Hari Jayaram

Human Müller Glia with Stem Cell Characteristics Differentiate into Retinal Ganglion Cell (RGC) Precursors In Vitro and Partially Restore RGC Function In Vivo Following Transplantation

Müller glia with stem cell characteristics have been identified in the adult human eye, and although there is no evidence that they regenerate retina in vivo, they can be induced to grow and differentiate into retinal neurons in vitro. We differentiated human Müller stem cells into retinal ganglion cell (RGC) precursors by stimulation with fibroblast growth factor 2 together with NOTCH inhibition using the γ‐secretase inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT). Differentiation into RGC precursors was confirmed by gene and protein expression analysis, changes in cytosolic [Ca2+] in response to neurotransmitters, and green fluorescent protein (GFP) expression by cells transduced with a transcriptional BRN3b‐GFP reporter vector. RGC precursors transplanted onto the inner retinal surface of Lister hooded rats depleted of RGCs by N‐methyl‐d‐aspartate aligned onto the host RGC layer at the site of transplantation but did not extend long processes toward the optic nerve. Cells were observed extending processes into the RGC layer and expressing RGC markers in vivo. This migration was observed only when adjuvant anti‐inflammatory and matrix degradation therapy was used for transplantation. RGC precursors induced a significant recovery of RGC function in the transplanted eyes as determined by improvement of the negative scotopic threshold response of the electroretinogram (indicative of RGC function). The results suggest that transplanted RGC precursors may be capable of establishing local interneuron synapses and possibly release neurotrophic factors that facilitate recovery of RGC function. These cells constitute a promising source of cells for cell‐based therapies to treat retinal degenerative disease caused by RGC dysfunction.

Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve

The 3 most common causes of visual impairment and legal blindness in developed countries (age-related macular degeneration, glaucoma, and diabetic retinopathy) share 1 end point: the loss of neural cells of the eye. Although recent treatment advances can slow down the progression of these conditions, many individuals still suffer irreversible loss of vision. Research is aimed at developing new treatment strategies to rescue damaged photoreceptors and retinal ganglion cells (RGC) and to replace lost cells by transplant. The neuroprotective and regenerative potential of stem and progenitor cells from a variety of sources has been explored in models of retinal disease and ganglion cell loss. Continuous intraocular delivery of neurotrophic factors via stem cells (SC) slows down photoreceptor cells and RGC loss in experimental models. Following intraocular transplantation, SC are capable of expressing proteins and of developing a morphology characteristic of photoreceptors or RGC. Recently, recovery of vision has been achieved for the first time in a rodent model of retinal dystrophy, using embryonic SC differentiated into photoreceptors prior to transplant. This indicates that clinically significant synapse formation and acquisition of the functional properties of retinal neurons, and restoration of vision, are distinct future possibilities.

Transplantation of Photoreceptors Derived From Human Müller Glia Restore Rod Function in the P23H Rat

Müller glia possess stem cell characteristics that have been recognized to be responsible for the regeneration of injured retina in fish and amphibians. Although these cells are present in the adult human eye, they are not known to regenerate human retina in vivo. Human Müller glia with stem cell characteristics (hMSCs) can acquire phenotypic and genotypic characteristics of rod photoreceptors in vitro, suggesting that they may have potential for use in transplantation strategies to treat human photoreceptor degenerations. Much work has been undertaken in rodents using various sources of allogeneic stem cells to restore photoreceptor function, but the effect of human Müller glia‐derived photoreceptors in the restoration of rod photoreceptor function has not been investigated. This study aimed to differentiate hMSCs into photoreceptor cells by stimulation with growth and differentiation factors in vitro to upregulate gene and protein expression of CRX, NR2E3, and rhodopsin and various phototransduction markers associated with rod photoreceptor development and function and to examine the effect of subretinal transplantation of these cells into the P23H rat, a model of primary photoreceptor degeneration. Following transplantation, hMSC‐derived photoreceptor cells migrated and integrated into the outer nuclear layer of the degenerated retinas and led to significant improvement in rod photoreceptor function as shown by an increase in a‐wave amplitude and slope using scotopic flash electroretinography. These observations suggest that hMSCs can be regarded as a cell source for development of cell‐replacement therapies to treat human photoreceptor degenerations and may also offer potential for the development of autologous transplantation.

