 2009;Zaverucha-do-Valle et al. 2014;Park et al. 2017;Yazdanyar et al. 2020), including photoreceptor degenerations (Otani et al. 2004;Lu et al. 2010;Tzameret et al. 2014;Moisseiev et al. 2016;Park et al. 2017;Di Pierdomenico et al. 2020a). Because up to now the survival of the cells has been limited, it has been proposed that these beneficial effects are due to a paracrine trophic effect achieved through their sustained release of neurotrophic, anti-angiogenic and immunomodulatory factors (Park et al. 2017;Puertas-Neyra et al. 2020;Garcia-Ayuso et al. 2022). ...
... Because up to now the survival of the cells has been limited, it has been proposed that these beneficial effects are due to a paracrine trophic effect achieved through their sustained release of neurotrophic, anti-angiogenic and immunomodulatory factors (Park et al. 2017;Puertas-Neyra et al. 2020;Garcia-Ayuso et al. 2022). Moreover, it has been documented that these transplants decrease retinal inflammation (Di Pierdomenico et al. 2020a) and this immunosuppressive effect may be also beneficial, as inflammation plays a role in RP and AMD (Tzameret et al. 2014;Tzameret et al. 2015;Moisseiev et al. 2016;Park et al. 2017;Di Pierdomenico et al. 2020a). Thus, its immunosuppressive effect may be key to the treatment of these diseases (Di Pierdomenico et al. 2020a;Garcia-Ayuso et al. 2022). ...
... The RCS rat suffers a mutation of the MERTK gene that affects the ability of the RPE to phagocytose (LaVail 1981;LaVail et al. 2018) and the P23H-1 rat suffers one of the commonest rhodopsin mutations found in human RP (LaVail et al. 2018). Both mutations lead to early and progressive photoreceptor loss (Garcia-Ayuso et al. 2013;Di Pierdomenico et al. 2017;Garcia-Ayuso et al. 2019a;Garcia-Ayuso et al. 2019b) and thus these animal models have been used extensively to study photoreceptor neuroprotection (Green et al. 2001;Inoue et al. 2007;Tzameret et al. 2014;Tzameret et al. 2015;Di Pierdomenico et al. 2017;Fernandez-Sanchez et al. 2017;Qu et al. 2017;Lax et al. 2019). ...
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Purpose: To study and compare effects of syngeneic bone marrow mononuclear stem cells (BM-MNCs) transplants on inherited retinal degeneration in two animal models with different etiologies: the RCS and the P23H-1 rats. To compare the safety and efficacy of two methods of intraocular delivery: subretinal and/or intravitreal. Methods: A suspension of BM-MNCs was injected subretinally or intravitreally in the left eyes of P23H-1 and RCS rats at post-natal day (P) 21. At different survival intervals after the injection: 7, 15, 30 or 60 days, the retinas were cross-sectioned, and photoreceptor survival and glial cell responses were investigated using immunodetection of cones (anti-cone arrestin), synaptic connections (anti-bassoon), microglia (anti-Iba-1), astrocytes and Müller cells (anti-GFAP). Electroretinographic function was also assessed longitudinally. Results: Intravitreal injections (IVIs) or subretinal injections (SRIs) of BM-MNCs did not produce adverse effects. The transplanted cells survived for up to 15 days but did not penetrate the retina. Both IVIs and SRIs increased photoreceptor survival, decreased synaptic degeneration and glial fibrillary acidic protein (GFAP) expression in Müller cells but did not modify microglial cell activation and migration or the electroretinographic responses. Conclusions: Intravitreal and subretinal syngeneic BM-MNCs transplantation decreases photoreceptor degeneration and shows anti-gliotic effects on Müller cells but does not ameliorate retinal function. Moreover, syngeneic BM-MNCs transplants are more effective than the xenotransplants of these cells. BM-MNC transplantation has potential therapeutic effects that merit further investigation.
... Cultured mesenchymal stem cells have been injected intravitreally or subretinally in animal models of retinal degeneration or ischemia. Preclinical studies indicate that the subretinal administration of mesenchymal stem cells can have protective effects in eyes with retinal degeneration (Arnhold et al., 2007;Tzameret et al., 2014). The protective effect is less obvious following intravitreal administration of the cells in eyes with retinal degeneration (Tzameret et al., 2014). ...
... Preclinical studies indicate that the subretinal administration of mesenchymal stem cells can have protective effects in eyes with retinal degeneration (Arnhold et al., 2007;Tzameret et al., 2014). The protective effect is less obvious following intravitreal administration of the cells in eyes with retinal degeneration (Tzameret et al., 2014). For retinal ischemia, intravitreal administration of mesenchymal stem cells has been shown to have a protective effect in preclinical studies (Li et al., 2009). ...
... For retinal ischemia, intravitreal administration of mesenchymal stem cells has been shown to have a protective effect in preclinical studies (Li et al., 2009). Some of these injected mesenchymal stem cells integrate into the retinal surface and stimulate gliosis while others can form a cellular clump in the vitreous cavity (Li et al., 2009;Tzameret et al., 2014). In NOD-SCID mice, intravitreal administration of cultured human mesenchymal stem cells resulted in abnormal cellular 5. Intravitreal injection of human CD34 + stem cells from bone marrow in rd1 mice with retinal degeneration results in rapid homing and integration of these human cells to the surface layers of the retina (Moisseiev et al., 2016). ...
Article
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Stem cell transplantation holds great promise as a potential treatment for currently incurable retinal degenerative diseases that cause poor vision and blindness. Recently, safety data have emerged from several Phase I/II clinical trials of retinal stem cell transplantation. These clinical trials, usually run in partnership with academic institutions, are based on sound preclinical studies and are focused on patient safety. However, reports of serious adverse events arising from cell therapy in other poorly regulated centers have now emerged in the lay and scientific press. While progress in stem cell research for blindness has been greeted with great enthusiasm by patients, scientists, doctors and industry alike, these adverse events have raised concerns about the safety of retinal stem cell transplantation and whether patients are truly protected from undue harm. The aim of this review is to summarize and appraise the safety of human retinal stem cell transplantation in the context of its potential to be developed into an effective treatment for retinal degenerative diseases.
... Mouse BM-MSCs transplanted into rhodopsin knockout mice integrated into the RPE and the neuroretina, which lead to prolonged photoreceptor survival [14]. Human (h) BM-MSCs injected into the subretinal space of a retinal dystrophy rat model showed significant and extensive photoreceptor rescue in transplanted eyes [15]. On the other hand, tail vein injection of rat MSCs preserved visual function in a rat model of RP [16]. ...
... In a previous study, a-and b-wave amplitudes were higher in dark-adapted RCS rats when treated with hBM-MSCs than untreated animals. However, the amplitudes continued to decrease over time [15]. Another study reported a combination of hBM-MSCs and fetal RPCs was used to treat retinal degeneration and showed an increase in the a-and b-wave amplitudes of dark-adapted RCS rats compared to untreated animals [17]. ...
... In general, xenogeneic expression was more significant in RPC than in pMSC transplanted animals. Although several reports studied the effectiveness of cells in treating RDD in animal models [15,56,58], xenogenic expression of neuroprotective and neurogenesis markers has not been well investigated. It is conceivable that retinogenesis may in part be due to the differentiation of transplanted cells. ...
Article
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Background Currently, there is no treatment for retinal degenerative diseases (RDD) such as retinitis pigmentosa (RP). Stem cell-based therapies could provide promising opportunities to repair the damaged retina and restore vision. Thus far, primarily adult mesenchymal stem cells (MSCs) have been investigated in preclinical and clinical studies, and the results have not been convincing. We applied a new approach in which primitive (p) MSC-derived retinal progenitor cells (RPCs) were examined to treat retinal degeneration in an rd12 mouse model of RP. Methods Well-characterized pMSCs and RPCs labeled with PKH26 were intravitreally injected into rd12 mice. The vision and retinal function of transplanted animals were analyzed using electroretinography. Animals were killed 4 and 8 weeks after cell transplantation for histological, immunological, molecular, and transcriptomic analyses of the retina. Results Transplanted RPCs significantly improved vision and retinal thickness as well as function in rd12 mice. pMSCs and RPCs homed to distinct retinal layers. pMSCs homed to the retinal pigment epithelium, and RPCs migrated to the neural layers of the retina, where they improved the thickness of the respective layers and expressed cell-specific markers. RPCs induced anti-inflammatory and neuroprotective responses as well as upregulated the expression of genes involved in neurogenesis. The transcriptomic analysis showed that RPCs promoted neurogenesis and functional recovery of the retina through inhibition of BMP and activation of JAK/STAT and MAPK signaling pathways. Conclusions Our study demonstrated that RPCs countered inflammation, provided retinal protection, and promoted neurogenesis resulting in improved retinal structure and physiological function in rd12 mice.
... SC can be obtained from different accessible tissues such as bone marrow, blood and adipose tissue [10,[37][38][39]. Two types of cells, mesenchymal stem cells or mononuclear/CD34+ stem cells, can be harvested from the bone marrow aspirate and have shown promising results in different animal models of neuronal degeneration [40][41][42][43][44], including photoreceptor degeneration [39,43,[45][46][47][48]. In this article, we use adult human bone-marrow-derived mononuclear/CD34+ stem cells (hBM-MSCs), a fraction that contains a small percentage of hematopoietic, mesenchymal and endothelial stem cells but also monocytes and lymphocytes, between other cells although more than 70% of these cells are CD34+ [43]. ...
... Based on these results, there have been a small number of clinical trials studying hBM-MSC transplantation to the eye to treat retinal degenerative diseases [9,43]. Some of them have shown modest visual improvements and lack of adverse effects [55][56][57][58][59]. Traditionally, studies investigating the effects of BM-MSCs therapies for retinal degenerations have used two means of cell delivery: (i) intravitreal injections, in which cells are delivered into the vitreous close to the internal retina, have shown promising retinal ganglion cell rescue effects [46,55,58,] and (ii) subretinal injections, in which cells are delivered under the retina and thus close to the outer retina and photoreceptors, and have shown variable cell rescue outcomes [46,39,60]. In addition, the results with animal models are highly variable depending on several factors such as the delivery method, the age of the animals, the strain used or the method of analysis [45,48,53,54]. ...
... We used two different methods of injection in dystrophic animals-IVI and SRI-and document the safety of both delivery methods because we did not observe adverse effects. We also document that the transplanted cells persist in the eye next to the inner limiting membrane (ILM) or under the retina for at least 15 days after IVI or SRI, respectively, and this is in accordance with previously published work using similar delivery methods [46]. However, we did not find migration of the injected cells and integration within the retinal layers, in accordance with previous studies [65,68]. ...
Article
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Inherited photoreceptor degenerations are not treatable diseases and a frequent cause of blindness in working ages. In this study we investigate the safety, integration and possible rescue effects of intravitreal and subretinal transplantation of adult human bone-marrow-derived mononuclear stem cells (hBM-MSCs) in two animal models of inherited photoreceptor degeneration, the P23H-1 and the Royal College of Surgeons (RCS) rat. Immunosuppression was started one day before the injection and continued through the study. The hBM-MSCs were injected in the left eyes and the animals were processed 7, 15, 30 or 60 days later. The retinas were cross-sectioned, and Land Scones , microglia, astrocytes and Müller cells were immunodetected. Transplantations had no local adverse effects and the CD45+ cells remained for up to 15 days forming clusters in the vitreous and/or a 2-3-cells-thick layer in the subretinal space after intravitreal or subretinal injections, respectively. We did not observe increased photoreceptor survival nor decreased microglial cell numbers in the injected left eyes. However, the injected eyes showed decreased GFAP immunoreactivity. We conclude that intravitreal or subretinal injection of hBM-MSCs in dystrophic P23H-1 and RCS rats causes a decrease in retinal gliosis but does not have photoreceptor neuroprotective effects, at least in the short term. However, this treatment may have a potential therapeutic effect that merits further investigation.
... Our group has developed a new method for drug delivery into this compartment. In previous studies we demonstrated the long term safety and efficacy of delivering stem cells into the SCS in rats and rabbits in vivo [35,36]. In addition, we demonstrated the efficacy and short term safety of delivering IO/HSA NPs into the SCS compartment of rabbits in vivo [37]. ...
... Gradual thinning of the outer nuclear layer in RCS rats due to photoreceptor degeneration has been extensively studied by our group and others [35,38,43]. As shown in Fig. 3, no significant changes in retinal structure were observed in the injected eyes compared with noninjected contralateral eyes. ...
