Defined conditions for differentiation of functional retinal ganglion cells from human pluripotent stem cells

Junwon Lee, Sang Hwi Choi, Young Beom Kim, Ikhyun Jun, Jin Jea Sung, Dongjin R. Lee, Yang In Kim, Myung Soo Cho, Suk Ho Byeon, Dae-Sung Kim, Dong Wook Kim

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

PURPOSE. We aimed to establish an efficient method for retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs) using defined factors. METHODS. To define the contribution of specific signal pathways to RGC development and optimize the differentiation of hPSCs toward RGCs, we examined RGC differentiation in three stages: (1) eye field progenitors expressing the eye field transcription factors (EFTFs), (2) RGC progenitors expressing MATH5, and (3) RGCs expressing BRN3B and ISLET1. By monitoring the condition that elicited the highest yield of cells expressing stage-specific markers, we determined the optimal concentrations and combinations of signaling pathways required for efficient generation of RGCs from hPSCs. RESULTS. Precise modulation of signaling pathways, including Wnt, insulin growth factor-1, and fibroblast growth factor, in combination with mechanical isolation of neural rosette cell clusters significantly enriched RX and PAX6 double-positive eye field progenitors from hPSCs by day 12. Furthermore, Notch signal inhibition facilitated differentiation into MATH5-positive progenitors at 90% efficiency by day 20, and these cells further differentiated to BRN3B and ISLET1 double-positive RGCs at 45% efficiency by day 40. RGCs differentiated via this method were functional as exemplified by their ability to generate action potentials, express microfilament components on neuronal processes, and exhibit axonal transportation of mitochondria. CONCLUSIONS. This protocol offers highly defined culture conditions for RGC differentiation from hPSCs and in vitro disease model and cell source for transplantation for diseases related to RGCs.

Original languageEnglish
Pages (from-to)3531-3542
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume59
Issue number8
DOIs
Publication statusPublished - 2018 Jul 1

Fingerprint

Pluripotent Stem Cells
Retinal Ganglion Cells
Fibroblast Growth Factor 1
Cell Differentiation
Wnt Signaling Pathway
Cell Transplantation
Actin Cytoskeleton
Action Potentials
Signal Transduction
Mitochondria
Transcription Factors
Insulin

Keywords

  • Defined factors
  • Differentiation
  • Human pluripotent stem cells
  • Induced pluripotent stem cells
  • Retinal ganglion cells

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Defined conditions for differentiation of functional retinal ganglion cells from human pluripotent stem cells. / Lee, Junwon; Choi, Sang Hwi; Kim, Young Beom; Jun, Ikhyun; Sung, Jin Jea; Lee, Dongjin R.; Kim, Yang In; Cho, Myung Soo; Byeon, Suk Ho; Kim, Dae-Sung; Kim, Dong Wook.

In: Investigative Ophthalmology and Visual Science, Vol. 59, No. 8, 01.07.2018, p. 3531-3542.

Research output: Contribution to journalArticle

Lee, Junwon ; Choi, Sang Hwi ; Kim, Young Beom ; Jun, Ikhyun ; Sung, Jin Jea ; Lee, Dongjin R. ; Kim, Yang In ; Cho, Myung Soo ; Byeon, Suk Ho ; Kim, Dae-Sung ; Kim, Dong Wook. / Defined conditions for differentiation of functional retinal ganglion cells from human pluripotent stem cells. In: Investigative Ophthalmology and Visual Science. 2018 ; Vol. 59, No. 8. pp. 3531-3542.
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T1 - Defined conditions for differentiation of functional retinal ganglion cells from human pluripotent stem cells

AU - Lee, Junwon

AU - Choi, Sang Hwi

AU - Kim, Young Beom

AU - Jun, Ikhyun

AU - Sung, Jin Jea

AU - Lee, Dongjin R.

AU - Kim, Yang In

AU - Cho, Myung Soo

AU - Byeon, Suk Ho

AU - Kim, Dae-Sung

AU - Kim, Dong Wook

PY - 2018/7/1

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N2 - PURPOSE. We aimed to establish an efficient method for retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs) using defined factors. METHODS. To define the contribution of specific signal pathways to RGC development and optimize the differentiation of hPSCs toward RGCs, we examined RGC differentiation in three stages: (1) eye field progenitors expressing the eye field transcription factors (EFTFs), (2) RGC progenitors expressing MATH5, and (3) RGCs expressing BRN3B and ISLET1. By monitoring the condition that elicited the highest yield of cells expressing stage-specific markers, we determined the optimal concentrations and combinations of signaling pathways required for efficient generation of RGCs from hPSCs. RESULTS. Precise modulation of signaling pathways, including Wnt, insulin growth factor-1, and fibroblast growth factor, in combination with mechanical isolation of neural rosette cell clusters significantly enriched RX and PAX6 double-positive eye field progenitors from hPSCs by day 12. Furthermore, Notch signal inhibition facilitated differentiation into MATH5-positive progenitors at 90% efficiency by day 20, and these cells further differentiated to BRN3B and ISLET1 double-positive RGCs at 45% efficiency by day 40. RGCs differentiated via this method were functional as exemplified by their ability to generate action potentials, express microfilament components on neuronal processes, and exhibit axonal transportation of mitochondria. CONCLUSIONS. This protocol offers highly defined culture conditions for RGC differentiation from hPSCs and in vitro disease model and cell source for transplantation for diseases related to RGCs.

AB - PURPOSE. We aimed to establish an efficient method for retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs) using defined factors. METHODS. To define the contribution of specific signal pathways to RGC development and optimize the differentiation of hPSCs toward RGCs, we examined RGC differentiation in three stages: (1) eye field progenitors expressing the eye field transcription factors (EFTFs), (2) RGC progenitors expressing MATH5, and (3) RGCs expressing BRN3B and ISLET1. By monitoring the condition that elicited the highest yield of cells expressing stage-specific markers, we determined the optimal concentrations and combinations of signaling pathways required for efficient generation of RGCs from hPSCs. RESULTS. Precise modulation of signaling pathways, including Wnt, insulin growth factor-1, and fibroblast growth factor, in combination with mechanical isolation of neural rosette cell clusters significantly enriched RX and PAX6 double-positive eye field progenitors from hPSCs by day 12. Furthermore, Notch signal inhibition facilitated differentiation into MATH5-positive progenitors at 90% efficiency by day 20, and these cells further differentiated to BRN3B and ISLET1 double-positive RGCs at 45% efficiency by day 40. RGCs differentiated via this method were functional as exemplified by their ability to generate action potentials, express microfilament components on neuronal processes, and exhibit axonal transportation of mitochondria. CONCLUSIONS. This protocol offers highly defined culture conditions for RGC differentiation from hPSCs and in vitro disease model and cell source for transplantation for diseases related to RGCs.

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