Differences between the neurogenic and proliferative abilities of Müller glia with stem cell characteristics and the ciliary epithelium from the adult human eye

Much controversy has arisen on the nature and sources of stem cells in the adult human retina. Whilst ciliary epithelium has been thought to constitute a source of neural stem cells, a population of Müller glia in the neural retina has also been shown to exhibit neurogenic characteristics. This study aimed to compare the neurogenic and proliferative abilities between these two major cell populations. It also examined whether differences exist between the pigmented and non-pigmented ciliary epithelium (CE) from the adult human eye. On this basis, Müller glia with stem cell characteristics and pigmented and non-pigmented CE were isolated from human neural retina and ciliary epithelium respectively. Expression of glial, epithelial and neural progenitor markers was examined in these cells following culture under adherent and non-adherent conditions and treatments to induce neural differentiation. Unlike pigmented CE which did not proliferate, non-pigmented CE cells exhibited limited proliferation in vitro, unless epidermal growth factor (EGF) was present in the culture medium to prolong their survival. In contrast, Müller glial stem cells (MSC) cultured as adherent monolayers reached confluence within a few weeks and continued to proliferative indefinitely in the absence of EGF. Both MSC and non-pigmented CE expressed markers of neural progenitors, including SOX2, PAX6, CHX10 and NOTCH. Nestin, a neural stem cell marker, was only expressed by MSC. Non-pigmented CE displayed epithelial morphology, limited photoreceptor gene expression and stained strongly for pigmented epithelial markers upon culture with neural differentiation factors. In contrast, MSC adopted neural morphology and expressed markers of retinal ganglion cells and photoreceptors when cultured under similar conditions. This study provides the first demonstration that pigmented CE possess different proliferative abilities from non-pigmented CE. It also showed that although non-pigmented CE express genes of retinal progenitors, they do not differentiate into neurons in vitro, as that seen with Müller glia that proliferate indefinitely in vitro and that acquire markers of retinal neurons in culture under neural differentiation protocols. From these observations it is possible to suggest that Müller glia that express markers of neural progenitors and become spontaneously immortalized in vitro constitute a potential source of retinal neurons for transplantation studies and fulfil the characteristics of true stem cells due to their proliferative and neurogenic ability.

Adult Retinal Stem Cells Revisited

Recent advances in retinal stem cell research have raised the possibility that these cells have the potential to be used to repair or regenerate diseased retina. Various cell sources for replacement of retinal neurons have been identified, including embryonic stem cells, the adult ciliary epithelium, adult Müller stem cells and induced pluripotent stem cells (iPS). However, the true stem cell nature of the ciliary epithelium and its possible application in cell therapies has now been questioned, leaving other cell sources to be carefully examined as potential candidates for such therapies. The need for identification of the ontogenetic state of grafted stem cells in order to achieve their successful integration into the murine retina has been recognized. However, it is not known whether the same requirements may apply to achieve transplant cell integration into the adult human eye. In addition, the existence of natural barriers for stem cell transplantation, including microglial accumulation and abnormal extracellular matrix deposition have been demonstrated, suggesting that several obstacles need to be overcome before such therapies may be implemented. This review addresses recent scientific developments in the field and discusses various strategies that may be potentially used to design cell based therapies to treat human retinal disease.

Complications and management of cosmetic anterior chamber iris implants

PURPOSE To report the management of complications due to cosmetic iris implantation. DESIGN Interventional case report. METHODS A 27-year-old female underwent bilateral anterior chamber NewColorIris implants in Panama to cosmetically change her eye colour. Two weeks later she presented as an eye emergency in London with corneal decompensation, raised intraocular pressure and significantly reduced vision. Bilateral explantation was performed with good postoperative recovery. RESULTS Three months following explantation, visual acuity had normalized and intraocular pressure was within the normal range without medication. CONCLUSIONS Cosmetic iris implantation can lead to potentially sight threatening complications. This is the first case to be reported in the United Kingdom. There is a lack of published safety data and their cosmetic application should be cautioned against.

Evaluation of the effect of elevated intraocular pressure and reduced ocular perfusion pressure on retinal capillary bed filling and total retinal blood flow in rats by OMAG/OCT

PURPOSE To determine if retinal capillary filling is preserved in the face of acutely elevated intraocular pressure (IOP) in anesthetized rats, despite a reduction in total retinal blood flow (RBF), using optical microangiography/optical coherence tomography (OMAG/OCT). METHODS OMAG provided the capability of depth-resolved imaging of the retinal microvasculature down to the capillary level. Doppler OCT was applied to measure the total RBF using an enface integration approach. The microvascular pattern, capillary density, and total RBF were monitored in vivo as the IOP was increased from 10 to 100mmHg in 10mmHg intervals and returned back to 10mmHg. RESULTS In animals with mean arterial pressure (MAP) of 102±4mmHg (n=10), when IOP was increased from 0 to 100mmHg, the capillary density remained at or above 80% of baseline for the IOP up to 60mmHg [or ocular perfusion pressure (OPP) at 40mmHg]. This was then decreased, achieving 60% of baseline at IOP 70mmHg and OPP of 30mmHg. Total RBF was unaffected by moderate increases in IOP up to 30mmHg, beyond which total RBF decreased linearly, reaching 50% of baseline at IOP 60mmHg and OPP 40mmHg. Both capillary density and total RBF were totally extinguished at 100mmHg, but fully recovered when IOP returned to baseline. By comparison, a separate group of animals with lower MAP (mean=75±6mmHg, n=7) demonstrated comparable decreases in both capillary filling and total RBF at IOPs that were 20mmHg lower than in the initial group. Both were totally extinguished at 80mmHg, but fully recovered when IOP returned to baseline. Relationships of both parameters to OPP were unchanged. CONCLUSION Retinal capillary filling and total RBF responses to IOP elevation can be monitored non-invasively by OMAG/OCT and both are influenced by OPP. Retinal capillary filling was relatively preserved down to a perfusion pressure of 40mmHg, despite a linear reduction in total RBF.