... h hours, W weeks 45]. We and others have shown that ERG a-and b-wave amplitudes gradually diminish in RCS rats with age as photoreceptors degenerate [35,43,46,47]. As shown in Fig. 4, no significant differences were observed between a-wave and b-wave amplitudes recorded in injected and control non-injected contralateral eyes in all time points following injection (all p values > 0.1). ...
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Background Retinal degeneration diseases affect millions of patients worldwide and lead to incurable vision loss. These diseases are caused by pathologies in the retina and underlying choroid, located in the back of the eye. One of the major challenges in the development of treatments for these blinding diseases is the safe and efficient delivery of therapeutics into the back of the eye. Previous studies demonstrated that narrow size distribution core–shell near infra-red fluorescent iron oxide (IO) nanoparticles (NPs) coated with human serum albumin (HSA, IO/HSA NPs) increase the half-life of conjugated therapeutic factors, suggesting they may be used for sustained release of therapeutics. In the present study, the in vivo tracking by MRI and the long term safety of IO/HSA NPs delivery into the suprachoroid of a rat model of retinal degeneration were assessed. Results Twenty-five Royal College of Surgeons (RCS) pigmented rats received suprachoroidal injection of 20-nm IO/HSA NPs into the right eye. The left eye was not injected and used as control. Animals were examined by magnetic resonance imaging (MRI), electroretinogram (ERG) and histology up to 30 weeks following injection. IO/HSA NPs were detected in the back part of the rats’ eyes up to 30 weeks following injection by MRI, and up to 6 weeks by histology. No significant differences in retinal structure and function were observed between injected and non-injected eyes. There was no significant difference in the weight of IO/HSA NP-injected animals compared to non-injected rats. Conclusions MRI could track the nanoparticles in the posterior segment of the injected eyes demonstrating their long-term persistence, and highlighting the possible use of MRI for translational studies in animals and in future clinical studies. Suprachoroidal injection of IO/HSA NPs showed no sign of adverse effects on retinal structure and function in a rat model of retinal degeneration, suggesting that suprachoroidal delivery of IO/HSA NPs is safe and that these NPs may be used in future translational and clinical studies for extended release drug delivery at the back of the eye. Electronic supplementary material The online version of this article (10.1186/s12951-018-0438-y) contains supplementary material, which is available to authorized users.
... trophic support, immunomodulation, and enhanced neuronal plasticity of stem or progenitor cells) [2]. Several populations of stem cells or progenitor cells, including retinal stem/progenitor cells (RSCs/RPCs), bone marrow mesenchymal stem cells (BMSCs), neural stem cells, and embryonic stem cells have been used for retinal transplantation [3][4][5][6]. In particular, RPCs of rodents, pigs, and humans (hRPCs) have been identified and can be cultured in serum-free media, which allows the survival of neuronal cultures with very few glia [6][7][8][9][10]. ...
... MSCs are the conceptual progenitors of most derivatives from mesoderm and have been identified from several different tissues. Both RPCs and MSCs show great potential in the treatment of retinal degenerative disease [5,23]. In this study, we investigated the growth kinetics and in vitro and in vivo characteristics of SF and S-hRPCs. ...
... When injected into the subretinal space of RCS-p + rats, compared with SF-hRPCs, S-hRPCs showed a stronger capacity in maintaining the thickness of ONL and prolonging the functional integrity of the retina in a way similar to human BMSCs. Transplantion of S-hRPCs causing slowed photoreceptor cell loss may be attributed to the improved circulation and secretion of trophic factors essential for photoreceptor survival [4,5,25]. It is reported that organ-matched mesenchyme permits progenitor proliferation and self-renewal in vitro and in vivo [26]. ...
Article
Retinal progenitor cells (RPCs) have a potential role in the treatment of retinal degenerative diseases. This study is to investigate in vitro and in vivo characteristics and retinal transplantation of RPCs cultured in media with or without serum. Progenitor cells obtained from the neural retina of human eyes at 6-16 weeks gestation were cultured in serum-free media (SF-hRPCs) or in media containing 10% fetal bovine serum (FBS) (S-hRPCs). The differences were characterized between the cells cultured in vitro and transplanted (retinal transplantation) into Royal College of Surgeons (RCS) rats. The functional status of the rats was examined by flash-electroretinogram recordings. The result was that S-hRPCs exhibited higher proliferative dynamics in vitro. On the basis of outer nuclear layer thickness and flash-electroretinograms, S-hRPCs were more efficacious in slowing the progression of retinal degeneration following transplantation compared with SF-hRPCs. Moreover, retinal mesenchymal-like stem cells were isolated and identified from the S-hRPCs cultures. Our study demonstrated the potential of retinal MSCs for the treatment of retinal degeneration.
... Stem cells from human exfoliated deciduous teeth (SHEDs) possess characteristics typical of MSCs including neural differentiation [5]; they express ESC markers [6] and have immunomodulatory action [7]. Reports have confirmed that transplantation of human bone marrow MSCs prolong retinal function in animals with retinal degeneration [8][9][10]. The reparative activity of MSCs in restoring retinal function includes two mechanisms: one is cell replacement, based on neural differentiation, and the other is their paracrine actions that have favorable effects such as neurotropic protection, immunomodulation, antiapoptosis, anti-inflammation, and regulation of angiogenesis [11]. ...
... In vivo bioluminescent luciferase imaging showed 3 weeks of good survival after xenogenous transplantation, but further histological analysis suggested at least 3 months of sustainability in vivo. Similarly, there are reports of MSC sustainability on the basis of histological analysis for 2 weeks [9], or 6 weeks [10], or even 6 months [50] after transplantation in rodents with retinal degeneration. The difference of cell sustainability in histological analysis and in vivo bioluminescence imaging may be due to the methodology. ...
... Transplanted SHEDs decreased considerably with time in vivo. It has been reported that retinal function improves and photoreceptors are rescued for 3 months [8,9] or 5 months [10] even after transplanted MSCs are no longer detectable. The reasons for the therapeutic effects apparently lasting longer than transplanted cell survival in vivo is unclear so far and may be related to the multifunctional effects of MSCs such as neurotrophic, immunomodulatory, antiapoptotic, and angiogenic effects [11]. ...
Article
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Retinal degeneration is characterized by the progressive loss of photoreceptors, and stem cell therapy has become a promising strategy. Many studies have reported that mesenchymal stem cell transplantation can sustain retinal structure and prolong retinal functions based on two mechanisms. One is cell replacement, and the other is the paracrine action of stem cells. Cells from human exfoliated deciduous teeth (SHEDs) show characteristics typical of mesenchymal stem cells. They are derived from the neural crest and are a potential cellular source for neural regeneration in stem cell therapy. In this study, we explored the potential of SHEDs to be induced towards the retinal photoreceptor phenotype and to be sustainable in an animal model of retinal degeneration. A factor-cocktail protocol was used to induce SHEDs towards retinal photoreceptors for 24 days, and the characteristics of the induced cells were identified in terms of morphological changes, biomarker expression and subcellular distribution, and calcium influx. SHEDs were labeled with firefly luciferase for in vivo tracking by bioluminescent imaging and then transplanted into the subretinal space of mice. Our results showed that SHEDs successfully transdifferentiated into photoreceptor-like cells, which displayed neuron-like morphology, and expressed specific genes and proteins associated with retinal precursors, photoreceptor precursors, and mature photoreceptors. In addition, calcium influx was significantly greater in the retinal-induced than in noninduced SHEDs. In vivo tracking confirmed at least 2 weeks of good survival by bioluminescent imaging and 3 months of sustainability of SHEDs by histological analysis. We conclude that SHEDs have the potential to transdifferentiate into retinal photoreceptor-like cells in vitro and maintain good viability in vivo after transplantation into mice with a normal immune system. This demonstrates preliminary success in generating photoreceptor-like cells from SHEDs and applying SHEDs in treating retinal degeneration.
... Thus, allogeneic or autologous cell transplantation of MSCs shows promises for potential therapeutic applications in RDDs. Indeed, several pre-clinical trials of MSCs in the treatment of rodent RDDs (such as streptozotocin or STZ-induced diabetic rodent models, rodent retinal degeneration models, and rodent glaucoma and retinal ischemia models) generated encouraging results (Lund et al., 2007;Guan et al., 2013;Tzameret et al., 2014;Ezquer et al., 2016;Mead et al., 2016;Roth et al., 2016). More than 40% of clinical trials of stem cell therapy for retinal diseases are also using bone marrow or umbilical cord-derived stem cells (Park et al., 2017;Shen, 2020). ...
... It has been demonstrated in vitro that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that secreted factor(s) from MSCs promote photoreceptor cell survival (Inoue et al., 2007). Subretinal or intravitreally injected human BM-MSCs into RCS rat can delay photoreceptor death for about 12-20 weeks (Tzameret et al., 2014). Subretinal transplantation of rat MSCs or engineered erythropoietin (EPO)expression rat MSCs into a sodium iodate (SI)-induced rat model of retinal degeneration protected RPE and retinal neurons; EPO expression MSCs had an even greater effect (Guan et al., 2013). ...
... Indeed, several pre-clinical studies injecting human MSCs into the eyes of rodent disease models (xenotransplantation) without using immunosuppressant have not observed obvious rejection (Lund et al., 2007;Tzameret et al., 2014;Li et al., 2016a;Elshaer et al., 2018). Subretinally administered human adult bone marrow-derived somatic cells (hABM-SCs) can achieve similar therapeutic benefits to protect the rods with or without cyclosporine A in the RCS rats (Lu et al., 2010). ...
Article
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Retinal degenerative diseases (RDDs) are a group of diseases contributing to irreversible vision loss with yet limited therapies. Stem cell-based therapy is a promising novel therapeutic approach in RDD treatment. Mesenchymal stromal/stem cells (MSCs) have emerged as a leading cell source due to their neurotrophic and immunomodulatory capabilities, limited ethical concerns, and low risk of tumor formation. Several pre-clinical studies have shown that MSCs have the potential to delay retinal degeneration, and recent clinical trials have demonstrated promising safety profiles for the application of MSCs in retinal disease. However, some of the clinical-stage MSC therapies have been unable to meet primary efficacy end points, and severe side effects were reported in some retinal “stem cell” clinics. In this review, we provide an update of the interaction between MSCs and the RDD microenvironment and discuss how to balance the therapeutic potential and safety concerns of MSCs' ocular application.
... Numerous studies demonstrated a direct correlation between retinal structure and visual function in animals and humans (2)(3)(4)(5)(6)(7). Traditionally, retinal structure was determined in translational regenerative studies by histological analysis which required sacrificing the animals, removing the eyes for sectioning, hematoxylin and eosin staining, quantification of retinal layer thickness, and immunofluorescence staining for specific cell markers or ultrastructural analysis of the specific retinal cells by electron microscopy (3,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). ...
... Numerous studies demonstrated a direct correlation between retinal structure and visual function in animals and humans (2)(3)(4)(5)(6)(7). Traditionally, retinal structure was determined in translational regenerative studies by histological analysis which required sacrificing the animals, removing the eyes for sectioning, hematoxylin and eosin staining, quantification of retinal layer thickness, and immunofluorescence staining for specific cell markers or ultrastructural analysis of the specific retinal cells by electron microscopy (3,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Histological analysis requires a large number of animals, does not enable longitudinal follow-up, easily permits transient changes to be missed, and most importantly cannot be employed in human clinical trials. ...
Article
Regenerative translational studies must include a longitudinal assessment of the changes in retinal structure and function that occur as part of the natural history of the disease and those that result from the studied intervention. Traditionally, retinal structural changes have been evaluated by histological analysis which necessitates sacrificing the animals. In this review, we describe key imaging approaches such as fundus imaging, optical coherence tomography (OCT), OCT-angiography, adaptive optics (AO), and confocal scanning laser ophthalmoscopy (cSLO) that enable noninvasive, non-contact, and fast in vivo imaging of the posterior segment. These imaging technologies substantially reduce the number of animals needed and enable progression analysis and longitudinal follow-up in individual animals for accurate assessment of disease natural history, effects of interventions and acute changes. We also describe the benefits and limitations of each technology, as well as outline possible future directions that can be taken in translational retinal imaging studies.
... The purpose of our current study was to investigate the effects of human CD34+ cells on degenerating retina using an animal model with much slower rate of retinal degeneration than rd1 mice. Royal College of Surgeons (RCS) rats was selected since it is a commonly used animal model for evaluating the protective effects of therapy on retinal degeneration (21). In order to optimize the regenerative potential of CD34+ BMSCs, we evaluated subretinal administration of human CD34+ BMSCs alone and in combination with exosomes harvested from human MSCs. ...
