Abstract
Purpose :
Three-dimensional optic vesicle-like structures are capable of being derived from human pluripotent stem cells (hPSCs). Highly enriched for retinal progenitor cells, these populations express a number of transcription factors associated with retinal development. Among these is the homeobox transcription factor SIX6, which is highly expressed in retinal progenitor cells yet its precise role in early retinogenesis remains largely unclear. In the current study, efforts were undertaken to elucidate the role(s) of SIX6 in the formation of hPSC-derived three-dimensional optic vesicle-like structures.
Methods :
hPSCs were differentiated to yield three-dimensional optic vesicle-like structures following existing protocols. Following differentiation, optic vesicle-like structures were readily identified and isolated apart from non-retinal forebrain populations of cells. The retinal progenitor nature of these cells was confirmed by both immunocytochemistry and qRT-PCR. To identify a role for SIX6, the gene was either overexpressed or knocked down in hPSCs using a lentiviral vector and its effects on proliferation and differentiation were characterized.
Results :
Within the first thirty days of differentiation, hPSC-derived optic vesicle-like structures were isolated and the retinal progenitor nature of these cells was confirmed by their expression of developmental transcription factors including CHX10, RAX, and PAX6. Analysis of the expression of SIX6 within these cells demonstrated a specific localization within hPSC-derived optic vesicle-like structures, in which nearly all cells were SIX6-positive. Further analysis of the role of SIX6 by overexpression and knockdown demonstrated important roles in the proliferation and differentiation of hPSC-derived optic vesicle-like structures.
Conclusions :
Overall, the results of this study build a foundation for understanding the role of SIX6 in retinal development. SIX6 was highly expressed specifically within optic vesicle-like structures, indicating an important role in the establishment of a retinal fate, as well as the differentiation of retinal cell types. Such results help to establish hPSCs as a valuable in vitro model system for studies of human retinogenesis, with important implications for the differentiation of specific cell types for translational applications.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.