TY - JOUR
T1 - Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells
AU - Sluch, Valentin M.
AU - Chamling, Xitiz
AU - Liu, Melissa M.
AU - Berlinicke, Cynthia A.
AU - Cheng, Jie
AU - Mitchell, Katherine L.
AU - Welsbie, Derek S.
AU - Zack, Donald J.
N1 - Funding Information:
We thank Hans Bjornsson for allowing us to use his MiSeq instrument, Li Zhang for providing training and help with the RNA-sequencing experiment, and Gabrielle Cannon and Loyal Goff for assistance with the RNA-sequencing run. This research was supported by grants from the Maryland Stem Cell Research Fund (2014-MSCRFI-0774), NIH (T32–90040730, 1R01EY02268001, 5T32EY007143, 5P30EY001765, and R01EY023754), BrightFocus Foundation, and unrestricted funds from Research to Prevent Blindness, Inc., and generous gifts from the Guerrieri Family Foundation and from Mr. and Mrs. Robert and Clarice Smith.
Publisher Copyright:
© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press
PY - 2017/11
Y1 - 2017/11
N2 - Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972–1986.
AB - Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972–1986.
KW - Biotechnology
KW - Cell differentiation
KW - Clustered regularly interspaced short palindromic repeats
KW - Retinal ganglion cells
KW - Stem cells
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U2 - 10.1002/sctm.17-0059
DO - 10.1002/sctm.17-0059
M3 - Article
C2 - 29024560
AN - SCOPUS:85032487583
SN - 2157-6564
VL - 6
SP - 1972
EP - 1986
JO - Stem cells translational medicine
JF - Stem cells translational medicine
IS - 11
ER -