... In this study, eyes treated with subretinal CD34+ cells showed significantly greater nuclei counts in the ONL at the superior retina near the subretinal injection site at 4 weeks after injection, whereas those treated with intravitreal CD34 cells showed comparable number of nuclei to untreated contralateral control eyes. The observation parallels the finding of a recent study in RCS rats showing a more pronounced and longer lasting rescue effect following subretinal injection of human MSCs (21). It can be hypothesized that the paracrine trophic effect of CD34+ cells may be greater when the cells were placed closer to the damaged cells in the outer retina, i.e., photoreceptor and RPE cells presumably due to limited migration of the cells or secreted factors. ...
Article
Background: To evaluate whether subretinal or intravitreal injection of human CD34+ bone marrow-derived stem cells (BMSC) can have protective effects on retinal degeneration that may be enhanced by coadministration of exosomes harvested from human bone marrow mesenchymal stem cells (MSCs). Methods: Human CD34+ cells were harvested from the mononuclear cell fraction of bone marrow using magnetic beads and labeled with EGFP. Exosomes were harvested from cultured human MSCs under hypoxic conditions. Royal College of Surgeons (RCS) 3-weeks-old rats, immunosuppressed with cyclosporine A, received subretinal or intravitreal injection of CD34+ cells (50,000 cells), CD34+ cells with exosomes (50,000 cells+10 µg), exosomes alone (10 µg), or PBS. Retinal function was examined using electroretinography (ERG), and the eyes were harvested for histologic and immunohistochemical analysis. Results: The b-wave amplitude of ERG at 2 weeks after injection was significantly higher in eyes with subretinal or intravitreal CD34+ BMSC alone or in combination with exosomes when compared to PBS injected eyes or untreated contralateral eyes. At 4 weeks after injection, the ERG signal decreased in all groups but eyes with subretinal CD34+ BMSCs alone or combined with exosomes showed partially preserved ERG signal and preservation of the outer nuclear layer of the retina near the injection site on histology when compared to eyes with PBS injection. Immunohistochemical analysis identified the human cells in the outer retina. Subretinal or intravitreal exosome injection had no effect on retinal degeneration when administered alone or in combination with CD34+ cells. Conclusions: Both subretinal and intravitreal injection of human CD34+ BMSCs can provide functional rescue of degenerating retina, although the effects were attenuated over time in this rat model. Regional preservation of the outer retina can occur near the subretinal injection site of CD34+ cells. These results suggest that CD34+ cells may have therapeutic potential in retinal degeneration.
... If this integrity is compromised and the BRB is breached during surgery, the relative immune privilege of the subretinal space is lost, and immunosuppressive therapy is more likely to be necessary. However, the overall therapeutic effects of correctly performed subretinal transplantation appear to be greater and to last longer than those of intravitreal injections, especially in the context of PR degeneration [207]. Compared to the epiretinal approach, the subretinal microenvironment can better support and promote the differentiation of precursor cells towards PRs [57,206]. ...
... Compared to the epiretinal approach, the subretinal microenvironment can better support and promote the differentiation of precursor cells towards PRs [57,206]. Moreover, compared to intravitreal injections, subretinal grafts are generally associated with better migration and integration, also owing to the closer proximity of the transplant site to the injured retinal layers [207]. ...
Chapter
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Millions of people worldwide suffer from visual disabilities as a result of retinal degeneration. Due to the poor regenerative capability of the central nervous system, retinal cell loss is essentially irreversible. Currently available therapies can only decelerate the degenerative process at late stages and are largely ineffective. However, the possibility of using stem cell-based therapy as cell rescue or cell replacement therapy is broadly being explored. While cell rescue is based on the secretion of biologically active molecules by the transplanted cells, cell replacement refers to the possibility of injected stem cells replacing the defective ones either via direct differentiation, transdifferentiation of the transplanted cells, or via cell fusion-mediated reprogramming of retinal cells.
... Thus, the immunosuppressive properties of MSCs permit an immunological acceptance of cells transplanted into the vitreous cavity of the eye. After a transplantation into the eyes of animals with slow retinal degeneration, such as Royal College of Surgeons (RCS) rats or mouse model of retinitis pigmentosa, MSCs sparsely differentiated into cells with glial characteristics, but not to mature photoreceptors or RPE cells [36]. However, they exerted the protective effects on endogenous photoreceptors and RPE cells [37]. ...
... These results are consistent with previous studies demonstrating that in laser-induced retinal injury, transplanted BM-derived MSCs migrated into the retina where they remained in the proliferative state [54]. We, and other researchers, also observed that after intravitreal injection, the MSC cluster is maintained in the vitreous body for several weeks [40,55], although a small number of MSCs do migrate into the retinal layers, and they are neither tumorigenic nor exhibit uncontrolled growth [36]. ...
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This study aimed to investigate whether the transplantation of genetically engineered bone marrow-derived mesenchymal stromal cells (MSCs) to overexpress brain-derived neurotrophic factor (BDNF) could rescue the chronic degenerative process of slow retinal degeneration in the rd6 (retinal degeneration 6) mouse model and sought to identify the potential underlying mechanisms. Rd6 mice were subjected to the intravitreal injection of lentivirally modified MSC-BDNF or unmodified MSC or saline. In vivo morphology, electrophysiological retinal function (ERG), and the expression of apoptosis-related genes, as well as BDNF and its receptor (TrkB), were assessed in retinas collected at 28 days and three months after transplantation. We observed that cells survived for at least three months after transplantation. MSC-BDNF preferentially integrated into the outer retinal layers and considerably rescued damaged retinal cells, as evaluated by ERG and immunofluorescence staining. Additionally, compared with controls, the therapy with MSC-BDNF was associated with the induction of molecular changes related to anti-apoptotic signaling. In conclusion, BDNF overexpression observed in retinas after MSC-BDNF treatment could enhance the neuroprotective properties of transplanted autologous MSCs alone in the chronically degenerated retina. This research provides evidence for the long-term efficacy of genetically-modified MSC and may represent a strategy for treating various forms of degenerative retinopathies in the future.
... Mice were sacrificed at 13 weeks of age, and their eyes were fixed in 4% formaldehyde, as previously described [39,40]. Retinal paraffin sections were deparaffinized and rehydrated. ...
... All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Group size was determined based on previous studies [40,44]. ...
Article
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The aim of this study was to characterize the distribution of the thrombin receptor, protease activated receptor 1 (PAR1), in the neuroretina. Neuroretina samples of wild-type C57BL/6J and PAR1−/− mice were processed for indirect immunofluorescence and Western blot analysis. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to determine mRNA expression of coagulation Factor X (FX), prothrombin (PT), and PAR1 in the isolated neuroretina. Thrombin activity following KCl depolarization was assessed in mouse neuroretinas ex vivo. PAR1 staining was observed in the retinal ganglion cells, inner nuclear layer cells, and photoreceptors in mouse retinal cross sections by indirect immunofluorescence. PAR1 co-localized with rhodopsin in rod outer segments but was not expressed in cone outer segments. Western blot analysis confirmed PAR1 expression in the neuroretina. Factor X, prothrombin, and PAR1 mRNA expression was detected in isolated neuroretinas. Thrombin activity was elevated by nearly four-fold in mouse neuroretinas following KCl depolarization (0.012 vs. 0.044 mu/mL, p = 0.0497). The intrinsic expression of coagulation factors in the isolated neuroretina together with a functional increase in thrombin activity following KCl depolarization may suggest a role for the PAR1/thrombin pathway in retinal function.
... MSC implantation under the retina, and not into the vitreous cavity, was more beneficial and resulted in improved vision in a rat model of RP [38]. ...
Article
Some ocular diseases, such as dystrophies, retinal and macular degeneration, optic nerve atrophy, and Stargardt disease, are progressive and irreversible. In this review, we focus on the use of mesenchymal stem cells (MSCs) in the treatment of these diseases. In animal studies, MSC transplantation significantly delayed retinal degeneration, led to the regeneration of cone cells, and supported the survival of retinal ganglion cells and axon regeneration. In clinical practice, patients with Behcet's disease with retinal vasculitis who received MSC injections experienced a decrease in retinal vasculitis but no improvement in vision acuity. Nonetheless, there is no evidence that MSCs are carcinogenic, and they even reduce the size of tumors in vitro. Furthermore, MSCs do not trigger the immune response.
... The pupil was dilated using tropicamide (Alcon, Canada) and the eye lid was kept open using a lid speculum. Cell transplantation was performed under a surgical microscope (Ocular Instruments, China) [41]. For subretinal injection, the peritomy was made 2.0 mm posterior to the limbus in the superotemporal quadrant of each eyeball. ...
Preprint
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Background: Retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSCs) exhibit great promise in treating retinal degenerative diseases. Here, we would explore the feasibility of non-colony dissociated hiPSCs to differentiate into functional RPE cells (hiPSC-RPE), and offer an alternative transplantation method based on cell spheroids. Methods: hiPSC-RPE cells were identified using reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence assay, Western blotting, and flow cytometry assay. The functions of hiPSC-RPE cells in vitro and in vivo were assessed by fluorescein leakage test, transepithelial electrical resistance (TEER) assay, atomic force microscopy observation, POS phagocytosis assay, frozen tissue sections, live/dead assay, SA-β-Gal staining, and immunocytochemistry. Results: hiPSC-RPE cells positively expressed biomarkers of RPE cells but not iPSCs, such as CRALBP (97.4%), EMMPRIN (93.8%), Oct4 (2.1%), and Sox2 (2.0%). hiPSC-RPE cells displayed RPE-like characteristics including barrier function, phagocytic activity, and polarized membrane. The cells derived from hiPSC-RPE spheroids positively expressed Nestin and exhibited reduced SA-β-Gal staining. hiPSC-RPE cell spheroids could form monolayer on decellularized corneal matrixes (DCM). After one month of subretinal transplantation, hiPSC-RPE cell spheroids could survive and maintain segmental sheet growth in sodium iodate (NaIO3) induced RPE-degenerated chinchilla rabbits. Conclusion: This study suggested that non-colony dissociated hiPSCs were effectively differentiated into functional RPE cells, and hiPSC-RPE cell spheroids maintained segmental sheet growth in the subretinal of RPE degenerate chinchilla rabbits in vivo, which may lay the foundation for cell spheroid transplantation as an alternative method for RPE degenerative disease therapy in the future.
... HBMSCs were also used in SRS transplantation to treat retinal degenerative diseases. After transplantation, visual function could be improved by reducing the apoptosis of photoreceptors and increasing the electrophysiological response [25][26][27][28] . HBMSCs were believed to produce cytokines and neurotrophic factors, which were able to improve the living conditions of retinal cells and activate the resident stem cells within retina [29][30][31] . ...
... Interestingly, intravitreally injected BM-MSCs were found to integrate into the inner retina and differentiate into retinal glial cells and improve ERG amplitude thereby protecting vision [25], although another study did not find any benefit on ERG [22]. Despite these promising results, the efficacy and potential mechanism of BM-MSC therapy are questionable since a long-term safety study revealed that some of the human bone marrow cells integrated into other ocular structures and circumvented the blood-retinal barrier to migrate into non-target tissue in a similar DR rat model [26]. ...
Article
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Diabetic retinopathy (DR), a complication of diabetes, is one of the leading causes of blindness in working-age adults. The pathology of the disease prevents the endogenous stem cells from participating in the natural repair of the diseased retina. Current treatments, specifically stem cell therapeutics, have shown variable efficacy in preclinical models due to the multi-faceted nature of the disease. Among the various adult stem cells, mesenchymal stem cells, especially those derived from adipose tissue and bone marrow, have been explored as a possible treatment for DR. This review summarizes the current literature around the various adult stem cell treatments for the disease and outlines the benefits and limitations of the therapeutics that are being explored in the field. The paracrine nature of adipose stem cells, in particular, has been highlighted as a potential solution to the lack of a homing and conducive environment that poses a challenge to the implantation of exogenous stem cells in the target tissue. Various methods of mesenchymal stem cell priming to adapt to a hostile retinal microenvironment have been discussed. Current clinical trials and potential safety concerns have been examined, and the future directions of stem cell therapeutics in DR have also been contemplated.
