Efficient retinal ganglion cells transduction by retro-orbital venous sinus injection of AAV-PHP.eB in mature mice.

Gene therapy is one of the strategies that may reduce or reverse progressive neurodegeneration in retinal neurodegenerative diseases. However, efficiently delivering transgenes to retinal ganglion cells (RGCs) remains hard to achieve. In this study, we innovatively investigated transduction efficiency of adeno-associated virus (AAV)-PHP.eB in murine RGCs by retro-orbital venous sinus injection. Five doses of AAV-PHP.eB-EGFP were retro-orbitally injected in venous sinus in adult C57/BL6J mice. Two weeks after administration, RGCs transduction efficiency was quantified by retinal flat-mounts and frozen section co-labeling with RGCs marker Rbpms. In addition, safety of this method was evaluated by RGCs survival rate and retinal morphology. To conform efficacy of this new method, AAV-PHP.eB-CNTF was administrated into mature mice through single retro-orbital venous injection after optic nerve crush injury to evaluate axonal elongation. Results indicated that AAV- PHP.eB readily crossed the blood-retina barrier and was able to transduce more than 90% of RGCs when total dose of virus reached 5 × 1010 vector genomes (vg). Moreover, this technique did not affect RGCs survival rate and retinal morphology. Furthermore, retro-orbital venous delivery of AAV-PHP.eB-CNTF effectively transduced RGCs, robustly promoted axonal regeneration after optic nerve crush injury. Thus, novel AAV-PHP.eB retro-orbital injection provides a minimally invasive and efficient route for transgene delivery in treatment of retinal neurodegenerative diseases.

Biodegradable scaffolds facilitate epiretinal transplantation of hiPSC-Derived retinal neurons in nonhuman primates.

Transplantation of stem cell-derived retinal neurons is a promising regenerative therapy for optic neuropathy. However, significant anatomic differences compromise its efficacy in large animal models. The present study describes the procedure and outcomes of human-induced pluripotent stem cell (hiPSC)-derived retinal sheet transplantation in primate models using biodegradable materials. Stem cell-derived retinal organoids were seeded on polylactic-coglycolic acid (PLGA) scaffolds and directed toward a retinal ganglion cell (RGC) fate. The seeded tissues showed active proliferation, typical neuronal morphology, and electrical excitability. The cellular scaffolds were then epiretinally transplanted onto the inner surface of rhesus monkey retinas. With sufficient graft-host contact provided by the scaffold, the transplanted tissues survived for up to 1 year without tumorigenesis. Histological examinations indicated survival, further maturation, and migration. Moreover, green fluorescent protein-labeled axonal projections toward the host optic nerve were observed. Cryopreserved organoids were also able to survive and migrate after transplantation. Our results suggest the potential efficacy of RGC replacement therapy in the repair of optic neuropathy for the restoration of visual function. STATEMENT OF SIGNIFICANCE: In the present study, we generated a human retinal sheet by seeding hiPSC-retinal organoid-derived RGCs on a biodegradable PLGA scaffold. We transplanted this retinal sheet onto the inner surface of the rhesus monkey retina. With scaffold support, donor cells survive, migrate and project their axons into the host optic nerve. Furthermore, an effective cryopreservation strategy for retinal organoids was developed, and the thawed organoids were also observed to survive and show cell migration after transplantation.

Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid.

This study was conducted to determine the dynamic Islet1 and Brn3 (POU4F) expression pattern in the human fetal retina and human-induced pluripotent stem cell- (hiPSC-) derived retinal organoid. Human fetal eyes from 8 to 27 fetal weeks (Fwks), human adult retina, hiPSC-derived retinal organoid from 7 to 31 differentiation weeks (Dwks), and rhesus adult retina were collected for cyrosectioning. Immunofluorescence analysis showed that Islet1 was expressed in retinal ganglion cells in the fetal retina, human adult retina, and retinal organoids. Unexpectedly, after Fwk 20, Brn3 expression gradually decreased in the fetal retina. In the midstage of development, Islet1 was detected in bipolar and developing horizontal cells. As the photoreceptor developed, the Islet1-positive cone precursors gradually became Islet1-negative/S-opsin-positive cones. This study highlights the distinguishing characteristics of Islet1 dynamic expression in human fetal retina development and proposes more concerns which should be taken regarding Brn3 as a cell-identifying marker in mature primate retina.

