TY - JOUR
T1 - Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids
AU - Kim, Sangbae
AU - Lowe, Albert
AU - Dharmat, Rachayata
AU - Lee, Seunghoon
AU - Owen, Leah A.
AU - Wang, Jun
AU - Shakoor, Akbar
AU - Li, Yumei
AU - Morgan, Denise J.
AU - Hejazi, Andre A.
AU - Cvekl, Ales
AU - DeAngelis, Margaret M.
AU - Jimmy Zhou, Z.
AU - Chen, Rui
AU - Liu, Wei
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank Drs. R. Chuck for support, Peng Wu for expert advice in multiphoton microscopy, and J. Nathans for antibodies; M.R.G. Stem Cell Institute (supported by New York State Stem Cell Science Grant C029154); and the Analytical Imaging Facility at Albert Einstein College of Medicine (AECOM) for service (supported by Grant P30CA013330). This work was supported by BrightFocus Grant M2012044 (to W.L.); Retina Research Foundation (R.C.); National Eye Institue Grants R01EY022645 (to W.L.), R01EY012200, and R01EY014237 (to A.C.), EY014800 (to M.M.D.), R01EY018571 and R01EY022356 (to R.C.), and R01EY026065 and R01EY17353 (to Z.J.Z.); unrestricted grants from Research to Prevent Blindness to the Department of Ophthalmology and Visual Sciences at AECOM and the Department of Ophthalmology & Visual Sciences at The University of Utah; the Carl Marshal & Mildred Almen Reeves Foundation (M.M.D.); Macular Degeneration Foundation (M.M.D.); National Institute of General Medical Sciences Grant T32GM007491 (to A.L.); Marvin L. Sears Professorship (Z.J.Z.); and Yale Vision Science Core (supported by Grant P30EY026878). Bulk and single-cell RNA-Seq were performed at the Single Cell Genomics Core at Baylor College of Medicine partially supported by NIH shared instrument Grants S10OD018033 and S10OD023469 and P30EY002520 (to R.C.). Support for The Einstein Training Program in Stem Cell Research of Albert Einstein College of Medicine, Inc. is acknowledged from the Empire State Stem Cell Fund through New York State Department of Health Contract C30292GG.
Funding Information:
We thank Drs. R. Chuck for support, Peng Wu for expert advice in multiphoton microscopy, and J. Nathans for antibodies; M.R.G. Stem Cell Institute (supported by New York State Stem Cell Science Grant C029154); and the Analytical Imaging Facility at Albert Einstein College of Medicine (AECOM) for service (supported by Grant P30CA013330). This work was supported by BrightFocus Grant M2012044 (to W.L.); Retina Research Foundation (R.C.); National Eye Institue Grants R01EY022645 (to W.L.), R01EY012200, and R01EY014237 (to A.C.), EY014800 (to M.M.D.), R01EY018571 and R01EY022356 (to R.C.), and R01EY026065 and R01EY17353 (to Z.J.Z.); unrestricted grants from Research to Prevent Blindness to the Department of Ophthalmology and Visual Sciences at AECOM and the Department of Ophthalmology & Visual Sciences at The University of Utah; the Carl Marshal & Mildred Almen Reeves Foundation (M.M.D.); Macular Degeneration Foundation (M.M.D.); National Institute of General Medical Sciences Grant T32GM007491 (to A.L.); Marvin L. Sears Professorship (Z.J.Z.); and Yale Vision Science Core (supported by Grant P30EY026878). Bulk and single-cell RNA-Seq were performed at the Single Cell Genomics Core at Baylor College of Medicine partially supported by NIH shared instrument Grants S10OD018033 and S10OD023469 and P30EY002520 (to R.C.). Support for The Einstein Training Program in Stem Cell Research of Albert Einstein College of Medicine, Inc. is acknowledged from the Empire State Stem Cell Fund through New York State Department of Health Contract C30292GG.
Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.
PY - 2019/5/28
Y1 - 2019/5/28
N2 - Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium—retinal organoid—that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.
AB - Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium—retinal organoid—that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.
KW - Cone and rod photoreceptor
KW - RNA-seq
KW - Retinal differentiation
KW - Retinal organoid
KW - Single cell
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U2 - 10.1073/pnas.1901572116
DO - 10.1073/pnas.1901572116
M3 - Article
C2 - 31072937
AN - SCOPUS:85066287373
VL - 166
SP - 10824
EP - 10833
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 22
ER -