... In addition, it was proved by staining with antibody against rhodopsin that epiretinal transplantation of BMSCs preserved rod photoreceptor cell structure for up to 20 weeks. The ONL showed more layers of cells following stem cell transplantation [38] . ...
... Nevertheless, MSCs often fail to repair injured tissues upon transplantation. This is because of the limited number of donor cells that can be injected and because the rescue of retinal cells is restricted to areas adjacent to the injection site (Tzameret et al. 2014). Moreover, the survival of MSCs is often poor; MSCs succumb to inflammation, oxidative stress or nutrient starvation (Herberg et al. 2013). ...
Article
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Mesenchymal stem cells (MSCs) hold great potential for cell- and gene-based therapies for retinal degeneration. Limited survival is the main obstacle in achieving successful subretinal transplantation of MSCs. The present study sought to evaluate the effect of interleukin-13 (IL-13) gene modification on the phenotypic alteration of retinal microglia (RMG) and the survival of MSCs following subretinal grafting. In this study, LPS-activated RMG were cocultured with MSCs or IL-13-expressing MSCs (IL-13-MSCs) for 24 h, and activated phenotypes were detected in vitro. Western blotting was performed to quantify cytokine secretion by light-injured retinas following subretinal transplantation. The numbers of activated RMG and surviving grafted cells were analysed, and the integrity of the blood–retinal barrier (BRB) was examined in vivo. We found that, compared with normal MSCs, cocultured IL-13-MSCs suppressed the expression of pro-inflammatory factors and major histocompatibility complex II, promoted the expression of anti-inflammatory cytokines by activated RMG and simultaneously inhibited the proliferation of and phagocytosis by RMG. The subretinal transplantation of IL-13-MSCs increased the expression of neurotrophic factors, IL-13 and tight junction proteins in the host retina, decreased the number of phagocytic RMG and improved the survival of grafted cells. Furthermore, IL-13-MSCs alleviated BRB breakdown induced by subretinal injection. Our results demonstrate that IL-13-MSCs can polarize activated RMG to the neuroprotective M2 phenotype and enhance the survival of grafted MSCs against the damage stress induced by subretinal transplantation.
... Intraparenchymal delivered MSCs were proven to be safe, and significantly delayed the loss of motor neurons [144]. Tzameret et al. found that intravitreally injected MSCs ameliorate retinal degeneration by integrating into the neural layers of the damaged retina [145]. Moreover, analysis of tissues after MSC transplantation revealed cell fusion between transplanted MSCs and cells of the recipient, albeit at a low frequency. ...
Article
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Mesenchymal stem cells (MSCs) have been extensively investigated for the treatment of various diseases. The therapeutic potential of MSCs is attributed to complex cellular and molecular mechanisms of action including differentiation into multiple cell lineages and regulation of immune responses via immunomodulation. The plasticity of MSCs in immunomodulation allow these cells to exert different immune effects depending on different diseases. Understanding the biology of MSCs and their role in treatment is critical to determine their potential for various therapeutic applications and for the development of MSC-based regenerative medicine. This review summarizes the recent progress of particular mechanisms underlying the tissue regenerative properties and immunomodulatory effects of MSCs. We focused on discussing the functional roles of paracrine activities, direct cell–cell contact, mitochondrial transfer, and extracellular vesicles related to MSC-mediated effects on immune cell responses, cell survival, and regeneration. This will provide an overview of the current research on the rapid development of MSC-based therapies.
... The surgical method for MSC delivery also affects the survival time of MSCs in host eyes and the therapeutic effect of MSCs in RCS rats. In subretinal injection treated eyes, retinal function can be significantly better than in control eyes for up to 20 weeks, whereas this improvement can be found for only up to 12 weeks in intravitreally injected eyes (Tzameret et al., 2014). In RP animal models, transplantation of bone marrow-derived MSCs can preserve ONL cells, prolonging photoreceptor survival (Arnhold et al., 2007). ...
Article
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Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases, such as age-related macular degeneration (AMD), Stargardt's disease, retinitis pigmentosa (RP) and glaucoma. These diseases are all characterized by the degeneration of one or two retinal cell types that cannot regenerate spontaneously in humans. Aberrant retinal pigment epithelial (RPE) cells can be observed through optical coherence tomography (OCT) in AMD patients. In RP patients, the morphological and functional abnormalities of RPE and photoreceptor layers are caused by a genetic abnormality. Stargardt's disease or juvenile macular degeneration, which is characterized by the loss of the RPE and photoreceptors in the macular area, causes central vision loss at an early age. Loss of retinal ganglion cells (RGCs) can be observed in patients with glaucoma. Once the retinal cell degeneration is triggered, no treatments can reverse it. Transplantation-based approaches have been proposed as a universal therapy to target patients with various concomitant diseases. Both the replacement of dead cells and neuroprotection are strategies used to rescue visual function in animal models of retinal degeneration. Diverse retinal cell types derived from pluripotent stem cells, including RPE cells, photoreceptors, RGCs and even retinal organoids with a layered structure, provide unlimited cell sources for transplantation. In addition, mesenchymal stem cells (MSCs) are multifunctional and protect degenerating retinal cells. The aim of this review is to summarize current findings from preclinical and clinical studies. We begin with a brief introduction to retinal degenerative diseases and cell death in diverse diseases, followed by methods for retinal cell generation. Preclinical and clinical studies are discussed, and future concerns about efficacy, safety and immunorejection are also addressed.
... The pupil was dilated using tropicamide (Alcon, Canada) and the eye lid was kept open using a lid speculum. Cell transplantation was performed under a surgical microscope (Ocular Instruments, China) [46]. For subretinal injection, the peritomy was made 2. ...
Preprint
Full-text available
Background Retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSCs) exhibit great promise in treating retinal degenerative diseases. To develop transplantable and functional hiPSC-RPE cells, here, we used a novel differentiation protocol based on a non-colony-type monolayer (NCM) culture and injectable spheroids. Methods The derived hiPSC-RPE cells were identified using reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence assay, Western blotting, and flow cytometry assay. The functions of transplantable hiPSC-RPE cells in vitro and in vivo were also analyzed by fluorescein leakage test, transepithelial electrical resistance (TEER) assay, atomic force microscopy observation, POS phagocytosis assay, frozen tissue sections, live/dead assay, SA-β-Gal activity assay, and immunocytochemistry. Results The derived hiPSC-RPE cells positively expressed biomarkers of RPE cells but not iPSCs, such as CRALBP (97.4%), EMMPRIN (93.8%), Oct4 (2.1%), and Sox2 (2.0%). hiPSC-RPE cells displayed RPE-like characteristics including barrier function, phagocytic activity, and polarized membrane. hiPSC-RPE cell spheroids positively expressed Nestin and exhibited reduced SA-β-Gal staining. Injectable hiPSC-RPE cell spheroids could form monolayers on decellularized corneal matrixes (DCM). After subretinal transplantation for one month, hiPSC-RPE cell spheroids could survive and maintain segmental sheet growth in RPE-degenerated chinchilla rabbits. Conclusion This study realized that NCM dissociated hiPSCs were effectively differentiated into transplantable and functional RPE through the sequential addition of defined factors but not involving exogenous genes. This study may lay the foundation for the clinical transplantation of hiPSC-RPE cell spheroids as therapy for RPE degenerative diseases in the future.
... The in vitro study performed by Duan et al. has shown that bone marrowÀderived MSCs can be differentiated into cells with RPE features [182]. Subretinal injection of bone marrowÀderived MSCs has shown direct integration into the RPE layer and prolongs the survival of photoreceptor [183] and ameliorates the retinal degeneration in animal models [184]. Intravitreal injection of MSCs has been explored in preclinical studies. ...
Chapter
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Stem cells have the capacity for differentiation and self-renewal. In the eye, endogenous retinal cells have been identified to express stem cell characteristics, which are crucial for maintaining and repairing the eye during physiological condition. On the other hand, exogenous stem cells are examined as an alternative source for stem cell therapy. Here, we review the properties of endogenous retinal stem cells in particular Müller, ciliary-epithelial, corneal epithelial, and retinal pigment epithelial (RPE) cells, together with exogenous stem cells, including induced pluripotent stem cells (iPSCs)/embryonic stem cells, mesenchymal stem cells, and hematopoietic stem cells and discuss the potential advantages as cell therapies for retinal degeneration diseases.
... Morphologically, mNPC-exos decreased the amount of apoptosis in photoreceptors and delayed the thinning of the ONL. We observed a transient reduction of b-wave amplitudes in mNPC-exo and vehicle groups at days 2 and 4, which was most likely due to the acute injury caused by the injection procedure [43], while the apoptosis of photoreceptors in mNPC-exo group reduced markedly compared to that of the vehicle and the untreated group. The mNPC-exo group had higher amplitudes of both a-wave and b-wave than that of the vehicle and the untreated groups at days 7 and 14, indicating the visual-protective effects of mNPCexos, which was consistent with the anti-apoptosis results in Figure 1(h). ...
Article
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Retinal degeneration (RD) is one of the most common causes of visual impairment and blindness and is characterized by progressive degeneration of photoreceptors. Transplantation of neural stem/progenitor cells (NPCs) is a promising treatment for RD, although the mechanisms underlying the efficacy remain unclear. Accumulated evidence supports the notion that paracrine effects of transplanted stem cells is likely the major approach to rescuing early degeneration, rather than cell replacement. NPC-derived exosomes (NPC-exos), a type of extracellular vesicles (EVs) released from NPCs, are thought to carry functional molecules to recipient cells and play therapeutic roles. In present study, we found that grafted human NPCs (hNPCs) secreted EVs and exosomes in the subretinal space (SRS) of RCS rats, an RD model. And direct administration of mouse neural progenitor cell-derived exosomes (mNPC-exos) delayed photoreceptor degeneration, preserved visual function, prevented thinning of the outer nuclear layer (ONL), and decreased apoptosis of photoreceptors in RCS rats. Mechanistically, mNPC-exos were specifically internalized by retinal microglia and suppressed their activation in vitro and in vivo. RNA sequencing and miRNA profiling revealed a set of 17 miRNAs contained in mNPC-exos that markedly inhibited inflammatory signal pathways by targeting TNF-α, IL-1β, and COX-2 in activated microglia. The exosomes derived from hNPC (hNPC-exos) contained similar miRNAs to mNPC-exos that inhibited microglial activation. We demonstrated that NPC-exos markedly suppressed microglial activation to protect photoreceptors from apoptosis, suggesting that NPC-exos and their contents may be the mechanism of stem cell therapy for treating RD.
... We developed a minimally invasive method for drug delivery into this compartment using a blunt adjustable depth injection system. In previous studies, we demonstrated safe and efficient injection of various solutions containing dyes, nanoparticles and human cells into the EVSC compartment in rats and rabbits using this method [8][9][10]. The rabbits were monitored with fundus imaging, optical coherence tomography (OCT) and electroretinography (ERG) for up to 7 weeks following injection. ...
Article
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PurposeTo evaluate the efficacy and safety of injecting increasing volumes into the extravascular spaces of the choroid (EVSC) in rabbit eyes in vivo using a blunt adjustable depth injector.Methods Indocyanine green (ICG) was injected in the superior–temporal quadrant, 2 mm posterior to the limbus at increasing volumes (0.1–0.3 ml) into the EVSC of New Zealand rabbit eyes in vivo. Intraocular pressure (IOP) measurements, spectral domain optical coherence tomography (SD-OCT), fundus imaging and histology analysis were performed to assess the safety and efficacy of the injection.ResultsVolumes up to 0.3 ml were administered consistently. ICG injection was successfully monitored in vivo using infrared fundus imaging and SD-OCT. ICG was detected across the EVSC compartment, reaching the retinal pigment epithelium, optic nerve head and visual streak. Injection of 0.3 ml yielded maximal dye distribution with a coverage area of 61.8% ± 6.7% (mean ± standard error, SE) of the posterior segment. Maximal IOP elevation was recorded 5 min following injection of 0.2 and 0.3 ml ICG (+ 20.0 mmHg, + 19.4 mmHg, respectively). Twenty minutes post-injection, the IOP was < 15 mmHg in all injection volumes. No retinal detachment or hemorrhages were detected in any of the injected eyes.Conclusions This study demonstrates consistent and safe delivery of large volumes within the EVSC using a blunt adjustable depth injector that distributes the dye over 60% of the retinal surface. This injection system may offer a minimally invasive and easy way to deliver large volumes of pharmaceuticals into the posterior segment.
