Optimized Culture System to Induce Neurite Outgrowth From Retinal Ganglion Cells in Three-Dimensional Retinal Aggregates Differentiated From Mouse and Human Embryonic Stem Cells

Maekawa, Yuki; Onishi, Akishi; Matsushita, K; Koide, Naoshi; Mandai, Michiko; Suzuma, Kiyoshi; Kitaoka, Takashi; Kuwahara, Atsushi; Ozone, Chikafumi; Nakano, Tokushige; Eiraku, Mototsugu; Takahashi, Masayo

doi:10.3109/02713683.2015.1038359

Cited by 30 publications

(38 citation statements)

References 46 publications

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“…Although there are several reports on the function and neurite outgrowth of human iPSC-RGCs differentiated by an organotypic culture system, 25,30,31 little has been reported on purified RGCs from 3D retinal organoids. 24 It is difficult to identify RGCs induced by direct genomic reprogramming completely as they are restricted to the eye.…”

Section: Discussionmentioning

confidence: 99%

“…We have previously reported an explant culture procedure in which human ESC-derived retinal organoids exhibit significant neurite outgrowth. 25 Following this trend, we applied an immunopanning method designed for both rodent and human systems. We applied this to mouse retinas and mouse ESC-derived retinal organoids and compared the morphologic and immunohistochemical features of the purified cells.…”

Section: Discussionmentioning

confidence: 99%

“…3A). 25 Retinal optic vesicle-like structures with clear neural epithelial layers were observed from around DD 7 to 10 and expanded gradually until DD 20 (Fig. 3A).…”

Section: Mouse Rgcs Couldmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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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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“…En route to differentiating photoreceptors and other later-born retinal cells, differentiation of RGC-like cells has been reported, 43,44 but only a few protocols have focused on RGC differentiation. 45–47 …”

Section: Pluripotent Stem Cells As a Source Of Rgcsmentioning

confidence: 99%

“…In a 3D self-organization technique, intrinsic cellular programs drive self-organization of stem cells to form an optic cup, which can further differentiate into a multilayered structure containing various retinal cells. 48,49 By optimizing the 3D culture technique, Maekawa et al 47 reported successful RGC neurite outgrowth from the 3D optic vesicles.…”

Section: Pluripotent Stem Cells As a Source Of Rgcsmentioning

confidence: 99%

The Potential of Human Stem Cells for the Study and Treatment of Glaucoma

Chamling

Sluch

Zack

2016

Invest. Ophthalmol. Vis. Sci.

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PurposeCurrently, the only available and approved treatments for glaucoma are various pharmacologic, laser-based, and surgical procedures that lower IOP. Although these treatments can be effective, they are not always sufficient, and they cannot restore vision that has already been lost. The goal of this review is to briefly assess current developments in the application of stem cell biology to the study and treatment of glaucoma and other forms of optic neuropathy.MethodsA combined literature review and summary of the glaucoma-related discussion at the 2015 “Sight Restoration Through Stem Cell Therapy” meeting that was sponsored by the Ocular Research Symposia Foundation (ORSF).ResultsOngoing advancements in basic and eye-related developmental biology have enabled researchers to direct murine and human stem cells along specific developmental paths and to differentiate them into a variety of ocular cell types of interest. The most advanced of these efforts involve the differentiation of stem cells into retinal pigment epithelial cells, work that has led to the initiation of several human trials. More related to the glaucoma field, there have been recent advances in developing protocols for differentiation of stem cells into trabecular meshwork and retinal ganglion cells. Additionally, efforts are being made to generate stem cell–derived cells that can be used to secrete neuroprotective factors.ConclusionsAdvancing stem cell technology provides opportunities to improve our understanding of glaucoma-related biology and develop models for drug development, and offers the possibility of cell-based therapies to restore sight to patients who have already lost vision.

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Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration

et al. 2016

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Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies.

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Optimized Culture System to Induce Neurite Outgrowth From Retinal Ganglion Cells in Three-Dimensional Retinal Aggregates Differentiated From Mouse and Human Embryonic Stem Cells

Cited by 30 publications

References 46 publications

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

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

The Potential of Human Stem Cells for the Study and Treatment of Glaucoma

Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration

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