Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells: Applications for the Study and Treatment of Optic Neuropathies

Inherited optic neuropathies share visual impairment due to the degeneration of retinal ganglion cells (RGCs) as the hallmark of the disease. This group of genetic disorders are caused by mutations in nuclear genes or in the mitochondrial DNA (mtDNA). An impaired mitochondrial function is the underlying mechanism of these diseases. Currently, optic neuropathies lack an effective treatment, and the implementation of induced pluripotent stem cell (iPSC) technology would entail a huge step forward. The generation of iPSC-derived RGCs would allow faithfully modeling these disorders, and these RGCs would represent an appealing platform for drug screening as well, paving the way for a proper therapy. Here, we review the ongoing two-dimensional (2D) and three-dimensional (3D) approaches based on iPSCs and their applications, taking into account the more innovative technologies, which include tissue engineering or microfluidics.

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Single Cell Sequencing of Induced Pluripotent Stem Cell Derived Retinal Ganglion Cells (iPSC-RGC) Reveals Distinct Molecular Signatures and RGC Subtypes

Genes ◽

10.3390/genes12122015 ◽

2021 ◽

Vol 12 (12) ◽

pp. 2015

Author(s):

Harini V. Gudiseva ◽

Vrathasha Vrathasha ◽

Jie He ◽

Devesh Bungatavula ◽

Joan M. O’Brien ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Single Cell ◽

Retinal Ganglion Cells ◽

Pluripotent Stem Cell ◽

Ganglion Cells ◽

Induced Pluripotent Stem Cell ◽

Retinal Ganglion ◽

Single Cell Sequencing ◽

Induced Pluripotent

We intend to identify marker genes with differential gene expression (DEG) and RGC subtypes in cultures of human-induced pluripotent stem cell (iPSC)-derived retinal ganglion cells. Single-cell sequencing was performed on mature and functional iPSC-RGCs at day 40 using Chromium Single Cell 3’ V3 protocols (10X Genomics). Sequencing libraries were run on Illumina Novaseq to generate 150 PE reads. Demultiplexed FASTQ files were mapped to the hg38 reference genome using the STAR package, and cluster analyses were performed using a cell ranger and BBrowser2 software. QC analysis was performed by removing the reads corresponding to ribosomal and mitochondrial genes, as well as cells that had less than 1X mean absolute deviation (MAD), resulting in 4705 cells that were used for further analyses. Cells were separated into clusters based on the gene expression normalization via PCA and TSNE analyses using the Seurat tool and/or Louvain clustering when using BBrowser2 software. DEG analysis identified subsets of RGCs with markers like MAP2, RBPMS, TUJ1, BRN3A, SOX4, TUBB3, SNCG, PAX6 and NRN1 in iPSC-RGCs. Differential expression analysis between separate clusters identified significant DEG transcripts associated with cell cycle, neuron regulatory networks, protein kinases, calcium signaling, growth factor hormones, and homeobox transcription factors. Further cluster refinement identified RGC diversity and subtype specification within iPSC-RGCs. DEGs can be used as biomarkers for RGC subtype classification, which will allow screening model systems that represent a spectrum of diseases with RGC pathology.

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Human Pluripotent Stem Cell-Derived Neural Progenitor Cells Promote Retinal Ganglion Cell Survival and Axon Recovery in an Optic Nerve Compression Animal Model

International Journal of Molecular Sciences ◽

10.3390/ijms222212529 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12529

Author(s):

Mira Park ◽

Hyun-Mun Kim ◽

Hyun-Ah Shin ◽

Seung-Hyun Lee ◽

Dong-Youn Hwang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Optic Nerve ◽

Retinal Ganglion Cells ◽

Pluripotent Stem Cell ◽

Neural Progenitor Cells ◽

Ganglion Cells ◽

Retinal Ganglion ◽

Human Pluripotent Stem Cell ◽

Hypoxic Conditions

Human pluripotent stem cell-derived neural progenitor cells (NPCs) have the potential to recover from nerve injury. We previously reported that human placenta-derived mesenchymal stem cells (PSCs) have neuroprotective effects. To evaluate the potential benefit of NPCs, we compared them to PSCs using R28 cells under hypoxic conditions and a rat model of optic nerve injury. NPCs and PSCs (2 × 106 cells) were injected into the subtenon space. After 1, 2, and 4 weeks, we examined changes in target proteins in the retina and optic nerve. NPCs significantly induced vascular endothelial growth factor (Vegf) compared to age-matched shams and PSC groups at 2 weeks; they also induced neurofilaments in the retina compared to the sham group at 4 weeks. In addition, the expression of brain-derived neurotrophic factor (Bdnf) was high in the retina in the NPC group at 2 weeks, while expression in the optic nerve was high in both the NPC and PSC groups. The low expression of ionized calcium-binding adapter molecule 1 (Iba1) in the retina had recovered at 2 weeks after NPC injection and at 4 weeks after PSC injection. The expression of the inflammatory protein NLR family, pyrin domain containing 3 (Nlrp3) was significantly reduced at 1 week, and that of tumor necrosis factor-α (Tnf-α) in the optic nerves of the NPC group was lower at 2 weeks. Regarding retinal ganglion cells, the expressions of Brn3a and Tuj1 in the retina were enhanced in the NPC group compared to sham controls at 4 weeks. NPC injections increased Gap43 expression from 2 weeks and reduced Iba1 expression in the optic nerves during the recovery period. In addition, R28 cells exposed to hypoxic conditions showed increased cell survival when cocultured with NPCs compared to PSCs. Both Wnt/β-catenin signaling and increased Nf-ĸb could contribute to the rescue of damaged retinal ganglion cells via upregulation of neuroprotective factors, microglial engagement, and anti-inflammatory regulation by NPCs. This study suggests that NPCs could be useful for the cellular treatment of various optic neuropathies, together with cell therapy using mesenchymal stem cells.