... There are also several studies which have used human BM-MSC-EVs in ophthalmology, showing their beneficial effects in rat retinal and retinal ganglion cell cultures [100,101] and in animal models of glaucoma [102,103] and optic nerve crush [101]. As well as AT-MSC, BM-MSC have also been widely used in ophthalmology [104][105][106][107][108][109][110][111][112][113], including 8 out of 293 registered clinical trials with these cells (ClinicalTrials.gov, NCT01531348, NCT01562002 [114], NCT01920867 [115,116], NCT02325843, NCT02330978, NCT03011541 [117], NCT03173638 and NCT03967275). ...
Article
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In recent years, the interest in adipose tissue mesenchymal cell–derived extracellular vesicles (AT-MSC-EVs) has increasingly grown. Numerous articles support the potential of human AT-MSC-EVs as a new therapeutic option for treatment of diverse diseases in the musculoskeletal and cardiovascular systems, kidney, skin, and immune system, among others. This approach makes use of the molecules transported inside of EVs, which play an important role in cell communication and in transmission of macromolecules. However, to our knowledge, there is no database where essential information about AT-MSC-EVs cargo molecules is gathered for easy reference. The aim of this study is to describe the different molecules reported so far in AT-MSC- EVs, their main molecular functions, and biological processes in which they are involved. Recently, the presence of 591 proteins and 604 microRNAs (miRNAs) has been described in human AT-MSC-EVs. The main molecular function enabled by both proteins and miRNAs present in human AT-MSC-EVs is the binding function. Signal transduction and gene silencing are the biological processes in which a greater number of proteins and miRNAs from human AT-MSC-EVs are involved, respectively. In this review we highlight the therapeutics effects of AT-MSC-EVs related with their participation in relevant biological processes including inflammation, angiogenesis, cell proliferation, apoptosis and migration, among others. Graphical abstract
... Moreover, despite the feasibility of intravitreal administration of stem cells, which stands on paracrine effects of growth factors, subretinal transplantation resulted in more long-lasting effects on delaying retinal and photoreceptor degeneration. The underlying reason of this more favorable effect is that when delivered subretinally, the cells are transplanted in best proximity to the degenerating cells (Tzameret et al. 2014). ...
Article
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Retinal degenerative diseases such as retinitis pigmentosa (RP) are of the major causes of vision loss in developed countries. Despite the unclear pathophysiology, treatment methods have been investigated vastly in the past decades. This review article mainly discusses the advances in application of stem cell and progenitor transplantation for retinitis pigmentosa. Stem cell sources such as mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, neural stem cells, retinal progenitor cells, and olfactory ensheathing cells are discussed separately in addition to a brief description of two approaches for treatment of early-stage RP, including gene therapy and nutritional therapy.
... Mesenchymal stem cells (MSCs) of different origins have shown the greatest potential in the treatment of optical neuropathy [4][5][6]. For example, intravitreal transplantation of human umbilical cord (hUC) blood stem cells or dental pulp stem cells is neuroprotective for RGCs in rats with optic injury [6][7][8][9]. An emerging alternative is the use of hUC-MSCs, which are derived from Wharton's jelly, the particular connective tissue among the veins and arteries of the fetal cord [10][11][12][13][14]. hUC-MSCs have the biological features of both adult stem cells and embryonic stem (ES) cells but are closer to the original form and proliferative than are adult stem cells. ...
Article
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Glaucoma is the leading cause of irreversible blindness worldwide, and pathologically elevated intraocular pressure (IOP) is the major risk factor. Neuroprotection is one of the potential therapies for glaucomatous retinal damage. Intravitreal mesenchymal stem cell (MSC) transplantation provides a viable therapeutic option, and human umbilical cord- (hUC-) MSCs are attractive candidates for cell-based neuroprotection. Here, we investigated the ability of transplanted hUC-MSCs to survive and migrate within the vitreous cavity and their neuroprotective effects on chronic glaucomatous retina. For this, we developed a chronic ocular hypertension (COH) rat model through the intracameral injection of allogeneic Tenon’s fibroblasts. Green fluorescent protein-transduced hUC-MSCs were then injected into the vitreous cavity one week after COH induction. Results showed that a moderate IOP elevation lasted for two months. Transplanted hUC-MSCs migrated toward the area of damaged retina, but did not penetrate into the retina. The hUC-MSCs survived for at least eight weeks in the vitreous cavity. Moreover, the hUC-MSCs were efficient at decreasing the loss of retinal ganglion cells; retinal damage was attenuated through the inhibition of apoptosis. In this study, we have developed a novel COH rat model and demonstrated the prolonged neuroprotective potential of intravitreal hUC-MSCs in chronic glaucoma.
... It is difficult to know whether ESC survival and immunogenicity is species specific or a result of the particular environment in which they are injected. Evidence of this site specific immune-privilege can be found in the eye, where stem cell grafts in the subretinal space of the rat eye show much better cell integration and migration compared to that of the vitreous cavity (143,144). However, it must be noted that in a diseased state even so called immune privileged sites are often compromised due to breakdown of blood-tissue barriers (145). ...
Thesis
Tendon injuries occur commonly in equine athletes. Adult tendons undergo poor natural regeneration, resulting in scar-tissue which is prone to re-injury. Fetal tendons however are capable of completely scar-less regeneration, a property which is intrinsic to the fetal cells themselves. Novel cell therapies should therefore try to recapitulate this scar-less fetal tendon regeneration. This thesis builds on previous research into the use of horse embryonic stem cells (ESCs) to aid tendon regeneration. The aim of this thesis was to determine if tendon cells derived from ESCs were more similar to fetal or adult tendon cells, as well as try to understand if scleraxis (SCX), an essential gene in tendon formation, has different roles at different stages of tendon development. Equine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture was compared using RNA-sequencing. No qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome wide transcriptomic analysis using RNA- sequencing revealed that ESC-derived tenocytes transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this thesis adds to the growing evidence that monolayer cultured cells gene expression profiles converge, with adult and fetal tenocytes having only 10 differentially expressed (DE) genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in three- dimensional culture, large distinctions in gene expression between these two developmental stages were found, with 542 genes being DE. The effects of knocking down the expression of SCX on gene expression in adult, fetal and ESC-derived tenocytes was then determined using RNA-sequencing and qPCR. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes effected in adult tenocytes, indicating that scleraxis- dependent processes differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, novel genes that SCX differentially interacts with in adult and fetal tenocytes were identified. SCX knockdown in ESCs resulted in upregulation of cartilage markers, a result which still needs to be confirmed in further biological replicates. In summary, the data presented in this thesis provides an unprecedented insight into some of the differences between fetal regenerative and adult reparative tenocytes. It also indicated that ESC-derived tenocytes are more similar to fetal rather than adult tenocytes, highlighting their potential as a therapeutic cell source. The results presented also indicate a role for SCX in modulating ECM synthesis and breakdown and provides a useful dataset for further study into SCX gene regulation. Taken together this data is likely to be important for the future development of novel cellular or pharmacological therapeutics.
... Surgical procedures to access the SCS have been reported in different studies [27][28][29][30][31][32]. The surgical approach includes an ab externo incision through the sclera (a.k.a. ...
Article
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The suprachoroidal space (SCS), a potential space between the sclera and choroid, is becoming an applicable method to deliver therapeutics to the back of the eye. In recent years, a vast amount of research in the field has been carried out, with new discoveries in different areas of interest, such as imaging, drug delivery methods, pharmacokinetics, pharmacotherapies in preclinical and clinical trials and advanced therapies. The SCS can be visualized via advanced techniques of optical coherence tomography (OCT) in eyes with different pathologies, and even in healthy eyes. Drugs can be delivered easily and safely via hollow microneedles fitted to the length of the approximate thickness of the sclera. SCS injections were found to reach greater baseline concentrations in the target layers compared to intravitreal (IVT) injection, while agent clearance was faster with highly aqueous soluble molecules. Clinical trials with SCS injection of triamcinolone acetonide (TA) were executed with promising findings for patients with noninfectious uveitis (NIU), NIU implicated with macular edema and diabetic macular edema (DME). Gene therapy is evolving rapidly with viral and non-viral vectors that were found to be safe and efficient in preclinical trials. Here, we review these novel different aspects and new developments in clinical treatment of the posterior segment of the eye.
... Nakano et al [48] has described the formation of optic cup and stratified retina from hPSCs. Several successive studies have confirmed the possibility of creating retinal organoids and layers of differentiated photoreceptors, which can develop outer segment structures [117]. Organoids may prove valuable in producing specific retinal cell types or 3D retinal structures for transplantation. ...
Article
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Stem cell therapies are successfully used in various fields of medicine. This new approach of research is also expanding in ophthalmology. Huge investments, resources and important clinical trials have been performed in stem cell research and in potential therapies. In recent years, great strides have been made in genetic research, which permitted and enhanced the differentiation of stem cells. Moreover, the possibility of exploiting stem cells from other districts (such as adipose, dental pulp, bone marrow stem cells, etc.) for the treatment of ophthalmic diseases, renders this topic fascinating. Furthermore, great strides have been made in biomedical engineering, which have proposed new materials and threedimensional structures useful for cell therapy of the eye. The encouraging results obtained on clinical trials conducted on animals have given a significant boost in the creation of study protocols also in humans. Results are limited to date, but clinical trials continue to evolve. Our attention is centered on the literature reported over the past 20 years, considering animal (the most represented in literature) and human clinical trials, which are limiting. The aim of our review is to present a brief overview of the main
... Despite its beneficial effects, MSCs can induce adverse effects like reactive astrogliosis, probably by upregulation of lipocalin 2 (Lcn2) (Jin et al., 2014). Proinflammatory vitreous clumping of MSCs when injected in the vitreous (Tzameret et al., 2014) or thick epiretinal membrane formation (Kim et al., 2017) limits the use of MSCs. Grafting of neural stem cells (NSCs) may also be beneficial in the treatment of neuronal damage. ...
Article
Traumatic brain injury (TBI) is a major cause of mortality and morbidity in the USA as well as in the world. As a result of TBI, the visual system is also affected often causing complete or partial visual loss, which in turn affects the quality of life. It may also lead to ocular motor dysfunction, defective accommodation, and impaired visual perception. As a part of the therapeutic strategy, early rehabilitative optometric intervention is important. Orthoptic therapy, medication, stem cell therapy, motor and attention trainings are the available treatment options. Gene therapy is one of the most promising emerging strategies. Use of state-of-the-art nanomedicine approaches to deliver drug(s) and/or gene(s) might enhance the therapeutic efficacy of the present and future modalities. More research is needed in these fields to improve the outcome of this debilitating condition. This review focuses on different visual pathologies caused by TBI, advances in pre-clinical and clinical research, and available treatment options.
... We described two different outcomes observed after intravitreal injections of bone marrow MSCs in human eyes, highlighting the concerns related to this procedure's safety. Although MSCs therapy has been previously associated with improvements in the retinal function, as in experimental retinal dystrophy [9] and assessed by ERG in glaucoma [10], the safety of MSCs injections in humans was still unknown. After bone marrow-derived MSCs treatment, ERG outcomes showed no functional improvement that could have been detected, for instance, with an increase in the photopic-negative response amplitude. ...
Article
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PurposeTo report electroretinographic (ERG) findings in advanced glaucoma treated with a single intravitreal injection of bone marrow-derived mesenchymal stem cells (MSCs).Methods Intravitreal injection of autologous MSCs (1 × 106 cells) was performed in 2 eyes from 2 patients with open-angle glaucoma in advanced stage of optic neuropathy (ClinicalTrials.gov, NCT02330978, 01.05.2015): cup/disk ratio worse than 0.9, visual field mean deviation index lower than − 15 dB, visual acuity of light perception, but controlled intraocular pressure. ERG tests were recorded at baseline and week 1, 4 and 48 after injection, using DTL electrodes following the ISCEV standard: After dark adaptation, ERG was elicited using white flashes of 0.01 cd.s/m2 and 3.0 cd.s/m2, followed by 10-min light adaptation (30 cd/m2) and stimuli of 3.0 cd.s/m2 and 30 Hz flicker.ResultsPatients did not show improvement on visual acuity or visual field after treatment. At baseline, ERG responses showed typical findings for advanced glaucoma, with a- and b-wave amplitude and latency within normative range, but reduced photopic negative responses. No noteworthy changes were observed on ERG responses for both cases up to 1 week after treatment, but at day 15, one patient showed retinal detachment with proliferative vitreoretinopathy and was removed from the trial. The other patient kept ERG responses stable throughout study period.Conclusion Although no ERG response changes were observed after MSCs injection in one case, the complication observed on the second one, along with the lack of visual function improvement, warrants further studies involving modified MSCs to treat ocular disorders, including glaucoma.Trial registration: ClinicalTrials.gov, NCT02330978- missed in pdf
... Intravitreal administration reduces this possibility, but the inner limiting membrane may block the migration of donor cells [56]. Cellular cluster formation in the vitreous occurred in some cases of MSC intravitreal injection [166]. Less prominent and shorter therapeutic effects than those following subretinal injection have also been noticed [7]. ...
