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Transplantation of photoreceptors derived from human Muller glia restore rod function in the P23H rat.

ABSTRACT: 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.

SUBMITTER: Jayaram H

PROVIDER: S-EPMC3952927 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Transplantation of photoreceptors derived from human Muller glia restore rod function in the P23H rat.

Jayaram Hari H Jones Megan F MF Eastlake Karen K Cottrill Phillippa B PB Becker Silke S Wiseman Joseph J Khaw Peng T PT Limb G Astrid GA

Stem cells translational medicine 20140129 3

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 ...[more]

PMID: 24477073

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Project description:Upon degeneration of photoreceptors in the adult retina, interneurons, including bipolar cells, exhibit a plastic response leading to their aberrant rewiring. Photoreceptor reintroduction has been suggested as a potential approach to sight restoration, but the ability of deafferented bipolar cells to establish functional synapses with photoreceptors is poorly understood. Here we use photocoagulation to selectively destroy photoreceptors in adult rabbits while preserving the inner retina. We find that rods and cones shift into the ablation zone over several weeks, reducing the blind spot at scotopic and photopic luminances. During recovery, rod and cone bipolar cells exhibit markedly different responses to deafferentation. Rod bipolar cells extend their dendrites to form new synapses with healthy photoreceptors outside the lesion, thereby restoring visual function in the deafferented retina. Secretagogin-positive cone bipolar cells did not exhibit such obvious dendritic restructuring. These findings are encouraging to the idea of photoreceptor reintroduction for vision restoration in patients blinded by retinal degeneration. At the same time, they draw attention to the postsynaptic side of photoreceptor reintroduction; various bipolar cell types, representing different visual pathways, vary in their response to the photoreceptor loss and in their consequent dendritic restructuring.SIGNIFICANCE STATEMENT Loss of photoreceptors during retinal degeneration results in permanent visual impairment. Strategies for vision restoration based on the reintroduction of photoreceptors inherently rely on the ability of the remaining retinal neurons to correctly synapse with new photoreceptors. We show that deafferented bipolar cells in the adult mammalian retina can reconnect to rods and cones and restore retinal sensitivity at scotopic and photopic luminances. Rod bipolar cells extend their dendrites to form new synapses with healthy rod photoreceptors. These findings support the idea that bipolar cells might be able to synapse with reintroduced photoreceptors, thereby restoring vision in patients blinded by retinal degeneration.

Project description:We previously reported autophagic structures in rod photoreceptors expressing a misfolding RHO (rhodopsin) mutant (RHOP23H), suggesting that autophagy may play a role in degrading the mutant RHO and/or be involved in photoreceptor cell death. To further examine autophagy in normal and diseased rods, we generated transgenic Xenopus laevis tadpoles expressing the dually fluorescent autophagy marker mRFP-eGFP-LC3 in rods, which changes from green to yellow and finally red as autophagic structures develop and mature. Using transgenic lines with constitutive and inducible expression, we determined the time-course of autophagy in rod photoreceptors: autophagosomes last for 6 to 8 hours before fusing with lysosomes, and acidified autolysosomes last for about 28 hours before being degraded. Autophagy was diurnally regulated in normal rods, with more autophagic structures generated during periods of light, and this regulation was non-circadian. We also found that more autophagosomes were produced in rods expressing the misfolding RHOP23H mutant. The RHO chromophore absorbs photons to initiate phototransduction, and is consumed in this process; it also promotes RHO folding. To determine whether increased autophagy in light-exposed normal rods is caused by increased RHO misfolding or phototransduction, we used CRISPR/Cas9 to knock out the RPE65 and GNAT1 genes, which are essential for chromophore biosynthesis and phototransduction respectively. Both knockouts suppressed light-induced autophagy, indicating that although light and misfolded rhodopsin can both induce autophagy in rods, light-induced autophagy is not due to misfolding of RHO, but rather due to phototransduction. Abbreviations: CYCS: cytochrome c; bRHOP23H: bovine RHOP23H; Cas9: CRISPR associated protein 9; dpf: days post-fertilization; eGFP: enhanced green fluorescent protein; GNAT1: guanine nucleotide-binding protein G(t) subunit alpha-1 aka rod alpha-transducin; HSPA1A/hsp70: heat shock protein of 70 kilodaltons; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1A/1B light chain 3; mRFP: monomeric red fluorescent protein; RHO: rhodopsin; RP: retinitis pigmentosa; RPE65: retinal pigment epithelium-specific 65 kDa protein: sfGFP: superfolding GFP; sgRNA: single guide RNA; WGA: wheat germ agglutinin; RHOp: the Xenopus laevis RHO.2.L promoter.

Dataset Information

Transplantation of photoreceptors derived from human Muller glia restore rod function in the P23H rat.

Publications

Transplantation of photoreceptors derived from human Muller glia restore rod function in the P23H rat.

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