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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
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.
... 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). ...
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.
... 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. ...
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.
... 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). ...
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.
... 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]. ...
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.
... 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]. ...
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.
... 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). ...
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.
... The paracrine capacity of cells, more than their capacity to replace RPE, such as BM-MSCs, has included them among the possible options (still theoretical) for ocular neurodegenerative diseases (Mead et al. 2015;Park et al. 2017). Tzameret et al. have described how BM-MSCs determined an improvement of retinal function by preserving and improving cell viability; however, they did not find differentiation of these cells into mature retinal cells (Tzameret et al. 2014). ASCs have a great, well-known paracrine capacity for producing neurotrophic factors, as seen for disorders of the SNC (Park et al. 2017). ...
... However, most studies are still in the preliminary preclinical phases. At the same time, bioengineering technologies and nanotechnologies represent other possible therapeutic solutions (Nakano et al. 2012;Tzameret et al. 2014;Stern et al. 2018). Recent studies have used these novel techniques to create organoids and 3D structures of the eye, which can be implanted to replace the original damaged tissues. ...
The use of stem cells in medicine is becoming an exciting therapeutic option. The fascinating elements of these innovative therapies include the possibility of isolating, culturing, and differentiating multipotent cells and using them to replace damaged cells or act as enhanced cellular response modulators. The most significant challenges are stem cell isolation, culture, and transfer techniques, which can be favored by bioengineering, nanotechnology, and gene therapy. There are numerous medical fields in which stem cells have recently played a key role in treatment options, including ophthalmology. The possibility of exploiting stem cells derived from different tissues (i.e., blood, bone marrow, adipose tissue, etc.) to rebuild ocular tissues represents one of the areas of most significant interest in which research has invested considerable resources. The most frequently and easily used option considered in this type of research derives from adult stem cells (i.e., adipose tissue). These isolated and cultured cells have shown interesting preliminary results for ocular surface diseases. Modern animal trials have also evaluated the efficacy of stem cells in the optic nerve and retinal pathologies.
This chapter briefly overviews stem cell-based therapy in ophthalmology, discussing in-depth the fascinating and evolving therapeutic options reported in the literature. Moreover, our chapter highlights the limitations in this field of research, including the economic and practical inadequacies and lack of clinical studies in humans.
... 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]. ...
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.
... 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. ...
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.
... 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. ...
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.
... These cells did not migrate to subretinal space. For this reason, this route of administration was chosen even though cellular cluster formation of MSC in the vitreous can occur in some cases after intravitreal delivery [38], as it happened to one of our patients who presented a retrolental aggregate, as we had found experimentally that could well be an aggregate of the injected cells [9]. The aggregate disappeared between the 6-month visit and the one-year visit leaving a subcapsular opacification. ...
Background
An effective treatment for acute non-arteritic ischemic optic neuropathy (NA-AION) has not been known or proven yet. Previous studies have suggested a neuroprotective effect of allogeneic bone marrow-derived mesenchymal stem cells. This study aims to report the results of a clinical trial on patients with acute non-arteritic optic neuropathy (NA-AION) treated with an intravitreal injection of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) (MSV®).
Methods
We conducted a prospective, non-randomized, clinical phase-II study (Eudra CT number 2016-003029-40; ClinicalTrials.gov Registry NCT03173638) that included 5 patients with acute unilateral NA-AION diagnosed within 2 weeks after symptom onset and who received an intravitreal injection of allogeneic BM-MSCs (0.05 ml; cell concentration: 1.5 × 10⁶cells/mL). The patients underwent regular ophthalmological examinations and were followed for one year.
Results
In this trial, allogeneic BM-MSCs appeared to be safe as no patients developed signs of acute nor chronic intraocular inflammation or a significant change in intraocular pressure, although an epiretinal membrane was developed in one patient. A retrolental aggregate formed shortly after the injection spontaneously disappeared within a few weeks in another phakic patient, leaving a subcapsular cataract. Visual improvement was noted in 4 patients, and amplitudes of P100 on the visually evoked potentials recordings increased in three patients. The retinal nerve fiber layer and macular ganglion cell layer thicknesses significantly decreased during the follow-up.
