Transcriptomics,Genomics

Dataset Information

162

Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation


ABSTRACT: Generation of three-dimensional (3D) organoids with optic cup like structures from pluripotent stem cells has created opportunities for investigating mammalian retinal development in vitro. However, retinal organoids in culture do not completely reflect the developmental state and in vivo architecture of the rod-dominant mouse retina. To assess rod photoreceptor differentiation in retinal organoids, we took advantage of Nrl-GFP mice that show rod-specific expression of GFP directed by the promoter of leucine zipper transcription factor NRL. Using embryonic and induced pluripotent stem cells (ESCs and iPSCs, respectively) derived from the Nrl-GFP mouse, we were successful in establishing long-term retinal organoid cultures (up to day 35) using modified culture conditions (called High Efficiency Hypoxia Induced Generation of Photoreceptors in Retinal Organoids, or HIPRO). We demonstrate efficient differentiation of pluripotent stem cells to retinal structures, with over 70% of embryoid bodies forming optic vesicles at Day (D)7, >50% producing optic cups by D10, and a majority of these surviving until at least D35. The HIPRO organoids include distinct inner retina neurons in somewhat stratified architecture and mature Müller glia spanning the entire retina. Almost 70% of the cells in retinal organoids are rod photoreceptors that exhibit elongated cilia. Transcriptome profiles of GFP+ rod photoreceptors, purified from organoids at Day 25-35, demonstrate a high correlation to gene profiles of purified rods from mouse retina at postnatal day (P) 2 to 6, indicating their early state of differentiation. Our 3D retinal organoids thus closely mimic in vivo retinogenesis and provide an efficient in vitro model to investigate photoreceptor development and modeling disease pathology. Overall design: ESC clones were isolated from the inner cell mass (ICM) of 3.5-day blastocysts of Nrlp-GFP transgenic mice on feeders of mouse embryonic fibroblast (MEF). ESCs and iPSCs (passage 15-25) were differentiated using a modified SFEBq protocol. Flow sorted cells of GFP+ photoreceptors from 18, 21, 25, 28, and 35 days post differentiation initiation were collected for expression profiling. RNA-seq profiles of high quality total RNA (RNA Integrity number ≥7.5) from were obtained using Illumina platform. After trimming Illumina adapters, defined in TrueSeq3-PE-2.fa file, by Trimmomatic (version 0.36) from paired end data, each sample was quantified against Ensemble release 84 cDNA FASTA file by Kallisto (version 0.42.5) via a custom pipeline in Perl. The quantifications were then normalized and summarized in gene level by tximport with the option “countFromAbundance=lengthScaledTPM”. The count values from tximport were converted to count per million (CPM) via edgeR.

INSTRUMENT(S): Illumina HiSeq 2500 (Mus musculus)

SUBMITTER: Matthew J Brooks  

PROVIDER: GSE86790 | GEO | 2016-09-09

SECONDARY ACCESSION(S): PRJNA342505

REPOSITORIES: GEO

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