Abstract
Purpose :
In this study, we aim to determine the neurogenic potentials of the bHLH transcription factors ATOH7 and Neurog2/Ngn2, especially their abilities to influence retinal ganglion cell (RGC) development during early retinogenesis in 3D human retinal organoid cultures.
Methods :
Human pluripotent stem cells were used to generate optic vesicles, which resembled undifferentiated retinal epithelium. A recombinant lentivirus encoding a Tet-inducible promoter (TetO) upstream of human ATOH7 cDNA with a C-terminal epitope tag was constructed. A similar TetO lentiviral vector encoding Neurog2/Ngn2 and the control viral vector expressing EGFP were provided by T. C. Sudhof. Concentrated lentiviral stocks were used to infect 3D optic vesicles-derived from human ES and iPS cells prior to the onset of retinogenesis and induced during the period of RGC genesis. Effects of ATOH7 and Neurog2/Ngn2 expression on RGC production were analyzed at different time intervals after Dox induction using immunocytochemistry and known RGC markers. Flow cytometry analyses were performed to quantify impacts of bHLH factor induction on neuronal differentiation. Whole cell patch clamp recording was performed to assess the function of human RGCs in adhered cultures.
Results :
Lentiviral mediated expression of either bHLH neurogenic factors resulted in increased production of postmitotic neurons in 3D human retinal organoids. Quantitative analyses further revealed that induction of ATOH7 or Neurog2/Ngn2 yielded different neuronal marker profiles, suggesting distinct roles for the two factors during early retinogenesis.
Conclusions :
Our results demonstrate a useful strategy to enhance human RGC production from pluripotent stem cell-derived retinal epithelium, which can serve as a model to study human retinal development and pathology.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.