Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells

Gill, Katherine; Hewitt, Alex W.; Davidson, Kathryn; Pébay, Alice; Wong, Raymond Ching-Bong

doi:10.1167/tvst.3.3.7

Cited by 51 publications

(20 citation statements)

References 88 publications

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“…As in other fields of stem cell biology, much effort is being invested in developing cell replacement measures to treat these diseases. RGCs have been generated in vivo from embryonic stem cells, but often with low efficiency (Gill et al, 2014). Using mouse stem cells carrying both alleles or just Pou4f2 FlagtdTomato , it may be possible to directly monitor the generated of RGCs without other labeling techniques, which may streamline the process of developing new procedures and improving efficiencies.…”

Section: Discussionmentioning

confidence: 99%

Two new genetically modified mouse alleles labeling distinct phases of retinal ganglion cell development by fluorescent proteins

Cheng

et al. 2020

Preprint

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During development, retinal progenitor cells (RPCs) take on different trajectories for distinct cell fates. Previous studies have identified key regulators involved in two key steps of retinal ganglion cell (RGC) genesis; Atoh7 functions in a subpopulation of RPCs to render them competent for the RGC fate, whereas Pou4f2 and Isl1 function to specify the RGC fate and promote RGC differentiation. Extensive research has been performed on the roles of these transcription factors in RGC development, but properties of these two phases they represent and the cellular context in which these two factors function have not been thoroughly investigated, largely due to the cellular heterogeneity of developing retina. In this paper, we describe two novel knock-in mouse alleles, Atoh7 zsGreenCreERT2 and Pou4f2 FlagtdTomato , which enabled us to label retinal cells in the two phases of RGC development by fluorescent proteins. In addition, the Atoh7 zsGreenCreERT2 allele also allowed for indirect labeling of RGCs and other cell types upon tamoxifen induction in a dose-dependent manner. Further, these alleles could be used to purify retinal cells of these different phases by fluorescence assisted cell sorting (FACS). Thus, these two alleles can serve as very useful tools for studying the molecular and genetic mechanisms underlying RGC formation.

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Section: Discussionmentioning

confidence: 99%

Two new genetically modified mouse alleles labeling distinct phases of retinal ganglion cell development by fluorescent proteins

Cheng

et al. 2020

Preprint

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“…Instead of making EB, plating the clumps of human ESCs directly on Matrigel with the same differentiation media led to similar retinal differentiation in complete adherent cell culture conditions ( Lamba et al, 2010 ). This pioneer “Lamba protocol” led to other protocols ( Table 2 ) improving the generation of RPCs and mature RGCs ( Gill et al, 2014 ). Retinal structures containing RGCs identified by Brn3b and Neurofilament-200 have been obtained in similar adherent conditions in absence of Matrigel by addition of Noggin, FGF2, DKK-1, IGF-1, and FGF-9 in specific time windows ( Singh et al, 2015 ).…”

Section: Pluripotent-stem Cells-derived Rgcsmentioning

confidence: 99%

“…One explanation may be the challenging goal of optic nerve regeneration that may look daunting to some. However, important progress has been achieved in order to generate well characterized transplantable cells ( Gill et al, 2014 ; Tanaka et al, 2016 ; Teotia et al, 2016 ; Liu et al, 2017 ; Sluch et al, 2017 ; Langer et al, 2018 ) and to address the question of axonal regeneration ( Park et al, 2008 ; Sun et al, 2011 ; de Lima et al, 2012 ; Benowitz et al, 2017 ; Calkins et al, 2017 ; Laha et al, 2017 ). In this review, we discuss the feasibility of regenerative strategies applied to RGC disorders such as glaucoma and inherited optic neuropathies using PSCs.…”

Section: Introductionmentioning

confidence: 99%

Pluripotent Stem Cell-Based Approaches to Explore and Treat Optic Neuropathies

2018

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Sight is a major sense for human and visual impairment profoundly affects quality of life, especially retinal degenerative diseases which are the leading cause of irreversible blindness worldwide. As for other neurodegenerative disorders, almost all retinal dystrophies are characterized by the specific loss of one or two cell types, such as retinal ganglion cells, photoreceptor cells, or retinal pigmented epithelial cells. This feature is a critical point when dealing with cell replacement strategies considering that the preservation of other cell types and retinal circuitry is a prerequisite. Retinal ganglion cells are particularly vulnerable to degenerative process and glaucoma, the most common optic neuropathy, is a frequent retinal dystrophy. Cell replacement has been proposed as a potential approach to take on the challenge of visual restoration, but its application to optic neuropathies is particularly challenging. Many obstacles need to be overcome before any clinical application. Beyond their survival and differentiation, engrafted cells have to reconnect with both upstream synaptic retinal cell partners and specific targets in the brain. To date, reconnection of retinal ganglion cells with distal central targets appears unrealistic since central nervous system is refractory to regenerative processes. Significant progress on the understanding of molecular mechanisms that prevent central nervous system regeneration offer hope to overcome this obstacle in the future. At the same time, emergence of reprogramming of human somatic cells into pluripotent stem cells has facilitated both the generation of new source of cells with therapeutic potential and the development of innovative methods for the generation of transplantable cells. In this review, we discuss the feasibility of stem cell-based strategies applied to retinal ganglion cells and optic nerve impairment. We present the different strategies for the generation, characterization and the delivery of transplantable retinal ganglion cells derived from pluripotent stem cells. The relevance of pluripotent stem cell-derived retinal organoid and retinal ganglion cells for disease modeling or drug screening will be also introduced in the context of optic neuropathies.

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“…PSCs have the ability to self-renew and differentiate into all cell types of the body, thereby providing great potential for regenerative medicine and cell replacement therapies. Further, PSC-derived progeny allow investigating disease-affected cell types that are not readily accessible due to their anatomical location, such as retinal cells [7][8][9] . Utilising such disease-affected cells will also significantly improve the drug development pipeline through efficacy profiling and side effect or toxicity assessment 10 .…”

Section: Background and Summarymentioning

confidence: 99%

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

Daniszewski

Senabouth

Nguyen

et al. 2017

Preprint

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We used single cell sequencing technology to characterize the transcriptomes of 1,174 human embryonic stem cell-derived retinal ganglion cells (RGCs) 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 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 showed increased expression of genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature RGC phenotype.

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Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells

Cited by 51 publications

References 88 publications

Two new genetically modified mouse alleles labeling distinct phases of retinal ganglion cell development by fluorescent proteins

Two new genetically modified mouse alleles labeling distinct phases of retinal ganglion cell development by fluorescent proteins

Pluripotent Stem Cell-Based Approaches to Explore and Treat Optic Neuropathies

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

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