Conclusions
Besides the development of an epiretinal membrane in one patient, the intravitreal application of allogeneic BM-MSCs appeared to be intraocularly well tolerated. Consequently, not only NA-AION but also BM-MSCs deserve more clinical investigational resources and a larger randomized multicenter trial that would provide stronger evidence both about safety and the potential therapeutic efficacy of intravitreally injected allogeneic BM-MSCs in acute NA-AION.
Trial registration: Safety Assessment of Intravitreal Mesenchymal Stem Cells for Acute Non-Arteritic Anterior Ischemic Optic Neuropathy (NEUROSTEM). NCT03173638. Registered June 02, 2017 https://clinicaltrials.gov/ct2/show/NCT03173638.
... Similar findings were observed using the same injection method with smaller proof-of-concept devices, in rats, rabbits, and pigs. [35][36][37] Chen et al. 30 reported the injection of 150 μL ICG into the SC of rabbit eyes using a 30-gauge needle connected to a 250-μL Hamilton syringe. Injection of 150 μL ICG was done more posteriorly compared to our injection (9 mm behind the limbus, in the inferior quadrant) and resulted in more limited spreading of ICG in the SC, covering roughly 65% of the SC. ...
Purpose:
Evaluation of distribution and tolerance of suprachoroidal injection of indocyanine green (ICG) in nonhuman primates (NHPs) using a novel suprachoroidal (SC) delivery technology.
Methods:
Three live and three euthanized African green monkeys were injected with 150 or 200 µL ICG/eye into the SC space of both eyes, 2.5 mm posterior to the limbus in the inferior quadrant, utilizing a novel SC injector. Eyes were analyzed by imaging of scleral flatmounts. Live animals were observed for 24 hours for general health. Ophthalmic evaluation included slit-lamp biomicroscopy, tonometry, fundus imaging, confocal laser ophthalmoscopy, and spectral-domain optical coherence tomography (SD-OCT) before and at 10 minutes and 1, 3, and 24 hours post-injection.
Results:
SC dosing was successfully performed in all eyes. Infrared fundus imaging demonstrated ICG distribution throughout the posterior segment, reaching the macula within 24 hours post-injection. No inflammation, intravitreal penetration, SC blebs, retinal detachment, or hemorrhages were detected. No significant changes were observed in retinal thickness by SD-OCT (P = 0.267, ANOVA). A mild, statistically insignificant elevation in intraocular pressure was observed within 10 minutes post-injection (mean ± standard error: 7.28 ± 5.09 mmHg; P = 0.061) and was spontaneously resolved within the first hour after dosing.
Conclusions:
Suprachoroidal injection of 150 to 200 µL ICG dye was successfully performed and well tolerated in NHP eyes, with rapid distribution into the macular region and throughout the posterior pole.
Translational relevance:
This novel SC drug delivery system may potentially provide safe and effective delivery of therapeutics to the posterior pole region in humans.
... The secretome of BM-MSCs also plays a vital role by containing an array of neurotrophic factors like NGF, Neurotrophin-3, 4, 5, IGF1, FGF2, etc., which bind to their associative receptors, thereby enhancing axonal outgrowth, neural cell survival and attachment [69]. Nevertheless, on the other hand, research conducted on the long-term effects of BM-MSC therapy on animal models shows their migration to non-target tissue by crossing BRB upon its retinal integration making its efficiency doubtable [70]. ...
Diabetic retinopathy is a common yet complex microvascular disease, caused as a complication of diabetes mellitus. Associated with hyperglycemia and subsequent metabolic abnormalities, advanced stages of the disease lead to fibrosis, subsequent visual impairment and blindness. Though clinical postmortems, animal and cell models provide information about the progression and prognosis of diabetic retinopathy, its underlying pathophysiology still needs a better understanding. In addition to it, the loss of pericytes, immature retinal angiogenesis and neuronal apoptosis portray the disease treatment to be challenging. Indulged with cell loss of both vascular and neuronal type cells, novel therapies like cell replacement strategies by various types of stem cells have been sightseen as a possible treatment of the disease. This review provides insight into the pathophysiology of diabetic retinopathy, current models used in modelling the disease, as well as the varied aspects of stem cells in generating three-dimensional retinal models. Further outlook on stem cell therapy and the future directions of stem cell treatment in diabetic retinopathy have also been contemplated.
