Developing Rods Transplanted into the Degenerating Retina of Crx-Knockout Mice Exhibit Neural Activity Similar to Native Photoreceptors

Homma, Kyoji; Okamoto, Satoshi; Mandai, Michiko; Gotoh, Noriko; Rajasimha, Harsha; Chang, Yi‐Ping; Chen, Shan; Li, Wei; Cogliati, Tiziana; Swaroop, Anand; Takahashi, Masayo

doi:10.1002/stem.1372

Cited by 91 publications

(70 citation statements)

References 67 publications

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“…GSE52006). (51,52); however, photoreceptor cell death occurring in the Crx -/-host retinas may impair long-term survival of the transplanted cells. The unique phenotype of the Crx Rip/+ mutant should provide a better host retinal environment for exploring photoreceptor integration and functional assessment in cell replacement therapy.…”

Section: Discussionmentioning

confidence: 99%

OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness

Roger¹,

Hiriyanna²,

Gotoh³

et al. 2014

J. Clin. Invest.

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106

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Leber congenital amaurosis (LCA) encompasses a set of early-onset blinding diseases that are characterized by vision loss, involuntary eye movement, and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA, which is typically recessive; however, mutations in homeodomain transcription factor CRX lead to an autosomal dominant form of LCA. The mechanism of CRX-associated LCA is not understood. Here, we identified a spontaneous mouse mutant with a frameshift mutation in Crx (Crx Rip ). We determined that Crx Rip is a dominant mutation that results in congenital blindness with nonrecordable response by ERG and arrested photoreceptor differentiation with no associated degeneration. Expression of LCA-associated dominant CRX frameshift mutations in mouse retina mimicked the Crx Rip phenotype, which was rescued by overexpression of WT CRX. Whole-transcriptome profiling using deep RNA sequencing revealed progressive and complete loss of rod differentiation factor NRL in Crx Rip retinas. Expression of NRL partially restored rod development in Crx Rip/+ mice. We show that the binding of homeobox transcription factor OTX2 at the Nrl promoter was obliterated in Crx Rip mice and ectopic expression of OTX2 rescued the rod differentiation defect. Together, our data indicate that OTX2 maintains Nrl expression in developing rods to consolidate rod fate. Our studies provide insights into CRX mutation-associated congenital blindness and should assist in therapeutic design.

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Section: Discussionmentioning

confidence: 99%

OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness

Roger¹,

Hiriyanna²,

Gotoh³

et al. 2014

J. Clin. Invest.

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106

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“…Thy1-EGFP mouse ESCs were generated 20 and maintained as described, 21 and 3D retinal organoids were differentiated using the SFEBq protocol 9,22 with minor modifications. Briefly, embryoid bodies forming optic vesicle-like structures at differentiation day (DD) 9 were transferred to 10-cm culture dishes and maintained in culture.…”

Section: Differentiation Of 3d Retinal Organoids Derived From Thy1-egmentioning

confidence: 99%

Culture Systems of Dissociated Mouse and Human Pluripotent Stem Cell–Derived Retinal Ganglion Cells Purified by Two-Step Immunopanning

Kobayashi

Onishi

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. We aimed to establish purification and culture systems for retinal ganglion cells (RGCs) differentiated from mouse and human pluripotent stem cells (PSC) for in vitro and regenerative medicine studies. METHODS.We used a two-step immunopanning method to purify RGCs from mouse and human PSC-derived three-dimensional (3D) retinal organoids. To assess the method, we purified RGCs from 3D retinal organoids derived from embryonic stem cells (ESCs) generated from Thy1-EGFP transgenic (TG) mice. In addition, 3D retinal organoids differentiated from human induced PSCs (iPSCs) were cultured for up to differentiation day (DD) 120, and RGCs were purified by immunopanning. RGC marker expressions were confirmed by immunostaining and reverse transcription-quantitative PCR. The purified RGCs were cultured, and neurite outgrowth was measured and analyzed using an IncuCyte Zoom system. RESULTS.Mouse RGCs purified from Thy1-EGFP TG mouse retinas and the ESC-derived 3D retinas could be maintained for approximately 2 to 3 weeks, expressing the markers BRN3B and SMI-312. Purified RGCs from human iPSC-derived retinal organoids expressed RGC markers and could be maintained for up to 4 weeks. The RGCs collected at DD 90 to 110 extended longer neurites than those collected at younger stages.CONCLUSIONS. We successfully purified RGCs from mouse and human PSC-derived 3D retinal organoids cultured for approximately 120 days. RGCs from older retinal organoids would be useful for neurite tracking. This method would be effective not only for studying the pathology of human RGC diseases but also for therapeutic drug studies and RGC transplantation.

