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Kevin Huan Nguyen, Xiangmei Zhang, Xian-Jie Yang; Analyses of Early Stage Neurogenesis in Human ES Cell-Derived 3D Retinal Organoids by Single Cell RNA Sequencing. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3332.
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© ARVO (1962-2015); The Authors (2016-present)
Human pluripotent stem cell-derived 3D retinal organoids resemble the embryonic human retina, thus can serve as a model system to study retinogenesis. To promote early retinal neuron production, especially human retinal ganglion cell (RGC) genesis, we have elevated the expression of bHLH transcription factors in 3D retinal organoid cultures. Our previous immunocytochemistry and quantitative FACS analyses have detected differential neuronal marker expression driven by the neurogenic factor ATOH7 and Neurog2/Ngn2. In this study, we analyze single cell transcriptomes to further evaluate effects of these neurogenic transcription factors on retinal differentiation.
Human ES cells were used to generate 3D retinal organoids. Recombinant lentiviruses were produced that encode a Tet-inducible promoter (TetO) upstream of human ATOH7 cDNA or mouse Neurog2/Ngn2 as well as EGFP. Viral transduced retinal organoid cultures were treated with doxycycline to induce the expression of bHLH factors. Following EGFP-based cell sorting, single cell transcriptomes were captured via 10X Genomics Chromium system and underwent Illumina sequencing. Between 2000-3000 single cells were analyzed per sample at different developing stages to reach 30,000-50,000 reads per cell. Single cell RNA sequencing data were further analyzed using available software and compared.
Analyses of single cell RNA seq data, especially multi-gene co-expression profiling, enable us to identify progenitor cells and postmitotic neurons. As expected, cells from organoids infected by a control virus that expresses only EGFP contain both progenitors and postmitotic neurons. In contrast, elevation of bHLH factors promotes neurogenesis within 48 hours. However, depending on thresholding during cell sorting, retinal progenitors expressing EGFP can still be detected. The transcript of bHLH gene ASCL1 is mostly distributed among progenitors, whereas mRNAs of ATOH7 and Neurog2/Ngn2 are detected among a subset of progenitors as well as postmitotic neurons.
Current single cell RNA sequencing data reveal distinctive expression patterns among retinal progenitors and postmitotic neurons, thus providing insights on the dynamic process of human retinal progenitor divergence and subsequent differentiation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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