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As the human retina has no regenerative ability, stem cell interventions represent potential therapies for various blinding retinal diseases. This type of therapy has been extensively studied in the human eyes through decades of preclinical studies. The safety profiles shown in clinical trials thus far have indicated that these strategies should be further explored. There are still challenges with regard to cell source, cell delivery, immuno-related adverse events and long-term maintenance of the therapeutic effects. Retinal stem cell therapy is likely to be most successful with a combination of multiple technologies, such as gene therapy. The purpose of this review is to present a synthetical and systematic coverage of stem cell therapies that target retinal diseases from bench to bedside, intending to appeal to both junior specialists and the broader community of clinical investigators alike. This review will only focus on therapies that have already been studied in clinical trials. This review summarizes key concepts, highlights the main studies in human patients and discusses the current challenges and potential methods to reduce safety concerns while enhancing the therapeutic effects.
... 3,4 Although promising. [5][6][7][8][9][10][11][12][13][14] SCTs require further development and optimization. In particular, cells transplanted into the eye do not efficiently migrate and integrate into the retina, 3,6,15-17 especially following intravitreal injection. ...
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Cell therapy approaches hold great potential for treating retinopathies, which are currently incurable. This study addresses the problem of inadequate migration and integration of transplanted cells into the host retina. To this end, we have identified the chemokines that were most upregulated during retinal degeneration and that could chemoattract mesenchymal stem cells (MSCs). The results were observed using a pharmacological model of ganglion/amacrine cell degeneration and a genetic model of retinitis pigmentosa, from both mice and human retinae. Remarkably, MSCs overexpressing Ccr5 and Cxcr6, which are receptors bound by a subset of the identified chemokines, displayed improved migration after transplantation in the degenerating retina. They also led to enhanced rescue of cell death and to preservation of electrophysiological function. Overall, we show that chemokines released from the degenerating retinae can drive migration of transplanted stem cells, and that overexpression of chemokine receptors can improve cell therapy-based regenerative approaches.
... It is recognized that MSCT maintains tissue homeostasis through either paracrine effects to establish beneficial microenvironments or through inhabitation in recipient tissues to replenish deficient cells [14,55]. After intravitreal transplantation, exogenous MSCs have been traced to remain in the vitreous body, in which no retinal incorporation has been reported [56][57][58]. Nevertheless, it has been claimed in MSCT treating a mouse model of RP that transplanted MSCs morphologically integrated into the RPE, while not being detected in other retinal layers [10]. ...
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Photoreceptor apoptosis is recognized as one key pathogenesis of retinal degeneration, the counteraction of which represents a promising approach to safeguard visual function. Recently, mesenchymal stem cell transplantation (MSCT) has demonstrated immense potential to treat ocular disorders, in which extracellular vesicles (EVs), particularly exosomes, have emerged as effective ophthalmological therapeutics. However, whether and how MSCT protects photoreceptors against apoptotic injuries remains largely unknown. Here, we discovered that intravitreal MSCT counteracted photoreceptor apoptosis and alleviated retinal morphological and functional degeneration in a mouse model of photoreceptor loss induced by N-methyl-N-nitrosourea (MNU). Interestingly, effects of MSCT were inhibited after blockade of exosomal generation by GW4869 preconditioning. Furthermore, MSC-derived exosomal transplantation (EXOT) effectively suppressed MNU-provoked photoreceptor injury. Notably, therapeutic efficacy of MSCT and EXOT on MNU-induced retinal degeneration was long-lasting as photoreceptor preservance and retinal maintenance were detected even after 1-2 months post to injection for only once. More importantly, using a natural occurring retinal degeneration model caused by a nonsense mutation of Phosphodiesterase 6b gene (Pde6b mut), we confirmed that MSCT and EXOT prevented photoreceptor loss and protected long-term retinal function. In deciphering therapeutic mechanisms regarding potential exosome-mediated communications, we identified that miR-21 critically maintained photoreceptor viability against MNU injury by targeting programmed cell death 4 (Pdcd4) and was transferred from MSC-derived exosomes in vivo for functional regulation. Moreover, miR-21 deficiency aggravated MNU-driven retinal injury and was restrained by EXOT. Further experiments revealed that miR-21 mediated therapeutic effects of EXOT on MNU-induced photoreceptor apoptosis and retinal dysfunction. These findings uncovered the efficacy and mechanism of MSCT-based photoreceptor protection, indicating exosomal miR-21 as a therapeutic for retinal degeneration.
... Copyright 2013 American Chemical Society. U.B. Kompella et al. line (Tassoni et al., 2015;Tzameret et al., 2014;Dominici et al., 2006;Kim et al., 2017). Recently at least four clinical trials are in place evaluating stem cells for glaucoma, NCT02330978, NCT01920867, NCT03011541, and NCT02144103. ...
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Although once daily anti-glaucoma drug therapy is a current clinical reality, most therapies require multiple dosing and there is an unmet need to develop convenient, safe, and effective sustained release drug delivery systems for long-term treatment to improve patient adherence and outcomes. One of the first sustained release drug delivery systems was approved for the reduction of intraocular pressure in glaucoma patients. It is a polymeric reservoir-type insert delivery system, Ocusert™, placed under the eyelid and on the ocular surface for zero-order drug release over one week. The insert, marketed in two strengths, released Pilocarpine on the eye surface. While many clinicians appreciated this drug product, it was eventually discontinued. No similar sustained release non-invasive drug delivery system has made it to the market to date for treating glaucoma. Drug delivery systems under development include punctal plugs, ring-type systems, contact lenses, implants, microspheres, nanospheres, gels, and other depot systems placed in the extraocular, periocular, or intraocular regions including intracameral, supraciliary, and intravitreal spaces. This article discusses the advantages and disadvantages of the various routes of administration and delivery systems for sustained glaucoma therapy. It also provides the reader with some examples and discussion of drug delivery systems that could potentially be applied for glaucoma treatment. Interestingly, one intracamerally injected implant, Durysta™, was approved recently for sustained intraocular pressure reduction. However, long-term acceptance of such devices has yet to be established. The ultimate success of the delivery system will depend on efficacy relative to eye drop dosing, safety, reimbursement options, and patient acceptance. Cautious development efforts are warranted considering prior failed approaches for sustained glaucoma drug delivery. Neuroprotective approaches for glaucoma therapy including cell, gene, protein, and drug-combination therapies, mostly administered intravitreally, are also rapidly progressing towards assessment in humans.
Chapter
In developed countries, blindness and visual impairment are caused mainly by diseases affecting the retina. These retinal degenerative diseases, including age-related macular dystrophy (AMD) and inherited retinal diseases such as retinitis pigmentosa (RP), are the predominant causes of human blindness worldwide and are responsible for more than 1.5 million cases in France and more than 30 million cases worldwide. Global prevalence and disease burden projections for next 20 years are alarming (Wong et al., Lancet Glob Health 2(2):e106–e116, 2014) and strongly argue toward designing innovative eye-care strategies. At present, despite the scientific advances achieved in the last years, there is no cure for such diseases, making retinal degenerative diseases an unmet medical need.
Article
Purpose To test the in-vivo bio-distribution and safety of bevacizumab delivery into the suprachoroidal space (SCS) using a novel injection system in a large eye model. Methods Bevacizumab (1.25 mg) was injected into the vitreous (IVT, 50µL, n=12) or the SCS, (150µL, n=37) of live rabbits. Immunofluorescence and ELISA were used to assess bevacizumab distribution. Intraocular pressure (IOP) measurements, SD-OCT and fundus imaging, electroretinogram, and histology analysis were performed for safety assessment. Results Bevacizumab was observed throughout the choroid layers up to the retinal pigment epithelium (RPE), within 1 hour following SCS injection. The Cmax of bevacizumab in the retina/choroid was 1043 ± 597 μg/gr tissue (mean± standard error), 40-fold higher than in IVT injected eyes (p=0.0339). One day following SCS injection, bevacizumab was detected throughout the posterior pole with a two-fold lower concentration. One week post-SCS injection, bevacizumab concentration in the retina/choroid dropped to 2.36 ± 1.32 μg/gr tissue (p=0.034 vs. 1 hour), with a half-life of 20 hours. No suprachoroidal blebs, retinal detachment, hemorrhages, inflammation or changes in retinal function were observed up to 2 months following SCS injection. Elevated IOP (+16 mmHg) was observed two minutes post-SCS injection and spontaneously returned to baseline levels within 10 minutes. Conclusions The novel injection system enabled a minimally invasive, safe, and consistent delivery of bevacizumab with rapid distribution throughout the choroid layers up to the RPE in large eyes. Large volumes of anti-angiogenic are delivered in close proximity to the retina due to the high volume distribution.
Chapter
Inherited retinal diseases (IRDs) result in progressive vision loss usually in both eyes. Stem cell therapy and gene therapy are promising therapeutic approaches for treatment of retinal degenerative conditions including IRDs. Since the eye is a small, enclosed organ with immune privilege and optical clarity, the effects of stem cell or gene therapy on the retina can be accessed readily using a small amount of therapeutic agent which may be administered in the eye using intravitreal, subretinal or suprachoroidal routes. Stem cell therapies aim to rescue degenerating retina via tissue replacement or paracrine effects. Autologous and allogeneic cells derived from embryonic, fetal or induced pluripotent stem cells or from bone marrow stem cells are being explored. Single cell suspension and cellular sheets are different cellular preparations that are being investigated. Gene therapy aims to repair or replace specific genetic defects associated with IRDs by gene silencing, addition or editing. The recent FDA approval of gene therapy for IRD associated with RPE65 has resulted in a great increase in research in this area. Several early phase clinical trials have started or are recently completed for both stem cell and gene therapy to treat IRDs. This chapter provides a listing of these clinical trials and relevant preclinical and clinical observations that form the basis for exploring specific therapies. This important on-going area of research may yield findings that help individuals with vision loss from IRDs and improve our understanding of tissue regeneration which may be applied to degenerative conditions affecting the retina and beyond.
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Preserving of vision is the main goal in vision research. The presented research evaluates the preservation of visual function in Royal College of Surgeon (RCS) rats using a depth perception test. Rats were placed on a stage with one side containing an illusory steep drop (“cliff”) and another side with a minimal drop (“table”). Latency of stage dismounting and the percentage of rats that set their first foot on the “cliff” side were determined. Nondystrophic Long–Evans (LE) rats were tested as control. Electroretinogram and histology analysis were used to determine retinal function and structure. Four-week-old RCS rats presented a significantly shorter mean latency to dismount the stage compared with 6-week-old rats (mean ± standard error, 13.7 ± 1.68 vs. 20.85 ± 6.5 s, P = 0.018). Longer latencies were recorded as rats aged, reaching 45.72 s in 15-week-old rats ( P < 0.00001 compared with 4-week-old rats). All rats at the age of 4 weeks placed their first foot on the table side. By contrast, at the age of 8 weeks, 28.6% rats dismounted on the cliff side and at the age of 10 and 15 weeks, rats randomly dismounted the stage to either table or cliff side. LE rats dismounted the stage faster than 4-week-old RCS rats, but the difference was not statistically significant (7 ± 1.58 s, P = 0.057) and all LE rats dismounted on the table side. The latency to dismount the stage in RCS rats correlated with maximal electroretinogram b-wave under dark and light adaptation (Spearman’s rho test = −0.603 and −0.534, respectively, all P < 0.0001), outer nuclear layer thickness (Spearman’s rho test = −0.764, P = 0.002), and number of S- and M-cones (Spearman’s rho test = −0.763 [ P = 0.002], and −0.733 [ P = 0.004], respectively). The cliff avoidance test is an objective, quick, and readily available method for the determination of RCS rats’ visual function.
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The article present a case report of orbital granulomatous inflammation after a retrobulbar injection of allogenous stem cells. Experimental treatment resulted in an orbital tumor that required surgical excision. Lymphogranulomatous inflammation with a secondary abscess was verified by morphological and immunohistochemical analyses. This case demonstrates the possible dangerous complications of the "off-label" therapy amid the rising popularity of stem cells treatment.
