title: Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.
abstract: Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine Baltimore, MD 21287.
authors.forename: Valentin M
authors.initials: VM
authors.lastname: Sluch
authors.affiliation: Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, MD, 21205.
authors.forename: Chung-ha O
authors.initials: CH
authors.lastname: Davis
authors.affiliation: Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
authors.forename: Vinod
authors.initials: V
authors.lastname: Ranganathan
authors.affiliation: Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Kerr
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205.|Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
authors.forename: Kellin
authors.initials: K
authors.lastname: Krick
authors.affiliation: Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21287.|Department of Materials Science and Engineering, Whiting School of Engineering, and Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218.
authors.forename: Russ
authors.initials: R
authors.lastname: Martin
authors.affiliation: Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Berlinicke
authors.affiliation: Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, MD, 21205.|The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Marsh-Armstrong
authors.affiliation: Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Diamond
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205.|Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21287.|Department of Materials Science and Engineering, Whiting School of Engineering, and Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218.
authors.forename: Hai-Quan
authors.initials: HQ
authors.lastname: Mao
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine Baltimore, MD 21287.|Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287.|The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205.|Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018800:Thy-1 Antigens / Q000378:metabolism; D013997:Time Factors; D050820:Transcription Factor Brn-3B / Q000235:genetics / Q000378:metabolism
chemical_list: D018800:Thy-1 Antigens; D050820:Transcription Factor Brn-3B