Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal
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ABSTRACT:
PROVIDER: PRJNA530144 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8822670_1.fastq.gz | Fastqsanger.gz | |||
| SRR8822670_2.fastq.gz | Fastqsanger.gz | |||
| SRR8822680_1.fastq.gz | Fastqsanger.gz | |||
| SRR8822680_2.fastq.gz | Fastqsanger.gz | |||
| SRR8822690_1.fastq.gz | Fastqsanger.gz |
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Project description:Differentiation of neural retina in organoid cultures from human pluripotent stem cells (PSCs) is highly variable and requires an extended period of as many as 200 days. To provide objective assessment of different protocols and culture conditions, we performed transcriptome analysis and molecular staging using RNA-seq data from developing and adult human retina. Using a modified protocol with 9-cis retinal (9CRAL) instead of widely-used all-trans retinoic acid (ATRA) in the culture media, we show comparable differentiation of human embryonic and induced PSCs into a polarized, laminated architecture with all retinal cell types in retinal organoids. Transcriptome comparisons of multiple developing organoids to that of human fetal and adult retina have permitted molecular staging of the differentiation state and uncovered expedited rod photoreceptor maturation by 9CRAL. A direct comparison with the ATRA-organoid cultures revealed higher expression of rhodopsin and other rod genes in 9CRAL-organoids. Our studies thus provide a framework for transcriptome-based staging of retinal organoids to facilitate advances in human disease modeling and evaluation of therapies.
2019-12-17 | GSE129104 | GEO
Project description:PURPOSE:Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS:We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS:We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION:Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.
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Project description:While dysfunction and/or death of light-detecting photoreceptor cells underlies most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids using induced pluripotent stem cells (iPSC) derived from a patient with genetic photoreceptor disease, an isogenic control, and an unrelated control. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation; up to 260 days of in vitro differentiation. Using single-cell transcriptomics data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint in development unique to the disease state that gives rise to a divergent rod photoreceptor cell population. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including expression of the light-sensitive protein rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes at both the transcript and protein level. Using joint multimodal single-cell sequencing on late-stage retinal organoids, we further identify the specific putative regulatory sites where rod-specific factors act to steer rod and cone photoreceptor cell development. Importantly, these findings are strikingly different than that observed in rodent models of disease. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation.
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Project description:Due to the high energy demands and characteristic morphology, retinal photoreceptor cells require the specialized lipid metabolism for survival and functions. In this study, we focus on the roles of saturated fatty acids and their metabolism in these processes. Frame-shift mutation of lysophosphatidylcholine acyltransferase 1 (Lpcat1), which introduces saturated fatty acid into lysophosphatidylcholine to produce disaturated phosphatidylcholine (PC), has been reported as a causative for spontaneous retinal degeneration in mice (rd11 mice). However, the molecular basis of retinal degeneration caused by Lpcat1 mutation remains unclear. Here, we report that Lpcat1 deficiency induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 affects not only disaturated PC production, but also the proper cellular fatty acid flux presumably through altering saturated fatty acyl-CoA availabilities. Furthermore, we demonstrated that Lpcat1 deletion increased mitochondrial reactive oxygen species (ROS) levels in photoreceptor cells, but not in other retinal cells, without affecting the OS structure and trafficking of OS localized proteins. These results suggested that LPCAT1-dependent production of disaturated PC is critical for metabolic adaptation during photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.
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