Silke Becker

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.

Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release

•  Hypoxic pulmonary vasoconstriction (HPV) is a mechanism by which pulmonary arteries maintain blood oxygenation during alveolar hypoxia. •  HPV is generally studied using a vasoconstricting co‐stimulus that amplifies the HPV but may also distort its properties; we therefore characterised HPV in isolated rat intrapulmonary arteries during 40 min hypoxic challenges in the absence of any such stimulus. •  Immediate (phase 1) and sustained (phase 2) components of HPV were unaffected by blocking voltage‐gated Ca2+ channels but were abolished by depletion of sarcoplasmic reticulum Ca2+. Phase 2 was attenuated by blockade of store‐operated Ca2+ entry (SOCE), although it largely persisted in Ca2+‐free physiological saline solution. •  HPV was associated with an increase in the intrapulmonary artery ratio of oxidised to reduced glutathione and was inhibited by antioxidants. •  HPV resulted primarily from intracellular Ca2+ release, with SOCE making a contribution, particularly to phase 2. Sustained HPV involves oxidation of the pulmonary artery redox state.

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.

Genetic background and light-dependent progression of photoreceptor cell degeneration in Prominin-1 knockout mice.

PURPOSE Mutations in the Prominin-1 (Prom1) gene are known to cause retinitis pigmentosa and Stargardt disease, both of which are associated with progressive photoreceptor cell death. There are no effective therapies for either disorder. The aim of this study was to investigate the mechanism of the retinal degeneration in Prom1-deficient mouse models. METHODS We constructed Prom1 knockout mice with two distinct genetic backgrounds of C57BL/6 and C57BL/6xCBA/NSlc, and investigated the photoreceptor degeneration by means of histology and functional tests.. In addition, we examined the effect of light on the Prom1(-/-) retina by rearing the mice in the normal light/dark cycle and completely dark conditions. Finally, we investigated if the retinoic-acid derivative Fenretinide slowed the pace of retinal degeneration in these mouse models. RESULTS The Prom1(-/-)-knockout mice with both backgrounds developed photoreceptor degeneration after eye opening, but the CB57/BL6-background mice developed photoreceptor cell degeneration much faster than the C57BL/6xCBA/NSlc mice, demonstrating genetic background dependency.. Interestingly, our histologic and functional examination showed that the photoreceptor cell degeneration of Prom1-knockout mice was light-dependent, and was almost completely inhibited when the mutant mice were kept in the dark. The Prom1-knockout retina showed strong downregulation of expression of the visual cycle components, Rdh12 and Abca4. Furthermore, administration of Fenretinide, which lowers the level of the toxic lipofuscin, slowed the degeneration of photoreceptor cells. CONCLUSIONS These findings improve our understanding of the mechanism of cell death in Prominin-1-related disease and provide evidence that fenretinide may be worth studying in human disease.

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.

Stem Cell Based Therapies for Glaucoma

Glaucoma remains one of the leading causes of worldwide blindness. In England and Wales glaucoma is a major or contributory factor for 12-14% of all registrations for blindness and partial sight, second only to macular degeneration (Bunce et al., 2010). The worldwide burden is more significant, with glaucoma being the second leading cause of global blindness after cataract (Resnikoff et al., 2004). It has been estimated that 60.5 million people worldwide would be affected by glaucoma by 2010, with the figure expected to rise to 80 million by 2020 (Quigley and Broman, 2006). Current treatments for glaucoma comprise the lowering of intraocular pressure by eye drops, laser procedures or drainage surgery. However, as implied by the statistics above, many patients experience significant visual loss due to degeneration of retinal ganglion cells (RGCs) despite the advances in the treatments currently available. The need for novel therapies exists for such patients, in particular those with end stage glaucoma, where maintenance of a small number of surviving RGCs may yet permit a reasonable quality of life (Much et al., 2008). Stem cell therapies developed in the laboratory and translated to clinical practice provide an exciting and realistic hope for those affected by degenerative retinal diseases including glaucoma. This chapter will discuss three mechanisms by which stem cell therapies may potentially offer hope to patients with glaucoma, namely local retinal ganglion cell replacement, retina and optic nerve regeneration and stem cell mediated neuroprotection.

Short Communication: Contribution of Voltage-Gated Sodium Channels to the Rabbit Cone Electroretinograms

ABSTRACT Purpose: Although the rabbit eye is of a similar size to the human eye, our limited understanding of the differences in retinal physiology to other species hinders its use in retinal research. The role of voltage-gated sodium channels (Nav) in the propagation of excitatory potentials along bipolar cells remains unclear, as conflicting data have been reported in the rabbit. The present study assesses the relative contributions of Nav to the scotopic and photopic flash ERGs as well as the wavelength-dependence of Nav blockade on the rabbit flicker ERG. Materials and Methods: Tetrodotoxin (TTX, 1 μM) was injected into the vitreous cavity of Chinchilla bastard rabbits. Scotopic ERGs were evoked by white flashes ranging from 10−5 to 10 cds m−2, photopic ERGs on a background of 25 cdm−2 using flash intensities of 0.032–25 cds m−2. Flicker ERGs (3–50 Hz) were elicited by blue, green and yellow stimuli at 2.34 cds m−2 on a white background of 30 cdm−2. Results: The a- and b-waves of the scotopic ERG were unaffected by intravitreal injection of the Nav blocker TTX. In contrast, the b-wave, but not the a-wave, of the photopic ERG was selectively blocked by TTX. The reduction by TTX of the flicker ERG was greater for blue than for green and yellow stimuli. Discussion: The data suggest that Nav selectively contribute to the generation of the photopic b-wave in the rabbit, indicating that they play an important role in the propagation of excitatory signals on bipolar cells in the cone, but not rod pathways. Importantly, the present study resolves conflicting previous reports into the role of Nav in the retinal function of the rabbit.

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.