Chapter
A vast body of preclinical research on retinal regeneration through stem cell transplantation has laid the foundation for numerous phase I/II clinical trials of retinal cell therapy that are currently in progress. Approaches of retinal stem cell therapy include “rescue” and “replace” concepts. In the former concept, therapy is directed at retarding the rate of functional decline, whereas in the latter concept, the aim is to restore visual function. Cell types that have been investigated for these purposes include retinal pigment epithelium cells, neural progenitor cells, photoreceptor precursor cells, bone marrow cells, and umbilical tissue-derived cells. Cell sources include primary cells, embryonic stem cells (ESC), and induced pluripotent stem cells (iPSC). Retinal cell therapy research has been facilitated by the use of numerous small and large animal models including mice, rats, rabbits, pigs, and nonhuman primates that model different aspects of the target diseases and therapy delivery. Animal data on retinal cell therapy are reviewed in this chapter. The recent discovery of cell-cell fusion between exogenous and recipient photoreceptor cells—resulting in the transfer of fluorescent reporter molecules giving rise to labeling artifacts following transplantation in animal models—has called previous assumptions of retinal cellular integration into question, and indicates the presence of multiple mechanisms underlying the regenerative effects of retinal cell transplantation.
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Purpose: Development of a method for noninvasive longitudinal follow-up of retinal degeneration in the whole retina for Royal College of Surgeons (RCS) rats, a commonly used model of retinitis pigmentosa associated with mutations in the MER-proto-oncogene tyrosine kinase (MERTK) gene. Methods: Pigmented RCS rats at postnatal (p) days p28 to p84 were subjected to a biweekly spectral-domain optical coherence tomography (SD-OCT), blue laser fundus autofluorescence (BL-FAF) imaging, and multicolor fundus imaging. Wild-type (WT; Long Evans) rats were tested as control. Results: Hyperautofluorescence developed throughout the fundus at p42, concomitant with a significant increase in SD-OCT thickness and reflectivity of the debris zone (DZ) layer as well as thinning of the photoreceptor outer nuclear layer (ONL). From p56 to p84, discrete hypofluorescent lesions surrounded by hyperfluorescent flecks were demonstrated around the optic disc that gradually spread throughout the retina. The hypofluorescent lesions were associated with loss of ONL and gradual thinning of the DZ layer. No hypofluorescent BL-FAF lesions were observed in WT rats. Conclusions: This study suggests that BL-FAF imaging may present a new method for noninvasive longitudinal follow-up of retinal degeneration in nearly the whole retina in RCS rats. Translational relevance: A clinical test was developed that may be implemented in translational studies in the RCS rat model of MERTK-associated retinitis pigmentosa.
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The retinoid cycle is the enzymatic pathway that regenerates the vision chromophore, 11-cis retinal, after it is bleached during light absorption. Genetic insults to this cycle result in incurable blinding retinal dystrophies such as retinitis pigmentosa and Leber congenital amaurosis. Previous studies demonstrated that oral treatment with 9-cis-β-carotene rich Dunaliella bardawil powder significantly improved visual and retinal function in patients with fundus albipunctatus night blindness and retinitis pigmentosa. Here we examined the effect of oral treatment with the Dunaliella powder on retinal function and structure in RPE65rd12 mice, a model of a genetic defect in the retinoid cycle. Mice were fed with a control diet, vitamin A deficient diet (VAD) or VAD diet supplemented with Dunaliella powder for 13 months. Mice fed with Dunaliella presented significantly higher dark-adapted (35.7 μV ± 3.1 vs. 6.9 μV ± 2.5, p < 0.001 for VAD and vs. 4.3 μV ± 1.1, p < 0.001 for control) and light-adapted (35.1 μV ± 4.3 vs. 6.2 μV ± 3.0, p < 0.001 for VAD and vs. 4.9 μV ± 1.3, p < 0.001 for control) maximal electroretinogram a-wave amplitudes. The Dunaliella group also presented higher dark- and light-adapted maximal electroretinogram b-wave amplitudes compared with the control diet (86.5 μV ± 9.4 vs. 28.7 μV ± 6.3, p < 0.001 and 79.2 μV ± 10.4 vs. 28.3 μV ± 4.7, p = 0.001, respectively), but comparable results to the VAD group. A significantly higher number of M-cone photoreceptors was identified in the retinas of DUNA treated mice. Taken together, our study suggests that 9CBC-rich Dunaliella bardawil powder may present an effective treatment for retinal dystrophies caused by defects in the retinoid cycle.
Chapter
In the aggregate, retinal degenerative diseases such as age-related macular degeneration, retinitis pigmentosa, and Stargardt disease are major causes of vision loss and blindness worldwide. Damage to retinal pigment epithelial cells and photoreceptors eventually leads to their death, and, in contrast to some species such as zebrafish, humans lack an endogenous system for robust spontaneous replacement or repair. Cell-based therapies provide a rational option to restore function, particularly when there is substantial loss of neuronal tissue. A number of important issues remain unresolved, however, including the differentiation and integration of cells after implantation. For stem cell therapies, the retina provides an excellent environment, not only because of its ease of access for surgical procedures but also because of the ability to observe transplanted cells directly through clear ocular media using innovative imaging approaches. The improvements in the clinical use of progenitor cells in the last decade have resulted in significant advances for the treatment of degenerative retinal disease. In this chapter, a brief discussion is given of the main sources of stem cells used for photoreceptor and/or retinal pigment epithelium recovery/replacement.
Article
Aims The degeneration of retinal neurons which occurs in many neurodegenerative diseases of retina such as retinitis pigmentosa and aged-related macular degeneration, is a progressive phenomenon and leads to permanent visual disability. Aside from their economic and social impact, those who suffer from these diseases have a poor quality of life due to the lack of cures. Researchers have turned to stem cell therapies as a potential solution to this global health crisis. Mesenchymal stem cells (MSCs) and their paracrine agents such as conditioned medium (CM) and exosomes (Exo) have been applied to treat different retinal disorders. This study compared the therapeutic effects of human adipose mesenchymal stem cells (hADSCs) and their secretome on an in vivo model of sodium iodate retinal neurodegeneration. Main methods We analyzed the expression of retinal cells' specific mRNAs by RT-PCR and proteins by immunostaining as well as performing visual cliff avoidance test as a functional evaluation technique. There were four therapeutic groups in this study: hADSC, hADSC-CM, hADSC-Exo and hADSC-Exo + CM. Key findings Although all groups showed different therapeutic effects on various retinal cells, the results of hADSC-CM were most striking, especially in terms of photoreceptor regeneration and retinal function. Significance The findings of present study demonstrated the different effects of MSC-based therapies on various retinal cells which could be helpful in designing more precise treatments that suit to each neurodegenerative disease mechanism and the cells involved. It also suggests that CM might be a better choice due to its multifactorial characteristic.
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Vision researchers have been at the forefront of translational medicine and therapeutics at least in part due to the accessibility of ocular structures, the relative immune privilege of the ocular environment, well defined objective and subjective endpoints and sophisticated imaging modalities that harness the optical clarity of ocular media, permitting real-time submicron resolution of ocular structures and direct visualisation of the central nervous system. Despite these significant advances, primary open angle glaucoma remains the leading cause of irreversible blindness worldwide, where traditional IOP-lowering therapies are often not sufficient to prevent progression to blindness even for patients with access to high quality healthcare. Neuroprotection strategies, which aim to boost the ability of target cells to withstand a pathological insult, have shown significant promise in animal models but none have shown clinically relevant efficacy in human clinical trials to date. Here we outline the current status of neuroprotection clinical trials for glaucoma, including how refinements in clinical trial design may improve the prospects for ongoing and future glaucoma neuroprotection trials. We also consider how lessons learned from trials for other diseases may be used to guide the development of future glaucoma therapies. Finally, we discuss how advances in our understanding of the glaucomatous degenerative process may aid future neuroprotective strategies and tailoring of treatment according to an individual’s risk, thereby improving outcomes for our patients.
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Retinitis pigmentosa (RP) is a hereditary disease characterized by degeneration and the loss of photoreceptors. Stem cell based therapy has emerged as a promising strategy for treating RP. Stem cells from exfoliated deciduous teeth (SHEDs), a type of mesenchymal stem cell from human exfoliated deciduous teeth have the potential to differentiate into photoreceptor-like cells under specific induction in vitro. It has been confirmed that through paracrine secreta, SHEDs exert neurotrophic, angiogenic, immunoregulatory, and anti-apoptotic functions in injured tissues. This study was designed to determine whether retinal-differentiated SHEDs and the conditioned medium derived from SHEDs (SHED-CM) have therapeutic effects in a mouse model of RP. The results showed that both SHEDs and SHED-CM improved electroretinogram (ERG) responses, ameliorated photoreceptor degeneration, and maintained the structure of the outer segments of photoreceptors. The therapeutic effects were related to anti-apoptotic activity of SHEDs and SHED-CM. Thus, SHEDs may be a promising stem cell source for treating retinal degeneration.
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Stem cell-based therapy raises hopes for a better approach to promoting tissue repair and functional recovery. However, transplanted stem cells show a high death percentage, creating challenges to successful transplantation and prognosis. Thus, it is necessary to investigate the mechanisms underlying stem cell death, such as apoptotic cascade activation, excessive autophagy, inflammatory response, reactive oxygen species, excitotoxicity, and ischemia/hypoxia. Targeting the molecular pathways involved may be an efficient strategy to enhance stem cell viability and maximize transplantation success. Notably, a more complex network of cell death receives more attention than one crucial pathway in determining stem cell fate, highlighting the challenges in exploring mechanisms and therapeutic targets. In this review, we focus on programmed cell death in transplanted stem cells. We also discuss some promising strategies and challenges in promoting survival for further study. ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
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A promising clinical application for stem and progenitor cell transplantation is in rescue therapy for degenerative diseases. This strategy seeks to preserve rather than restore host tissue function by taking advantage of unique properties often displayed by these versatile cells. In studies using different neurodegenerative disease models, transplanted human neural progenitor cells (hNPC) protected dying host neurons within both the brain and spinal cord. Based on these reports, we explored the potential of hNPC transplantation to rescue visual function in an animal model of retinal degeneration, the Royal College of Surgeons rat. Animals received unilateral subretinal injections of hNPC or medium alone at an age preceding major photoreceptor loss. Principal outcomes were quantified using electroretinography, visual acuity measurements and luminance threshold recordings from the superior colliculus. At 90-100 days postnatal, a time point when untreated rats exhibit little or no retinal or visual function, hNPC-treated eyes retained substantial retinal electrical activity and visual field with near-normal visual acuity. Functional efficacy was further enhanced when hNPC were genetically engineered to secrete glial cell line-derived neurotrophic factor. Histological examination at 150 days postnatal showed hNPC had formed a nearly continuous pigmented layer between the neural retina and retinal pigment epithelium, as well as distributed within the inner retina. A concomitant preservation of host cone photoreceptors was also observed. Wild type and genetically modified human neural progenitor cells survive for prolonged periods, migrate extensively, secrete growth factors and rescue visual functions following subretinal transplantation in the Royal College of Surgeons rat. These results underscore the potential therapeutic utility of hNPC in the treatment of retinal degenerative diseases and suggest potential mechanisms underlying their effect in vivo.
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Regeneration and plasticity refer to the ability of certain progenitor cells to produce cell lineages with specific morphological and functional settings. The pathway from a less delineated or immature phenotype to a mature or specialized one follows intricate routes where a monumental array of molecular elements, basically transcription factors and epigenetic regulators that turn off or on a specific phenotypic change, play a fundamental role. Nature itself offers procedures to healing strategies. Therapy approaches to pathologies in the realm of ophthalmology may benefit from the knowledge of the properties and mechanisms of activation of different routes controlling the pathways of cell definition and differentiation. Specification of cell identity, not only in terms of phenotypic traits, but also regarding the mechanisms of gene expression and epigenetic regulation, will provide new tools to manipulating cell fates and status, both forward and backwards. In the human eye, two main locations shelter stem cells: the limbus, which is situated in the limit of the cornea and the conjunctiva, and the ciliary body pars plana. Transplantation of limbal cells is currently used in certain pathologies where corneal epithelium is damaged. Therapeutic applications of retina progenitors are not yet fully developed due to the complexity of the cellular components of the multilayer retinal architecture. Animal models of Retinitis pigmentosa or Glaucoma offer an interesting approach to validate certain techniques, such as the direct injection of progenitors into the vitreal compartment, aimed to restoring retinal function.
