Karen Eastlake

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.

Müller glia as an important source of cytokines and inflammatory factors present in the gliotic retina during proliferative vitreoretinopathy

Retinal gliosis is characterized by biochemical and physiological changes that often lead to Müller glia proliferation and hypertrophy and is a feature of many neuro‐degenerative and inflammatory diseases such as proliferative vitreoretinopathy (PVR). Although Müller glia are known to release inflammatory factors and cytokines, it is not clear whether cytokine production by these cells mirrors the pattern of factors present in the gliotic retina. Lysates from normal cadaveric retina and gliotic retinal specimens from patients undergoing retinectomy for treatment of PVR, the Müller cell line MIO‐M1 and four human Müller glial cell preparations isolated from normal retina were examined for their expression of cytokines and inflammatory factors using semi‐quantitative dot blot antibody arrays and quantitative arrays. Comparative analysis of the expression of inflammatory factors showed that in comparison with normal retina, gliotic retina exhibited greater than twofold increase in 24/102 factors examined by semiquantitative arrays, and a significant increase in 19 out of 27 factors assessed by quantitative methods (Pu2009<u20090.05 to Pu2009<u20090.001). It was observed that with the exception of some chemotactic factors, the majority of cytokines and inflammatory factors were produced by Müller glia in vitro and included G‐CSF, MCP‐1, PDGF‐bb, RANTES, VEGF, and TGFβ2. These results showed that a large number of inflammatory factors expressed by Müller glia in vitro are upregulated in the gliotic retina, suggesting that targeting the production of inflammatory factors by Müller glia may constitute a valid approach to prevent neural damage during retinal gliosis and this merits further investigations. GLIA 2016;64:495–506

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

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

Gene expression and protein distribution of ADAMTSL-4 in human iris, choroid and retina

Background Mutations in ADAMTSL4 have recently been shown to be the major cause of autosomal recessive isolated ectopia lentis (IEL). However, the function and ocular localisation of the protein is yet to be fully established. We therefore aimed to confirm the expression of this gene and protein in normal ocular tissue. Methods Donor ocular tissue was obtained within 48u2005h post-mortem and iris, choroid and retina were isolated for analysis. Expression of mRNA coding for ADAMTSL4 was examined in four eyes using reverse transcription PCR. Protein coding for this molecule was also investigated in two eyes by western blot analysis. Furthermore, the in situ localisation of ADAMTSL4 was investigated in cryostat sections of whole eyes following immunostaining for this protein and confocal analysis of the stained tissue. Results mRNA and protein coding for ADAMTSL4 were both demonstrated to be expressed in iris and choroidal tissue but were absent from the neural retina. Confocal studies revealed ADAMTS-Like 4 to be present in the ciliary body and ciliary processes and also in the retinal pigment epithelium. Conclusions We have confirmed the gene and protein expression of ADAMTSL4 in human ocular tissue. The pattern of expression may suggest further functions of this gene beyond those suggested by its causative role in IEL.

Optimized feline vitrectomy technique for therapeutic stem cell delivery to the inner retina

Objective To describe an optimized surgical technique for feline vitrectomy which reduces bleeding and aids posterior gel clearance in order to facilitate stem cell delivery to the inner retina using cellular scaffolds. Procedures Three-port pars plana vitrectomies were performed in six-specific pathogen-free domestic cats using an optimized surgical technique to improve access and minimize severe intraoperative bleeding. Results The surgical procedure was successfully completed in all six animals. Lens sparing vitrectomy resulted in peripheral lens touch in one of three animals but without cataract formation. Transient bleeding from sclerotomies, which was readily controlled, was seen in two of the six animals. No cases of vitreous hemorrhage, severe postoperative inflammation, retinal detachment, or endophthalmitis were observed during postoperative follow-up. Conclusions Three-port pars plana vitrectomy can be performed successfully in the cat in a safe and controlled manner when the appropriate precautions are taken to minimize the risk of developing intraoperative hemorrhage. This technique may facilitate the use of feline models of inner retinal degeneration for the development of stem cell transplantation techniques using cellular scaffolds.