MicroRNA expression in the glaucomatous retina

PURPOSE MicroRNAs are small, endogenous noncoding RNAs that modulate posttranscriptional gene expression. Although the contribution of microRNAs to the pathogenesis of glaucomatous damage is unknown, supporting evidence from central nervous system (CNS) research suggests they may play a role. It was therefore hypothesized that microRNAs known to be altered in CNS injury are also altered in experimental glaucoma. METHODS Intraocular pressure (IOP) was elevated in rats by unilateral injection of hypertonic saline and IOP monitored for 5 weeks. After rats were killed, retrobulbar optic nerve sections were graded for damage. MicroRNA was extracted from whole retinae of eyes with advanced nerve damage (n = 8) and from normal, noninjected control eyes (n = 8). Quantitative PCRs were performed using a panel of 17 microRNAs, reported from CNS research to be implicated in mechanisms also linked to glaucomatous damage. Computationally and experimentally derived gene targets were identified for the differentially expressed microRNAs. These were then integrated with existing gene array data. Functional interpretation was performed using the Molecular Signatures Database and DAVID Functional Annotation Clustering. RESULTS Eight microRNAs were significantly downregulated in glaucomatous retinae compared with controls (miR-181c, miR-497, miR-204, let-7a, miR-29b, miR-16, miR106b, and miR-25); miR-27a was significantly upregulated. Enrichment of targets associated with extracellular matrix/cell proliferation, immune system, and regulation of apoptosis were observed. Cholesterol homeostasis and mTORC-1 pathways showed reduced expression. CONCLUSIONS MicroRNAs are differentially expressed in retinae of eyes with advanced glaucomatous damage compared with normal controls. Integrating microRNA with gene expression data may improve understanding of the complex biological responses produced by chronically elevated IOP.

Independent Adipogenic and Contractile Properties of Fibroblasts in Graves’ Orbitopathy: An In Vitro Model for the Evaluation of Treatments

Graves’ orbitopathy (GO) is a disfiguring and sometimes blinding disease, characterised by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. Little is known about the disease aetiology and the molecular mechanisms driving the phenotypic changes in orbital fibroblasts are unknown. Using fibroblasts isolated from the orbital fat of undiseased individuals or GO patients, we have established a novel in vitro model to evaluate the dual profile of GO cells in a three-dimensional collagen matrix; this pseudo-physiological 3D environment allows measurement of their contractile and adipogenic properties. GO cells contracted collagen matrices more efficiently than control cells following serum or TGFβ1 stimulation, and showed a slightly increased ability to proliferate in the 3D matrix, in accordance with a fibro-proliferative phenotype. GO cells, unlike controls, also spontaneously differentiated into adipocytes in 3D cultures - confirming an intrinsic adipogenic profile. However, both control and GO cells underwent adipogenesis when cultured under pathological pressure levels. We further demonstrate that a Thy-1-low population of GO cells underlies the adipogenic - but not the contractile - phenotype and, using inhibitors, confirm that the contractile and adipogenic phenotypes are regulated by separate pathways. In view of the current lack of suitable treatment for GO, we propose that this new model testing the duality of the GO phenotype could be useful as a preclinical evaluation for the efficacy of potential treatments.

Recent Advances towards the Clinical Application of Stem Cells for Retinal Regeneration.

Retinal degenerative diseases constitute a major cause of irreversible blindness in the world. Stem cell-based therapies offer hope for these patients at risk of or suffering from blindness due to the deterioration of the neural retina. Various sources of stem cells are currently being investigated, ranging from human embryonic stem cells to adult-derived induced pluripotent stem cells as well as human Müller stem cells, with the first clinical trials to investigate the safety and tolerability of human embryonic stem cell-derived retinal pigment epithelium cells having recently commenced. This review aims to summarize the latest advances in the development of stem cell strategies for the replacement of retinal neurons and their supportive cells, the retinal pigment epithelium (RPE) affected by retinal degenerative conditions. Particular emphasis will be given to the advances in stem cell transplantation and the challenges associated with their translation into clinical practice.