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Background: Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP. Methodology/principal findings: Animals received syngeneic MSCs (1x10(6) cells) by tail vein at an age before major photoreceptor loss. Principal results: both rod and cone photoreceptors were preserved (5-6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs. Conclusions/significance: These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases.
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Purpose. Retrograde neurotrophic factor transport blockade has been implicated in the pathophysiology of glaucoma. Stem cell transplantation appears to ameliorate some neurodegenerative conditions in the brain and spinal cord, in part by neurotrophic factor secretion. The present study was conducted to determine whether local or systemic bone marrow-derived mesenchymal stem cell (MSC) transplantation can confer neuroprotection in a rat model of laser-induced ocular hypertensive glaucoma. Methods. MSCs were isolated from the bone marrow of adult wild-type and transgenic rats that ubiquitously express green fluorescent protein. MSCs were transplanted intravitreally 1 week before, or intravenously on the day of, ocular hypertension induction by laser photocoagulation of the trabecular meshwork. Ocular MSC localization and integration were determined by immunohistochemistry. Optic nerve damage was quantified by counting axons within optic nerve cross-sections 4 weeks after laser treatment. Results. After intravitreal transplantation, MSCs survived for at least 5 weeks. Cells were found mainly in the vitreous cavity, though a small proportion of discrete cells migrated into the host retina. Intravitreal MSC transplantation resulted in a statistically significant increase in overall RGC axon survival and a significant decrease in the rate of RGC axon loss normalized to cumulative intraocular pressure exposure. After intravenous transplantation, MSCs did not migrate to the injured eye. Intravenous transplantation had no effect on optic nerve damage. Conclusions. Local, but not systemic, transplantation of MSCs was neuroprotective in a rat glaucoma model. Autologous intravitreal transplantation of MSCs should be investigated further as a potential neuroprotective therapy for glaucoma.
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Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.
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To evaluate the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. Adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts. To assess the integration and differentiation of BM-MSCs in laser-injured retina, host retinas were evaluated 2.4 and 8 weeks after injury/transplantation. Our results demonstrated that the grafted cells survived in the retina for at least 8 weeks and almost all BM-MSCs migrated and incorporated into the neural retina, specifically in the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) while a subset of grafted cells were found in the subretinal space posttransplantation. At 8 weeks immunohistochemical analysis with several retinal specific markers revealed that the majority of the grafted cells expressed rhodopsin, a rod photoreceptor marker, followed by parvalbumin, a marker for bipolar and amacrine cells. A few subsets of cells were able to express a glial marker, glial fibrillary acidic protein. However, grafted cells failed to express pan-cytokeratin, a retinal pigment epithelium marker. These results suggest the potential of BM-MSCs to differentiate into retinal neurons. Taken together, these findings might be clinically relevant for future mesenchymal stem cell therapy studies concerning retinal degeneration repair.
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The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K(+) conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K(+) in the retina. To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of the Kir4.1 gene (Kir4.1 -/-). Müller cells from Kir4.1 -/- mice were not labeled with an anti-Kir4.1 antibody, although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody. In contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes. In situ whole-cell patch-clamp recordings showed a 10-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells compared with Kir4.1 +/+ cells. The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K(+) fluxes through Müller cells, was totally absent in retinas from Kir4.1 -/- mice. The b-wave of the ERG, in contrast, was spared in the null mice. Overall, these results indicate that Kir4.1 is the principal K(+) channel subunit expressed in mouse Müller glial cells. The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K(+) in the mouse retina.
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In the retinas of Royal College of Surgeons (RCS) rats light induces an increase in distal extracellular potassium irrespective of the age, between days 19-24 and days 29-35 postpartum, but by days 29-35 the ERG b-wave has become reduced. The synaptic blocker 2-amino-4-phosphonobutyric acid (APB) causes the abolition of both the b-wave and the potassium increase at any age. MgCl2 greatly reduces the b-wave at all ages and abolishes the potassium increase in older rats, but in younger rats the potassium increase is enlarged. Since this increase occurs in the absence of the b-wave it is unlikely that the on-bipolar cells are the only sources of the b-wave. Because the NMDA receptor blocker ketamine reduces the b-wave, third order neurons, which possess NMDA receptors, could contribute to the b-wave.
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It has been 13 years since the discovery of human embryonic stem cells (hESCs). Our report provides the first description of hESC-derived cells transplanted into human patients. We started two prospective clinical studies to establish the safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium (RPE) in patients with Stargardt's macular dystrophy and dry age-related macular degeneration--the leading cause of blindness in the developed world. Preoperative and postoperative ophthalmic examinations included visual acuity, fluorescein angiography, optical coherence tomography, and visual field testing. These studies are registered with ClinicalTrials.gov, numbers NCT01345006 and NCT01344993. Controlled hESC differentiation resulted in greater than 99% pure RPE. The cells displayed typical RPE behaviour and integrated into the host RPE layer forming mature quiescent monolayers after transplantation in animals. The stage of differentiation substantially affected attachment and survival of the cells in vitro after clinical formulation. Lightly pigmented cells attached and spread in a substantially greater proportion (>90%) than more darkly pigmented cells after culture. After surgery, structural evidence confirmed cells had attached and continued to persist during our study. We did not identify signs of hyperproliferation, abnormal growth, or immune mediated transplant rejection in either patient during the first 4 months. Although there is little agreement between investigators on visual endpoints in patients with low vision, it is encouraging that during the observation period neither patient lost vision. Best corrected visual acuity improved from hand motions to 20/800 (and improved from 0 to 5 letters on the Early Treatment Diabetic Retinopathy Study [ETDRS] visual acuity chart) in the study eye of the patient with Stargardt's macular dystrophy, and vision also seemed to improve in the patient with dry age-related macular degeneration (from 21 ETDRS letters to 28). The hESC-derived RPE cells showed no signs of hyperproliferation, tumorigenicity, ectopic tissue formation, or apparent rejection after 4 months. The future therapeutic goal will be to treat patients earlier in the disease processes, potentially increasing the likelihood of photoreceptor and central visual rescue. Advanced Cell Technology.
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Visual impairment associated with photoreceptor degeneration is a largely untreatable condition affecting millions of people worldwide. Cellular therapies offer an attractive alternative for the treatment of retinal degeneration. Human adult bone marrow-derived somatic cells (hABM-SCs) present particular advantages for interventional therapy to the eye because they are non-immunogenic, effective at low dose, maintain a stable phenotype and secrete factors known to promote photoreceptor cell survival. Here we assess the potential of hABM-SCs (developed by Garnet BioTherapeutics) to sustain vision in a rodent model of human retinal disease-the Royal College of Surgeons (RCS) rat.
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PURPOSE. To investigate the effects and possible mechanisms of rat bone marrow mesenchymal stem cell (BMSC) transplantation on the light-damaged retinal structure and the apoptosis of photoreceptors. METHODS. DAPI-labeled BMSCs were transplanted into the subretinal space of light-damaged Sprague-Dawley rats 10 days after exposure. BMSCs were cultivated with the supernatant of homogenized retina (SHR). RESULTS. The outer nuclear layer (ONL) contained significantly more cells and the percentage of apoptotic ONL cells was significantly reduced in the BMSC transplantation group than in the phosphate-buffered solution injection group or the light damage group. Most DAPI-labeled BMSCs expressed brain-derived neurotrophic factor (BDNF). There was elevated basic fibroblast growth factor (bFGF) and BDNF immunoreactivity in the retinas of the BMSC transplantation group compared with the light damage group. In vitro culture showed that 10% of BMSCs changed from fusiform shape to multipolar shape. A fraction of cells expressed MAP2 or glial fibrillary acidic protein, and some cells expressed bFGF or BDNF when cultivated with light-damaged SHR for 7 days. CONCLUSIONS. BMSC subretinal transplantation could inhibit photoreceptor apoptosis and slow down retinal damage in light-damaged eyes. BMSCs could express bFGF (in vitro) and BDNF (in vitro and in vivo), pointing to potential trophic and protective effects on light-damaged retinas.
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Cell transplantation is a novel therapeutic strategy to restore visual responses to the degenerate adult neural retina and represents an exciting area of regenerative neurotherapy. So far, it has been shown that transplanted postmitotic photoreceptor precursors are able to functionally integrate into the adult mouse neural retina. In this review, we discuss the differentiation of photoreceptor cells from both adult and embryonic-derived stem cells and their potential for retinal cell transplantation. We also discuss the strategies used to overcome barriers present in the degenerate neural retina and improve retinal cell integration. Finally, we consider the future translation of retinal cell therapy as a therapeutic strategy to treat retinal degeneration.
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Assessments of safety and efficacy are crucial before human ESC (hESC) therapies can move into the clinic. Two important early potential hESC applications are the use of retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration and Stargardt disease, an untreatable form of macular dystrophy that leads to early-onset blindness. Here we show long-term functional rescue using hESC-derived RPE in both the RCS rat and Elov14 mouse, which are animal models of retinal degeneration and Stargardt, respectively. Good Manufacturing Practice-compliant hESC-RPE survived subretinal transplantation in RCS rats for prolonged periods (>220 days). The cells sustained visual function and photoreceptor integrity in a dose-dependent fashion without teratoma formation or untoward pathological reactions. Near-normal functional measurements were recorded at >60 days survival in RCS rats. To further address safety concerns, a Good Laboratory Practice-compliant study was carried out in the NIH III immune-deficient mouse model. Long-term data (spanning the life of the animals) showed no gross or microscopic evidence of teratoma/tumor formation after subretinal hESC-RPE transplantation. These results suggest that hESCs could serve as a potentially safe and inexhaustible source of RPE for the efficacious treatment of a range of retinal degenerative diseases.
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Stem cell research offers great promise for understanding basic mechanisms of human development and differentiation, as well as the hope for new treatments for diseases such as diabetes, spinal cord injury, Parkinson's disease, and myocardial infarction. However, human stem cell (hSC) research also raises sharp ethical and political controversies. The derivation of pluripotent stem cell lines from oocytes and embryos is fraught with disputes about the onset of human personhood. The reprogramming of somatic cells to produce induced pluripotent stem cells avoids the ethical problems specific to embryonic stem cell research. In any hSC research, however, difficult dilemmas arise regarding sensitive downstream research, consent to donate materials for hSC research, early clinical trials of hSC therapies, and oversight of hSC research. These ethical and policy issues need to be discussed along with scientific challenges to ensure that stem cell research is carried out in an ethically appropriate manner. This article provides a critical analysis of these issues and how they are addressed in current policies.
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To examine the survival, migration, integration, differentiation and the expression of various neurotrophic factors of bone-marrow mesenchymal stem cells (BMSCs) transplanted into the vitreous cavity of rats injured by ischemia/reperfusion(I/R). The BMSCs were separated from rat marrow using the wall-sticking method, and cultured in vitro to expand. Flow cytometry detected the surface antigens of BMSCs. Ninety-six rats were randomly divided into four groups: normal control injected PBS(C+P), normal control injected BMSCs (C+B), ischemic/reperfusion injected PBS(I/R+P)and ischemic/reperfusion injected BMSCs(I/R+B). After retinal I/R injury was induced in each group by increasing intraocular pressure, 10 microl PBS and BMSC suspensions labeled by red fluorescence CM-Dil were immediately injected into the vitreous cavity. We observed the survival, migration and integration of BMSCs using confocal microscopy. The differentiation and expression of basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) of CM-Dil-labeled BMSCs were detected by immunofluorescent labeling and reserved by confocal microscopy. The expression of mRNA and proteins of bFGF, BDNF and CNTF were assayed by RT-PCR and Western Blot respectively. After transplantation to normal eyes, BMSCs labeled by CM-Dil were mostly present in the vitreous cavity, and did not migrate. After transplantation to I/R eyes, BMSCs labeled by CM-Dil were mostly present along with the inner limiting membrane. Only a few cells were integrated into the ganglion cell layer. Two or 4 weeks after transplantation, a few BMSCs labeled by CM-Dil were observed to express markers of neuron- neurone specific enolase (NSE), neurofilament (NF) and various neurotrophic factors. The BMSC-injected I/R model eyes showed less reduction in the number of RGCs than that of the I/R eyes with PBS injection. BMSC transplantation is a valuable neuroprotection tool for the treatment of retina and optic nerve diseases.