... Human umbilical cord blood MSCs, injected into the subretinal space of RCS rats, significantly reduced the degree of PR degeneration and this was attributed to secretion of neurotrophic factors, such as FGF2 and BDNF [136]. Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) in subretinal [137], epiretinal [138], or comparatively subretinal/intravitreal spaces [139] increased PR cell survival and rescued retinal function in the rat model of retinal dystrophy. ...
Photoreceptors (PRs), as the most abundant and light-sensing cells of the neuroretina, are responsible for converting light into electrical signals that can be interpreted by the brain. PR degeneration, including morphological and functional impairment of these cells, causes significant diminution of the retina’s ability to detect light, with consequent loss of vision. Recent findings in ocular regenerative medicine have opened promising avenues to apply neuroprotective therapy, gene therapy, cell replacement therapy, and visual prostheses to the challenge of restoring vision. However, successful visual restoration in the clinical setting requires application of these therapeutic approaches at the appropriate stage of the retinal degeneration. In this review, firstly, we discuss the mechanisms of PR degeneration by focusing on the molecular mechanisms underlying cell death. Subsequently, innovations, recent developments, and promising treatments based on the stage of disorder progression are further explored. Then, the challenges to be addressed before implementation of these therapies in clinical practice are considered. Finally, potential solutions to overcome the current limitations of this growing research area are suggested. Overall, the majority of current treatment modalities are still at an early stage of development and require extensive additional studies, both pre-clinical and clinical, before full restoration of visual function in PR degeneration diseases can be realized.
Graphical Abstract
... One of the commonly used methods in clinical trials is to inject stem cells directly through the retina after pars plana vitrectomy [26]. In some preclinical studies, researchers successfully implanted the cells through the sclera and choroid into the subretinal space [27]. Without passing into the vitreous, the incidence rate of proliferative vitreoretinopathy is reduced because of less interference on the intraocular environment. ...
Retinal diseases are major causes of irreversible vision loss and blindness. Despite extensive research into their pathophysiology and etiology, pharmacotherapy effectiveness and surgical outcomes remain poor. Based largely on numerous preclinical studies, administration of mesenchymal stem cells (MSCs) as a therapeutic strategy for retinal diseases holds great promise, and various approaches have been applied to the therapies. However, hindered by the retinal barriers, the initial vision for the stem cell replacement strategy fails to achieve the anticipated effect and has now been questioned. Accumulating evidence now suggests that the paracrine effect may play a dominant role in MSC-based treatment, and MSC-derived extracellular vesicles emerge as a novel compelling alternative for cell-free therapy. This review summarizes the therapeutic potential and current strategies of this fascinating class of cells in retinal degeneration and other retinal dysfunctions.
... However, a long-term safety study has questioned the clinical appliance of BM-MSCs. This study using Royal College of Surgeon rats, an animal model of AMD, demonstrated that BM-MSCs have the potential to circumvent BRB and migrate into non-target tissues after administration, which is a crucial side effect for researchers to overcome [60]. ...
Diabetic retinopathy is the major blinding disease among working-age populations, which is becoming more significant due to the growth of diabetes. The metabolic-induced oxidative and inflammatory stress leads to the insult of neovascular unit, resulting in the core pathophysiology of diabetic retinopathy. Existing therapies focus on the inflammation, oxidation, and angiogenesis phenomena of diabetic retinopathy, without effect to radically cure the disease. This review also summarizes novel therapeutic attempts for diabetic retinopathy along with their advantages and disadvantages, mainly focusing on those using cellular and genetic techniques to achieve remission on a fundamental level of disease.
... Lu et al. showed that bone marrow mesenchymal stem cells (BM-MSCs) secrete various cytokines and growth factors that improved photoreceptors' function [16]. Another study demonstrated that BM-MSCs can integrate in retinal layers of a rat model of retinal dystrophy [17]. In a research on rat optic nerve injury, Li and colleagues saw improvements in the survival rate of retinal ganglion cells (RGCs) and a decline in apoptosis of retinal cells following the transplantation of adipose derived stem cells (ADSCs) [18]. ...
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.
... 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). ...
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. ...
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). ...
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. ...
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. ...
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
... 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). ...
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
... 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. ...
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
... 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. ...
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]. ...
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. ...
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.
... 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]. ...
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.
... 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. ...
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.
... 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. ...
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.
