Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids

Sangbae Kim, Albert Lowe, Rachayata Dharmat, Seunghoon Lee, Leah A. Owen, Jun Wang, Akbar Shakoor, Yumei Li, Denise J. Morgan, Andre A. Hejazi, Ales Cvekl, Margaret M. DeAngelis, Z. Jimmy Zhou, Rui Chen, Wei Liu

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)10824-10833
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Volume166
Issue number22
DOIs
StatePublished - May 28 2019

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Organoids
Retinal Cone Photoreceptor Cells
Gene Expression Profiling
RNA Sequence Analysis
Vertebrate Photoreceptor Cells
Transcriptome
Cysts
Stem Cells
Retinal Rod Photoreceptor Cells
Retinal Diseases
Photoreceptor Cells
Differentiation Antigens
Macular Degeneration
Alternative Splicing
Visual Acuity
Extracellular Matrix
Retina
Cell Differentiation
Microscopy
Electron Microscopy

Keywords

  • Cone and rod photoreceptor
  • Retinal differentiation
  • Retinal organoid
  • RNA-seq
  • Single cell

ASJC Scopus subject areas

  • General

Cite this

Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids. / Kim, Sangbae; Lowe, Albert; Dharmat, Rachayata; Lee, Seunghoon; Owen, Leah A.; Wang, Jun; Shakoor, Akbar; Li, Yumei; Morgan, Denise J.; Hejazi, Andre A.; Cvekl, Ales; DeAngelis, Margaret M.; Jimmy Zhou, Z.; Chen, Rui; Liu, Wei.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 166, No. 22, 28.05.2019, p. 10824-10833.

Research output: Contribution to journalArticle

Kim, S, Lowe, A, Dharmat, R, Lee, S, Owen, LA, Wang, J, Shakoor, A, Li, Y, Morgan, DJ, Hejazi, AA, Cvekl, A, DeAngelis, MM, Jimmy Zhou, Z, Chen, R & Liu, W 2019, 'Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids', Proceedings of the National Academy of Sciences of the United States of America, vol. 166, no. 22, pp. 10824-10833. https://doi.org/10.1073/pnas.1901572116
Kim, Sangbae ; Lowe, Albert ; Dharmat, Rachayata ; Lee, Seunghoon ; Owen, Leah A. ; Wang, Jun ; Shakoor, Akbar ; Li, Yumei ; Morgan, Denise J. ; Hejazi, Andre A. ; Cvekl, Ales ; DeAngelis, Margaret M. ; Jimmy Zhou, Z. ; Chen, Rui ; Liu, Wei. / Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids. In: Proceedings of the National Academy of Sciences of the United States of America. 2019 ; Vol. 166, No. 22. pp. 10824-10833.
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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.

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