Abstract
Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies.
Highlights
Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment
Several research groups have established that human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can be differentiated into multiple retinal cell types, including RGCs, retinal pigment epithelium (RPE) cells, and photoreceptors[20,21]
Multiple signaling pathways are known to regulate retinogenesis, including those modulated by bFGF45–48, Insulin-like Growth Factor (IGF)-149, sonic hedgehog (SHH)[47] activin[50,51], BMP46,48, Wnt[52], Nodal[53] and Notch[54]
Summary
Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. The enriched hESCRGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity This RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. Many current differentiation protocols yield a heterogeneous population of retinal cells, containing only a low proportion of RGCs22 This remains an issue for using hiPSCs for modeling optic neuropathies and development of RGC replacement therapy. This study provides a protocol for generating an enriched population of RGCs using hESCs which can be used for modeling of optic neuropathies and RGC replacement therapy
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