Human induced pluripotent stem cells generate light responsive retinal organoids with variable and nutrient dependent efficiency

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format which enhanced generation of retinal organoids with retinal pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. © AlphaMed Press 2018

Additional file 2: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

a-d. Gene Ontology analyses showing over and under-represented gene ontologies (biological process and molecular function) in genes differentially expressed between untreated time-points. Analyses were performed using the Panther classification system [73], with a Bonferroni correction for multiple testing. e. Genes differentially expressed between IGF-1 treated and untreated samples. List includes genes identified by comparision at each timepoint, and an all v all comparison. No significant genes were identified in the day 90 analysis. (XLSX 50 kb

Additional file 8: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

Sequence of all primers generated for this study, together with qPCR probes, amplification efficiencies, and primer/probe combinations used. Exon content of amplicons used for Northern analyses is also shown. For additional assays, see [7]. (XLSX 12 kb

Additional file 5: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

Gene Ontology analysis showing over and under-represented gene ontologies (biological process and molecular function) in parent loci of 239 significantly altered circRNAs. Analyses were performed using the Panther classification system [73], with a Bonferroni correction for multiple testing. (XLSX 11 kb

Additional file 6: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

FIRRE exon junctions confirmed by amplicon sequencing. All reads were mapped against hg19 without reference to transcript annotation: Amplicons using convergent primer pairs and divergent primer pairs (to amplify circRNAs only) are shown separately. Canonical junctions were mapped using MapSplice [67], circRNA (back-splice) junctions were mapped using PTESFinder [46]. For details, see methods. Exon number is according to schema in Fig. 6b. Junction position (hg19), amplicons of origin, and junction frequencies, are given for all junctions. Only splices with a frequency of 1% or higher in each amplicon, identified either by MapSplice or PTESfinder, are reported. Canonical junctions present within the current FIRRE annotation are show in blue. All others are not present within current annotation. Off target junctions (presumed to be generated by illegitimate primer binding) are also shown. Data is for confirmation of junction presence within transcripts only: Junction frequency is affected by position relative to primer, size dependent amplification bias during Nextera indexing, and size dependent bias in cluster formation/resolution efficiencies during MiSeq sequencing. (XLSX 71 kb

Additional file 7: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

Genomic co-ordinates of all annotated and novel FIRRE exons. The position of all junctions are shown with respect to hg19, together with their current annotations where appropriate. Splice acceptor and donor junctions identified by annotation-free mapping of day 0 samples against hg19 using MapSplice are shown, together with donor exons and acceptor exons, and junction numbers in brackets. Supporting evidence for each junction is colour coded. Minor / Others – additional junctions not involving annotated exons which are present at low frequency. (XLSX 12 kb

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests

Retinal pigment epithelium extracellular vesicles are potent inducers of age-related macular degeneration disease phenotype in the outer retina

Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells