1. Transcriptome Dynamics of Developing Photoreceptors in Three‐Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks
- Author
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Mahendra Rao, Koray Dogan Kaya, Kohei Homma, Jizhong Zou, Vijender Chaitankar, Matthew Brooks, Rossukon Kaewkhaw, and Anand Swaroop
- Subjects
genetic structures ,Cellular differentiation ,Human Embryonic Stem Cells ,Translational and Clinical Research ,Stem cells ,Biology ,Global gene profiling ,Retina ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Retinal Rod Photoreceptor Cells ,Next generation sequencing ,medicine ,Humans ,Induced pluripotent stem cell ,Eye Proteins ,030304 developmental biology ,Genetics ,Three‐dimensional organoid culture ,0303 health sciences ,Cell Differentiation ,Cell Biology ,Embryonic stem cell ,Antigens, Differentiation ,eye diseases ,Cell biology ,medicine.anatomical_structure ,Human rod and cone photoreceptors ,Rhodopsin ,biology.protein ,Retinal Cone Photoreceptor Cells ,Molecular Medicine ,Homeobox ,sense organs ,Transcriptome ,030217 neurology & neurosurgery ,Developmental Biology ,Visual phototransduction - Abstract
The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile, by RNA-seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures. Stem Cells 2015;33:3504–3518
- Published
- 2015