Title: Assessing hPSC-OV (Human Pluripotent Stem Cell-Optical Vesicle) Production and PR Maturation with a CRX Reporter Line.
Legend: A) Consecutive live images showing increased tdTomato fluorescence from one CRX+/tdTomato OV from day 28 to day 60 (identical exposure); B) Percent of OVs expressing tdTomato over time (n=48 OVs); C, D) Forebrain neurospheres served as a negative control and did not fluoresce (n=48 for panel D); E, F) Low magnification brightfield (E) and tdTomato fluorescence (F) images of a CRX+/tdTomato OV culture demonstrating fluorescence in all OVs at day 80.
Citation: Phillips MJ et al., A novel approach to single cell RNA-seq analysis facilitates in silico gene reporting of human pluripotent stem cell-derived retinal cell types. Stem Cells. 2017 (Accepted).
Abstract: Cell type-specific investigations commonly employ gene reporters or single-cell (sc) analytical techniques. However, reporter line development is arduous and generally limited to a single gene of interest, while scRNA-seq frequently yields equivocal results that preclude definitive cell identification. To examine gene expression profiles of multiple retinal cell types derived from hPSCs, we performed scRNA-seq on OVs cultured under cGMP-compatible conditions. However, efforts to apply traditional scRNA-seq analytical methods based on unbiased algorithms were unrevealing. Therefore, we developed a simple, versatile, and universally applicable approach that generates gene expression data akin to those obtained from reporter lines. This method ranks single cells by expression level of a bait gene and searches the transcriptome for genes whose cell-to-cell rank order expression most closely matches that of the bait. Moreover, multiple bait genes can be combined to refine datasets. Using this approach, we provide further evidence for the authenticity of hPSC-derived retinal cell types.
About the Lab: David Gamm’s laboratory at the Waisman Center uses stem cell technology to investigate the cellular and molecular events that occur during human retinal differentiation and to generate cells for use in human retinal disease modeling and cell-based rescue or replacement strategies. To meet these goals, Gamm utilizes a variety of human cell types, including ES and iPS cells, which have the capacity to mimic retinal development and disease, as well as to delineate the genetic “checkpoints” necessary to produce particular retinal cell types. By understanding the behavior of these cell types in vitro and in vivo, Gamm hopes to optimize strategies to delay or reverse the effects of blinding disorders such as retinitis pigmentosa and age–related macular degeneration.