Characteristics and vitreoretinal management of retinal detachment in eyes with Boston keratoprosthesis

Purpose To review the incidence and features of vitreoretinal complications of a permanent Boston keratoprosthesis and to report the use and outcomes of 23-gauge vitrectomy to manage vitreoretinal pathology. Design Retrospective non-comparative, interventional case series. Subject, Participants 27 eyes of 27 patients managed with a Boston keratoprosthesis at Moorfields Eye Hospital over a 3-year period. Methods All eyes that underwent pars plana vitrectomy (PPV) and had at least 6u2005months follow-up were analysed with a specific focus on the anatomical and histological characteristics of retinal detachment and outcomes of surgery. Main outcome measures Anatomical success and characteristics of retinal detachment over the follow-up period. Results 27 patients underwent Boston keratoprosthesis implantation over the study period. Of these, six (22%) required PPV for retinal detachment which demonstrated a specific pattern of serous elevation with subsequent severe anterior proliferative vitreoretinopathy (PVR). The mean follow-up period was 9u2005months (range 6–14u2005months). At final follow-up, visual acuity ranged from perception of light to 6/18, and five of six cases had attached retinae under the silicone oil. Histological analysis of a subretinal membrane demonstrated a predominantly glial/retinal pigment epithelium fibrocellular tissue, consistent with PVR. Conclusions The study showed that retinal detachment complicated by PVR, as demonstrated by the clinical and histological characteristics of this condition, is common in patients undergoing Boston keratoprosthesis. We also showed that 23-gauge vitrectomy can be effectively performed in patients with a permanent prosthesis. Visual acuity often remains poor, despite successful anatomical results.

Comparative proteomic analysis of normal and gliotic PVR retina and contribution of Müller glia to this profile

&NA; Müller glia are responsible for the neural retina regeneration observed in fish and amphibians throughout life. Despite the presence of these cells in the adult human retina, there is no evidence of regeneration occurring in humans following disease or injury. It may be possible that factors present in the degenerated retina could prevent human Müller glia from proliferating and neurally differentiating within the diseased retina. On this basis, investigations into the proteomic profile of these cells and the abundance of key proteins associated to Müller glia in the gliotic PVR retina, may assist in the identification of factors with the potential to control Müller proliferation and neural differentiation in vivo. Label free mass spectrometry identified 1527 proteins in Müller glial cell preparations, 1631 proteins in normal retina and 1074 in gliotic PVR retina. Compared to normal retina, 28 proteins were upregulated and 196 proteins downregulated by 2‐fold or more in the gliotic PVR retina. As determined by comparative proteomic analyses, of the proteins highly upregulated in the gliotic PVR retina, the most highly abundant proteins in Müller cell lysates included vimentin, GFAP, polyubiquitin and HSP90a. The observations that proteins highly upregulated in the gliotic retina constitute major proteins expressed by Müller glia provide the basis for further studies into mechanisms that regulate their production. In addition investigations aimed at controlling the expression of these proteins may aid in the identification of factors that could potentially promote endogenous regeneration of the adult human retina after disease or injury.

Biomaterials for repair and regeneration of the neural retina

Although major advances have been made in the treatment and prevention of retinal degenerative disorders, degeneration of the neural retina remains a significant cause of blindness. In cases of advanced disease or unresponsiveness to treatment, the only hope for restoration of vision is either neural cell replacement or induction of endogenous regeneration by resident stem cells. Various stem cell sources have been experimentally investigated for retinal transplantation with variable success, and refinement of protocols for cell delivery will undoubtedly benefit from the use of biomaterials. These could be used for the design of cellular scaffolds to facilitate cell engraftment, as well as for drug and growth factor delivery to promote immune protection and survival of transplanted cells. New scientific developments have yielded a wide range of biomaterials with high biodegradability and tissue compatibility, and the retina is an amenable tissue that could benefit from advances in this technology. However, much research is still needed before this approach can be safely translated into the clinic. This chapter addresses various aspects of retinal development and degeneration, sources of stem cells to repair the damaged retina, and recent advances in the development of biomaterials for use in retinal regeneration therapies.