... 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). ...
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.
... 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. ...
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.
... 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. ...
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.
... 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. ...
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.
... 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). ...
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.
... 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]. ...
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.
... 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] . ...
Because of their unique capacity for differentiation to a diversity of cell lineages and immunosuppressive properties, mesenchymal stem cells (MSC) are being looked at as a potential new treatment option in ophthalmology. The MSCs derived from all tissue sources possess immunomodulatory attributes through cell-to-cell contact and releasing a myriad of immunomodulatory factors (IL-10, TGF-β, growth-related oncogene (GRO), indoleamine 2,3 dioxygenase (IDO), nitric oxide (NO), interleukin 1 receptor antagonist (IL-1Ra), prostaglandin E2 (PGE2)). Such mediators, in turn, alter both the phenotype and action of all immune cells that serve a pathogenic role in the progression of inflammation in eye diseases. Exosomes from MSCs, as natural nano-particles, contain the majority of the bioactive components of parental MSCs and can easily by-pass all biological barriers to reach the target epithelial and immune cells in the eye without interfering with nearby parenchymal cells, thus having no serious side effects. We outlined the most recent research on the molecular mechanisms underlying the therapeutic benefits of MSC and MSC-exosome in the treatment of inflammatory eye diseases in the current article.
Rhodopsin is a light-sensitive G protein-coupled receptor that initiates the phototransduction cascade in rod photoreceptors. Mutations in the rhodopsin-encoding gene RHO are the leading cause of autosomal dominant retinitis pigmentosa (ADRP). To date, more than 200 mutations have been identified in RHO. The high allelic heterogeneity of RHO mutations suggests complicated pathogenic mechanisms. Here, we discuss representative RHO mutations as examples to briefly summarize the mechanisms underlying rhodopsin-related retinal dystrophy, which include but are not limited to endoplasmic reticulum stress and calcium ion dysregulation resulting from protein misfolding, mistrafficking, and malfunction. Based on recent advances in our understanding of disease mechanisms, various treatment methods, including adaptation, whole-eye electrical stimulation, and small molecular compounds, have been developed. Additionally, innovative therapeutic treatment strategies, such as antisense oligonucleotide therapy, gene therapy, optogenetic therapy, and stem cell therapy, have achieved promising outcomes in preclinical disease models of rhodopsin mutations. Successful translation of these treatment strategies may effectively ameliorate, prevent or rescue vision loss related to rhodopsin mutations.
The efficient migration, survival, and engraftment of transplanted cells are important initial steps toward effective cellular therapies for retinal degenerative diseases. The inner limiting membrane (ILM), which separates the retina from the vitreous cavity, is a major barrier for intravitreally transplanted cells. Focusing on the cellular components of the ILM, we found that the cell adhesion molecule N-cadherin (NCAD) at Müller glia (MG) endfeet helps maintain this barrier. To increase ILM cellular permeability, we modulated NCAD expression via two approaches: an inducible MG-specific knockout animal model and with intravitreal self-deliverable siRNA injections. We show that NCAD suppression enhances retinal migration of multiple cell types after intravitreal transplantation, including mouse MG, human induced pluripotent stem cell-derived retinal ganglion cells, and human dental pulp stem cells. Our study may contribute to the development of targeted approaches for recipient tissue modulation to improve cellular therapies for retinal disease.
Retinal pigment epithelium (RPE) transplants rescue photoreceptors in selected animal models of retinal degenerative disease. Early clinical studies of RPE transplants as treatment for age-related macular degeneration (AMD) included autologous and allogeneic transplants of RPE suspensions and RPE sheets for atrophic and neovascular complications of AMD. Subsequent studies explored autologous RPE–Bruch membrane–choroid transplants in patients with neovascular AMD with occasional marked visual benefit, which establishes a rationale for RPE transplants in late-stage AMD. More recent work has involved transplantation of autologous and allogeneic stem cell–derived RPE for patients with AMD and those with Stargardt disease. These early-stage clinical trials have employed RPE suspensions and RPE monolayers on biocompatible scaffolds. Safety has been well documented, but evidence of efficacy is variable. Current research involves development of better scaffolds, improved modulation of immune surveillance, and modification of the extracellular milieu to improve RPE survival and integration with host retina.
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.