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Platelet-derived growth factor-BB is involved in mesenchymal stem cell secretome-induced neuroprotection of retinal ganglion cells

Brain ◽

10.1093/brain/awu017 ◽

2014 ◽

Vol 137 (5) ◽

pp. e276-e276 ◽

Cited By ~ 2

Author(s):

Jian-Li He

Keyword(s):

Stem Cell ◽

Growth Factor ◽

Mesenchymal Stem Cell ◽

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Platelet Derived Growth Factor ◽

Retinal Ganglion

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Effects of Adult Müller Cells and Their Conditioned Media on the Survival of Stem Cell-Derived Retinal Ganglion Cells

Cells ◽

10.3390/cells9081759 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1759

Author(s):

Xandra Pereiro ◽

Adam M. Miltner ◽

Anna La Torre ◽

Elena Vecino

Keyword(s):

Stem Cell ◽

Cell Survival ◽

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Müller Cells ◽

Cell Types ◽

Conditioned Media ◽

Retinal Ganglion ◽

Muller Cells ◽

Müller Glia

Retinal neurons, particularly retinal ganglion cells (RGCs), are susceptible to the degenerative damage caused by different inherited conditions and environmental insults, leading to irreversible vision loss and, ultimately, blindness. Numerous strategies are being tested in different models of degeneration to restore vision and, in recent years, stem cell technologies have offered novel avenues to obtain donor cells for replacement therapies. To date, stem cell–based transplantation in the retina has been attempted as treatment for photoreceptor degeneration, but the same tools could potentially be applied to other retinal cell types, including RGCs. However, RGC-like cells are not an abundant cell type in stem cell–derived cultures and, often, these cells degenerate over time in vitro. To overcome this limitation, we have taken advantage of the neuroprotective properties of Müller glia (one of the main glial cell types in the retina) and we have examined whether Müller glia and the factors they secrete could promote RGC-like cell survival in organoid cultures. Accordingly, stem cell-derived RGC-like cells were co-cultured with adult Müller cells or Müller cell-conditioned media was added to the cultures. Remarkably, RGC-like cell survival was substantially enhanced in both culture conditions, and we also observed a significant increase in their neurite length. Interestingly, Atoh7, a transcription factor required for RGC development, was up-regulated in stem cell-derived organoids exposed to conditioned media, suggesting that Müller cells may also enhance the survival of retinal progenitors and/or postmitotic precursor cells. In conclusion, Müller cells and the factors they release promote organoid-derived RGC-like cell survival, neuritogenesis, and possibly neuronal maturation.

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Temporal expression of CD184(CXCR4) and CD171(L1CAM) identifies distinct early developmental stages of human retinal ganglion cells in embryonic stem cell derived retina

Experimental Eye Research ◽

10.1016/j.exer.2016.11.013 ◽

2017 ◽

Vol 154 ◽

pp. 177-189 ◽

Cited By ~ 9

Author(s):

J.G. Aparicio ◽

H. Hopp ◽

A. Choi ◽

J. Mandayam Comar ◽

V.C. Liao ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Retinal Ganglion Cells ◽

Developmental Stages ◽

Ganglion Cells ◽

Embryonic Stem ◽

Retinal Ganglion ◽

Temporal Expression ◽

Early Developmental Stages ◽

Early Developmental

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Single cell RNA sequencing of stem cell-derived retinal ganglion cells

Scientific Data ◽

10.1038/sdata.2018.13 ◽

2018 ◽

Vol 5 (1) ◽

Cited By ~ 21

Author(s):

Maciej Daniszewski ◽

Anne Senabouth ◽

Quan H. Nguyen ◽

Duncan E. Crombie ◽

Samuel W. Lukowski ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Retinal Ganglion ◽

Single Cell Rna Sequencing

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Transplantation of Retinal Ganglion Cells Derived from Male Germline Stem Cell as a Potential Treatment to Glaucoma

Stem Cells and Development ◽

10.1089/scd.2019.0060 ◽

2019 ◽

Vol 28 (20) ◽

pp. 1365-1375 ◽

Cited By ~ 3

Author(s):

Hoi Ching Suen ◽

Yan Qian ◽

Jinyue Liao ◽

Chun Shui Luk ◽

Wing Tung Lee ◽

...

Keyword(s):

Stem Cell ◽

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Germline Stem Cell ◽

Retinal Ganglion ◽

Potential Treatment ◽

Male Germline

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Single Cell RNA Sequencing of stem cell-derived retinal ganglion cells

10.1101/191395 ◽

2017 ◽

Author(s):

Maciej Daniszewski ◽

Anne Senabouth ◽

Quan Nguyen ◽

Duncan E. Crombie ◽

Samuel W. Lukowski ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Retinal Ganglion Cells ◽

Human Embryonic Stem Cell ◽

Ganglion Cells ◽

Embryonic Stem ◽

Retinal Ganglion ◽

Single Cell Rna Sequencing

ABSTRACTWe used human embryonic stem cell-derived retinal ganglion cells (RGCs) to characterize the transcriptome of 1,174 cells at the single cell level. The human embryonic stem cell line BRN3B-mCherry A81-H7 was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and subsequently prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells upregulated genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature phenotype.

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