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“…8 Mouse ES/iPS cells (Rx knock-in GFP ES line mESRxþ; Nrlp-eGFP transgenic iPS line) were maintained in Glasgow minimum essential medium (GMEM), 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids (NEAA; Invitrogen, Carlsbad, CA, USA), 0.1 mM 2-mercaptoethanol (2-ME), 100 U/mL penicillin and 100 mg/mL streptomycin supplemented with 1000 U/mL leukemia inhibitory factor (LIF), 3 lM CHIR99021, which is a glycogen synthase kinase 3b (GSK-3b) inhibitor, and 1 lM PD0325901, which is a mitogen activated protein kinase kinase (MEK or MAPKK) inhibitor, on gelatin-coated dishes. LIF, CHIR99021, and PD0325901 were freshly added to the culture medium at each medium change.…”

Section: Cell Culturementioning

confidence: 99%

Induction of Functional 3D Ciliary Epithelium–Like Structure From Mouse Induced Pluripotent Stem Cells

Kinoshita

Suzuma

Kaneko

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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Citation: Kinoshita H, Suzuma K, Kaneko J, Mandai M, Kitaoka T, Takahashi M. Induction of functional 3D ciliary epithelium-like structure from mouse induced pluripotent stem cells. Invest Ophthalmol Vis Sci. 2016;57:153-161. DOI:10.1167/ iovs.15-17610 PURPOSE. To generate ciliary epithelium (CE) from mouse induced pluripotent stem (iPS) cells.METHODS. Recently, a protocol for self-organizing optic cup morphogenesis in threedimensional culture was reported, and it was suggested that ocular tissue derived from neural ectoderm could be differentiated. We demonstrated that a CE-like double-layered structure could be induced in simple culture by using a modified Eiraku differentiation protocol.RESULTS. Differentiation of a CE-like double-layered structure could be promoted by glycogen synthase kinase 3b (GSK-3b) inhibitor. Connexin43 and aquaporin1 were expressed in both thin layers, and induced CE-like cells expressed ciliary marker genes, such as cyclinD2, zic1, tgfb2, aldh1a3, wfdc1, otx1, BMP4, and BMP7. Increases in cytoplasmic and nuclear bcatenin in aggregates of the CE-like double-layered structure were confirmed by Western blot analysis. In addition, tankyrase inhibitor prevented the induction of the CE-like double-layered structure by GSK-3b inhibitor. Dye movement from pigmented cells to nonpigmented cells in the mouse iPS cell-derived CE-like structure was observed in a fluid movement experiment, consistent with the physiological function of CE in vivo.CONCLUSIONS. We could differentiate CE from mouse iPS cells in the present study. In the future, we hope that this CE-like complex will become useful as a graft for transplantation therapy in pathologic ocular hypotension due to CE dysfunction, and as a screening tool for the development of drugs for diseases associated with CE function.Keywords: ciliary epithelium, iPS, GSK-3b, b-catenin T he ciliary body (CB) plays important roles in eye homeostasis. It secretes aqueous humor that maintains IOP, controls the lens shape through the ciliary muscle and ciliary zonules, and synthesizes glycoproteins of the vitreous body. The CB contains a double-layered epithelial structure comprising the inner nonpigmented epithelium (NPE), the outer pigmented epithelium (PE), and the underlying stroma. The NPE is continuous with the neural retina (NR) and the iris pigmented epithelium, while the PE lies between the retinal pigment epithelium (RPE) and the outer iris muscle. 1 The NPE and the PE, as well as the NR and RPE, are derived from neural ectoderm. During eye development, the ciliary epithelium (CE) folds to form the ciliary processes, while neural crest cellderived stroma cells underneath the CE also contribute to CB morphogenesis. 2 The PE rests on the connective tissue stroma, and the NPE is polarized with its basal lamina facing the posterior chamber of the eye. The PE and NPE interact via their apical membranes with gap junctions.Water can pass from PE to NPE via gap junctions following NaCl movement 3,4 and exit the NPE to the posterior chamber by using ba...

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Developing Rods Transplanted into the Degenerating Retina of Crx-Knockout Mice Exhibit Neural Activity Similar to Native Photoreceptors

Cited by 91 publications

References 67 publications

OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness

OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness

Culture Systems of Dissociated Mouse and Human Pluripotent Stem Cell–Derived Retinal Ganglion Cells Purified by Two-Step Immunopanning

Induction of Functional 3D Ciliary Epithelium–Like Structure From Mouse Induced Pluripotent Stem Cells

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