Diabetes Mellitus (DM) is a disease with increasing incidence rates and global awareness. Both type 1 (T1D) and type 2 (T2D) diabetes are classifications that require lifetime management. The dysfunction of β islet cells is a primary complication that requires treatment and may lead to several life-threatening complications including blindness, heart disease, and kidney failure. Mesenchymal stem cells (MSCs) are well known for their tissue regenerative action and have shown promising results for restoring β islet cell function as well as ameliorating sequelae of DM. There are several subtypes of MSCs, and each subtype is accompanied by a unique array of pros and cons. This review highlights 3 lineages of MSCs—bone marrow MSCs, adipose-derived (ADSCs), and umbilical cord (UBC-MSCs) - and summarizes the current feasibility and efficacy of each. On evaluation, current primary literature sources suggest that umbilical cord MSCs appear to have the most potential, with particular future implications for exosome research.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether bone marrow stem cells can differentiate into retinal neural cells in adult rats. PKH-67-labeled stem cell-enriched bone marrow cells (BMCs) were injected into the vitreous space of eyes in which the retinas had been mechanically injured using a hooked needle. Two weeks after the injection of these cells, immunohistochemical examinations were carried out. The stem cell-enriched BMCs had been incorporated and had differentiated into retinal neural cells in the injured retina. The stem cell-enriched BMCs had accumulated mainly in the outer nuclear layer around the injured sites. The incorporated cells expressed glial fibrillary acidic protein, calbindin, rhodopsin, and vimentin. These results raise the possibility that stem cell-enriched BMCs have the ability to differentiate into retinal neural cells, and that the injection of stem cell-enriched BMCs into the retina would help repair damaged retinal cells.
Retinal degenerations and dystrophies are the major causes of genetically inherited blindness that are characterized by the apoptotic death of the photoreceptor cell layer of the retina. To date, no treatment exists for these diseases and only recently have they been considered as candidates for gene and stem cell therapies. Here we report the ability of adult CD90+ marrow stromal cells (MSCs) to be induced by activin A, taurine, and EGF into cells (20-32%) expressing photoreceptor-specific markers rhodopsin, opsin, and recoverin in vitro. CD90+ cells were either transduced with recombinant adeno-associated virus expressing green fluorescent protein (GFP) or bromodeoxyuridine (BrdU) labeled and then injected into the subretinal space of adult Royal College of Surgeons rats. Fundus photography and angiography showed no adverse effects of CD90+ MSC transplantation. GFP-expressing cells or BrdU-positive cells covered approximately 30% of the entire retinal area. By 2 weeks after injection, CD90+ MSCs integrated into the host retina, forming structures similar to the photoreceptor layer and expressed a photoreceptor-specific marker. No teratoma formation was observed in the recipient retina. The subretinally delivered CD90+ MSCs did not stain for proliferating cell nuclear antigen, indicating that they primarily undergo differentiation rather than proliferation. In addition, we established that transplanted cells can attract synaptic vesicles and hence are potentially capable of signal transduction. This study demonstrates for the first time the partial differentiation of adult CD90+ MSCs into photoreceptors in vitro and in vivo. Our results establish a proof of concept for CD90+ MSC differentiation with autologous transplantation, which may provide a promising therapeutic strategy for the treatment of some forms of genetically inherited retinal degenerations.
Inherited retinal degenerations afflict 1 in 3,500 individuals and are a heterogeneous group of diseases that result in profound vision loss, usually the result of retinal neuronal apoptosis. Atrophic changes in the retinal vasculature are also observed in many of these degenerations. While it is thought that this atrophy is secondary to diminished metabolic demand in the face of retinal degeneration, the precise relationship between the retinal neuronal and vascular degeneration is not clear. In this study we demonstrate that whenever a fraction of mouse or human adult bone marrow-derived stem cells (lineage-negative hematopoietic stem cells [Lin- HSCs]) containing endothelial precursors stabilizes and rescues retinal blood vessels that would ordinarily completely degenerate, a dramatic neurotrophic rescue effect is also observed. Retinal nuclear layers are preserved in 2 mouse models of retinal degeneration, rd1 and rd10, and detectable, albeit severely abnormal, electroretinogram recordings are observed in rescued mice at times when they are never observed in control-treated or untreated eyes. The normal mouse retina consists predominantly of rods, but the rescued cells after treatment with Lin- HSCs are nearly all cones. Microarray analysis of rescued retinas demonstrates significant upregulation of many antiapoptotic genes, including small heat shock proteins and transcription factors. These results suggest a new paradigm for thinking about the relationship between vasculature and associated retinal neuronal tissue as well as a potential treatment for delaying the progression of vision loss associated with retinal degeneration regardless of the underlying genetic defect.
