Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells

Venkata R M Chavali,Jie He,Sergei Nikonov,Jason A Mills,Kamaljot Gill,Devin S Mcdougald,Teja Alapati,Thu T Duong,Vrathasha Vrathasha,Joan O’Brien,Roman Nikonov,Sonika Rathi,Naqi Haider

doi:10.1038/s41598-020-68811-8

Venkata R M Chavali, Jie He + Show 11 more

Open Access

https://doi.org/10.1038/s41598-020-68811-8

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Abstract

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-β (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity.

Highlights

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs)
Though the field was previously reliant on embryonic stem cells (ESCs), advances in stem cell research led to the discovery that overexpressing genes associated with “stemness” in somatic cells can reprogram them into induced pluripotent stem cells[12,13]
We detail a chemically defined in vitro technique for cultivating retinal progenitor cell (RPC) populations from induced pluripotent stem cells (iPSCs) that are directed toward the RGC lineage

Summary

Introduction

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). Many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Primary open-angle glaucoma (POAG), the most common form of glaucoma, is characterized by chronic and progressive optic nerve degeneration and corresponding visual field deficits in the presence of an open and normal iridocorneal chamber a ngle[3] This disease is the leading cause of irreversible blindness worldwide[4], with an estimated 11.1 million expected to become blind from POAG by 20205. Stem-cell based therapy holds promise as a method to restore vision in conditions of retinal cell loss; success of these treatment strategies hinges on de novo synthesis of RGCs with stable phenotypes from h PSCs15,16

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