BAM15 attenuates transportation-induced apoptosis in iPS-differentiated retinal tissue.

BACKGROUND: BAM15 is a novel mitochondrial protonophore uncoupler capable of protecting mammals from acute renal ischemic-reperfusion injury and cold-induced microtubule damage. The purpose of our study was to investigate the effect of BAM15 on apoptosis during 5-day transportation of human-induced pluripotent stem (hiPS)-differentiated retinal tissue. METHODS: Retinal tissues of 30 days and 60 days were transported with or without BAM15 for 5 days in the laboratory or by real express. Immunofluorescence staining of apoptosis marker cleaved caspase3, proliferation marker Ki67, and neural axon marker NEFL was performed. And expression of apoptotic-related factors p53, NFkappaB, and TNF-a was detected by real-time PCR. Also, location of ganglion cells, photoreceptor cells, amacrine cells, and precursors of neuronal cell types in retinal tissue was stained by immunofluorescence after transportation. Furthermore, cell viability was assessed by CCK8 assay. RESULTS: Results showed transportation remarkably intensified expression of apoptotic factor cleaved caspase3, p53, NFkappaB, and TNF-a, which could be reduced by supplement of BAM15. In addition, neurons were severely injured after transportation, with axons manifesting disrupted and tortuous by staining NEFL. And the addition of BAM15 in transportation was able to protect neuronal structure and increase cell viability without affecting subtypes cells location of retinal tissue. CONCLUSIONS: BAM15 might be used as a protective reagent on apoptosis during transporting retinal tissues, holding great potential in research and clinical applications.

[Transplantation of porcine type I atelocollagen into rabbit cornea].

OBJECTIVE: To investigate corneal wound healing in rabbit after lamellar keratoplasty with porcine type I atelocollagen. METHODS: One hundred Chinese white rabbits, 80 rabbits were randomly divided into collagen materials group (group A) and allograft group (group B), and group A and B were operated for the lamellar keratoplasty in one eye. Rest 20 rabbits' eyes (40 eyes) were used as the source of allograft for group B. After operation, the rabbits' eyes were observed by naked eyes and slit lamp, and recorded the score of the transparency and neovascularization of the cornea. The rabbits were sacrificed at 3rd, 14th day, the 1st, 3rd, and 6th month (each group eight eyes). The corneas were observed by histopathology, and the epithelial cells marker protein K3 were detected by immunohistochemistry. The results were analyzed by Rank sum test. RESULTS: The transparency of the cornea was increased gradually in A and B groups, and reached the peak on the 6th month (group A: χ(2) = 31.250, P = 0.000; group B: χ(2) = 32.566, P = 0.000). The difference had not statistics significance in the comparison of the transparency from the 1st to 6th month (Z = -1.414, 0.000, -0.743;P = 0.157, 1.000, 0.458). The two groups had corneal neovascularization after 7 days, the intensity of corneal neovascularization in A group increased gradually, reached the peak on day 14, then decreased gradually (χ(2) = 20.727, P = 0.001); the intensity of corneal neovascularization in B group increased gradually, reached the peak on day 14, and then decreased gradually (χ(2) = 25.562, P = 0.000). The difference had statistics significance in the comparison of the neovascularization of the cornea on the 6th month between A and B group (Z = -2.070, P = 0.038). Histopathology showed the collagen material nearly disappeared after 1 month of surgery and arrangement of collagen tends to be regular. On the 6th month, arrayed collagen was regular in general, but disordered partly. CONCLUSIONS: Porcine type I atelocollagen can promote corneal cells regeneration. But it needs more study to be a cornea substitute that can completely replace the corneal allograft.