To determine the potential of adenovirally transduced bone marrow stromal cells (BMSCs) to differentiate into retinal pigment epithelial-like cells and to evaluabe possible rescue effects after transplantation into the retinas of Royal College of Surgeons (RCS) rats.
Through a high-capacity adenoviral vector expressing either green fluorescent protein (GFP) or pigment epithelial-derived factor (PEDF), rat MSCs were transduced in vitro before subretinal transplantation into Wistar rats or, alternatively, RCS rats. Two months after cell injection, the rats were killed and the eyes enucleated. The eyes were then investigated light microscopically or processed for electron microscopic investigations. Cell differentiation and integration were analyzed immunocytochemically using antibodies against cytokeratin and the tight junction protein ZO-1. Electroretinography was performed 16 days after injection of cells, to check whether a functional rescue could be detected.
In vitro experiments in cocultured human MSCs and human RPE cells showed that MSCs adopted RPE-like characteristics. In grafting experiments, some rat MSCs integrate into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells express the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects can be demonstrated after grafting of vector-transduced and nontransduced MSCs in semithin sections of dystrophic retinas. Ultrastructurally, MSCs can be detected on top of host RPE and in close contact with photoreceptor outer segments phagocytosing rod outer segments.
Taken together, these results raise the possibility that MSCs have the potency to replace diseased RPE cells and deliver therapeutic proteins into the subretinal space to protect photoreceptor cells from degeneration.
Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.
Mesenchymal stem cells or multipotent stromal cells (MSCs) isolated from the bone marrow of adult organisms were initially characterized as plastic adherent, fibroblastoid cells with the capacity to generate heterotopic osseous tissue when transplanted in vivo. In recent years, MSCs or MSC-like cells have been shown to reside within the connective tissue of most organs, and their surface phenotype has been well described. A large number of reports have also indicated that the cells possess the capacity to transdifferentiate into epithelial cells and lineages derived from the neuroectoderm. The broad developmental plasticity of MSCs was originally thought to contribute to their demonstrated efficacy in a wide variety of experimental animal models of disease as well as in human clinical trials. However, new findings suggest that the ability of MSCs to alter the tissue microenvironment via secretion of soluble factors may contribute more significantly than their capacity for transdifferentiation in tissue repair. Herein, we critically evaluate the literature describing the plasticity of MSCs and offer insight into how the molecular and functional heterogeneity of this cell population, which reflects the complexity of marrow stroma as an organ system, may confound interpretation of their transdifferentiation potential. Additionally, we argue that this heterogeneity also provides a basis for the broad therapeutic efficacy of MSCs.
Disclosure of potential conflicts of interest is found at the end of this article.
As a follow-up to previous studies showing that human cortical neural progenitor cells (hNPC(ctx)) can sustain vision for at least 70 days after injection into the subretinal space of Royal College of Surgeons (RCS) rats, the authors examined how functional rescue is preserved over long periods and how this relates to retinal integrity and donor cell survival.
Pigmented dystrophic RCS rats (n = 15) received unilateral subretinal injections of hNPC(ctx) at postnatal day (P) 21; control rats (n = 10) received medium alone and were untreated. All animals were maintained on oral cyclosporine A. Function was monitored serially by measuring acuity (using an optomotor test) and luminance thresholds (recording from the superior colliculus) at approximately P90, P150, and P280. Eyes were processed for histologic study after functional tests.
Acuity and luminance thresholds were significantly better in hNPC(ctx)-treated animals than in controls (P < 0.001) at all time points studied. Acuity was greater than 90%, 82%, and 37% of normal at P90, P150, and P270, whereas luminance thresholds in the area of best rescue remained similar the whole time. Histologic studies revealed substantial photoreceptor rescue, even up to P280, despite progressive deterioration in rod and cone morphology. Donor cells were still present at P280, and no sign of donor cell overgrowth was seen.
Long-term rescue of function and associated morphologic substrates was seen, together with donor cell survival even in the xenograft paradigm. This is encouraging when exploring further the potential for the application of hNPC(ctx) in treating retinal disease.
To summarize current technique, indications, and pitfalls of electrophysiologic testing used in ophthalmology.
Visual evoked potentials (VEPs) may be useful as an objective measurement of refractive error in complicated patients. VEP P100 latency was found superior to color vision and visual field in early stages of hydroxychloroquine maculopathy. VEP results can be predictive of visual recovery in traumatic optic neuropathy. Multifocal electroretinogram (ERG) or VEP can provide an objective assessment of visual field defects not yet present on automated perimetry in patients with glaucomatous and nonglaucomatous optic neuropathies. In patients with intraocular lymphoma, reduced amplitudes of all ERG components can be recorded, with the b-wave amplitude being most significantly affected.
Various visual electrophysiologic tests are useful to the ophthalmologist, each with different indications. The flash ERG is most useful in diffuse retinal disorders, whereas the multifocal ERG is superior in localized retinal disease. VEPs can be valuable in diagnosing optic neuropathies, nonorganic visual loss, and assessing visual function in infants or children.
Unlabelled:
The vascular beds supplying the retina may sustain injury as a result of underlying disease such as diabetes, and/or the interaction of genetic predisposition, environmental insults, and age. The vascular pathologic features observed in different intraocular vascular diseases can be categorized broadly as proliferation, exemplified by proliferative diabetic retinopathy, leakage such as macular edema secondary to retinal vein occlusion, or a combination of proliferation and leakage, as seen in neovascular age-related macular degeneration (AMD). The World Health Organization has identified diabetic retinopathy and AMD as priority eye diseases for the prevention of vision loss in developed countries. The pathologic transformations of the retinal vasculature seen in intraocular vascular disease are associated with increased expression of vascular endothelial growth factor A (VEGF), a potent endothelial-specific mitogen. Furthermore, in model systems, VEGF alone is sufficient to trigger intraocular neovascularization, and its inhibition is associated with functional and anatomic improvements in the affected eye. Therapeutic interventions with effect on VEGF include intraocular capture and neutralization by engineered antibodies or chimeric receptors, downregulation of its expression with steroids, or alleviation of retinal ischemia, a major stimulus for VEGF expression, with retinal ablation by laser treatment. Data from prospective randomized clinical trials indicate that VEGF inhibition is a potent therapeutic strategy for intraocular vascular disease. These findings are changing clinical practice and are stimuli for further study of the basic mechanisms controlling intraocular angiogenesis.
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Proprietary or commercial disclosure may be found after the references.
As an alternative to a viral vector, the application of stem cells to transfer specific genes is under investigation in various organs. Using this strategy may provide more effective method to supply neurotrophic factor to the neurodegenerative diseases caused by neurotrophic factor deprivation. This study investigated the possibility and efficacy of stem cell-based delivery of the brain-derived neurotrophic factor (BDNF) gene to rat retina. Rat BDNF cDNA was transduced into rat bone marrow mesenchymal stem cells (rMSCs) using a retroviral vector. Its incorporation into the experimental rat retina and the expression of BDNF after intravitreal injection or subretinal injection were detected by real-time PCR, western blot analysis, and immunohistochemical staining. For the incorporated rMSCs, retinal-specific marker staining was performed to investigate the changes in morphology and the characteristics of the stem cells. Transduction of the rMSCs by retrovirus was effective, and the transduced rMSCs expressed high levels of the BDNF gene and protein. The subretinal injection of rMSCs produced rMSC migration and incorporation into the rat retina (about 15.7% incorporation rate), and retinal BDNF mRNA and protein expression was increased at 4 weeks after transplantation. When subretinal injection of rMSCs was applied to axotomized rat retina, it significantly increased the expression of BDNF until 4 weeks after transplantation. Some of the transplanted rMSCs exhibited morphological changes, but the retinal-specific marker stain was not sufficient to indicate whether neuronal differentiation had occurred. Using mesenchymal stem cells to deliver the BDNF gene to the retina may provide new treatment for glaucoma.
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.
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.
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.
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.
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.
Disclosure of potential conflicts of interest is found at the end of this article.
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.
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),