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Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal.

ABSTRACT: 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.

SUBMITTER: Kaya KD

PROVIDER: S-EPMC6857775 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

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

Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

Project description:Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

| PRJNA530144 | ENA

COCO enhances the efficiency of photoreceptor precursor differentiation in early human embryonic stem cell-derived retinal organoids.

Project description:BACKGROUND:Significant progress has been made in cell replacement therapy for neural retinal diseases using retinal cells differentiated from human pluripotent stem cells. Low tumorigenicity and the ability to mature to form synaptic junctions make precursor cells a promising donor source. Here, we attempted to improve the yield of photoreceptor precursor cells in three-dimensional retinal organoids from human embryonic stem cells (hESCs). METHODS:A CRX-tdTomato-tagged hESC line was generated to track retinal precursors in 3D retinal organoids. COCO, a multifunctional antagonist of the Wnt, TGF-?, and BMP pathways, was employed to 3D organoid differentiation schemes for enhanced photoreceptor precursor cells. Organoid fluorescence intensity measurement was used to monitor retinalization tendency with the number of precursors further checked by flow cytometry. Signature gene expression during organoid differentiation were assessed by qPCR and immunocytochemistry after COCO supplementation. RESULTS:CRX-positive cells can be spatiotemporally tracked by tdTomato without affecting retinalization during retinal organoid differentiation. Fluorescence intensity of organoids, which turned out highly consistent with flow cytometry measurement, allowed us to determine the differentiation efficiency of precursors during organoid culturing directly. Using COCO as an auxiliary supplement, rather than alone, can yield an increased number of photoreceptor precursors in the early stage of organoid differentiation. Over a longer time-frame, photoreceptor precursors enhanced their fate of cones and decreased fate of rods after treatment with COCO. CONCLUSIONS:Tracing with the CRX-reporter system showed that in retinal organoids derived from human pluripotent stem cells, COCO increased the differentiation efficiency of photoreceptor precursors and cones.

| S-EPMC7444242 | biostudies-literature

Investigating cone photoreceptor development using patient-derived NRL null retinal organoids.

Project description:Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa. While murine Nrl loss has been characterized, studies of human NRL can identify important insights for human retinal development and disease. We utilized iPSC organoid models of retinal development to molecularly define developmental alterations in a human model of NRL loss. Consistent with the function of NRL in rod fate specification, human retinal organoids lacking NRL develop S-opsin dominant photoreceptor populations. We report generation of two distinct S-opsin expressing populations in NRL null retinal organoids and identify MEF2C as a candidate regulator of cone development.

| S-EPMC7035245 | biostudies-literature

CRX haploinsufficiency compromises photoreceptor precursor translocation and differentiation in human retinal organoids.

Project description:BackgroundThe CRX-associated autosomal dominant retinopathies suggest a possible pathogenic mechanism of gene haploinsufficiency. However, based on reported human patient cases and studies with mouse models, it is hard to confirm the specific weight of haploinsufficiency in pathogenesis due to the interspecies gaps between gene expression and function.MethodsWe created monoallelic CRX by replacing one allele with tdTomato in human embryonic stem cells (hESCs) and subsequently dissect pathogenesis in hESCs-derived retinal organoids. We used transcriptome and immunofluorescence analyses to dissect phenotypic differences between CRX-monoallelic knockout and control wildtype organoids. For location analysis of CRX+ cells, a CRX-expression-tracing system was constructed in control hESCs. We implemented long-term live-cell imaging to describe the translocation of CRX+ cells between two groups in early organoid differentiation. The expression pattern of these dynamic differences was validated using RNA-seq and immunofluorescence assays.ResultsWe identified delayed differentiation of outer nuclear layer (ONL) stratification along with thinner ONL, serious loss of photoreceptor outer segments, as well as downregulated expression of gene for phototransduction and inner/outer segment formation. By live-cell imaging and immunostaining, we observed the overtension of actomyosin network and the arrested translocation of monoallelic CRX+ cells in the early stage of retinal differentiation.ConclusionsWe confirmed that gene haploinsufficiency is the mechanism for the dominant pathogenicity of CRX and discovered that CRX regulated postmitotic photoreceptor precursor translocation in addition to its specification of photoreceptor cell fates during human retinal development. These findings revealed a new underlying mechanism of CRX dominant pathogenesis and provided a new clue for the treatment of CRX-associated human retinopathies.

| S-EPMC10696917 | biostudies-literature

Accelerated and Improved Differentiation of Retinal Organoids from Pluripotent Stem Cells in Rotating-Wall Vessel Bioreactors.

Project description:Pluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV) bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25) reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.

| S-EPMC5768666 | biostudies-literature

Investigating cone photoreceptor development using patient-derived NRLnull retinal organoids

Project description:Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from an improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa. While murine Nrl loss has been characterized, studies of human NRL can identify important insights for human retinal disease. Here we utilized human organoid models of retinal development to molecularly define developmental alterations in a human model of NRL loss. Consistent with the function of NRL in rod fate specification, human retinal organoids lacking NRL develop S-opsin dominant photoreceptor populations. We report generation of two distinct S-opsin expressing populations in NRL null retinal organoids and identify MEF2C as a candidate regulator of cone development.

2020-01-15 | GSE143669 | GEO

Use of bioreactors for culturing human retinal organoids improves photoreceptor yields.

Project description:BACKGROUND:The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. METHODS:We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. RESULTS:Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. CONCLUSIONS:Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies.

| S-EPMC5998504 | biostudies-literature

Antioxidant and lipid supplementation improve the development of photoreceptor outer segments in pluripotent stem cell-derived retinal organoids.

Project description:The generation of retinal organoids from human pluripotent stem cells (hPSC) is now a well-established process that in part recapitulates retinal development. However, hPSC-derived photoreceptors that exhibit well-organized outer segment structures have yet to be observed. To facilitate improved inherited retinal disease modeling, we determined conditions that would support outer segment development in maturing hPSC-derived photoreceptors. We established that the use of antioxidants and BSA-bound fatty acids promotes the formation of membranous outer segment-like structures. Using new protocols for hPSC-derived retinal organoid culture, we demonstrated improved outer segment formation for both rod and cone photoreceptors, including organized stacked discs. Using these enhanced conditions to generate iPSC-derived retinal organoids from patients with X-linked retinitis pigmentosa, we established robust cellular phenotypes that could be ameliorated following adeno-associated viral vector-mediated gene augmentation. These findings should aid both disease modeling and the development of therapeutic approaches for the treatment of photoreceptor disorders.

| S-EPMC9023802 | biostudies-literature

Convolutional Neural Networks Can Predict Retinal Differentiation in Retinal Organoids.

Project description:We have developed a deep learning-based computer algorithm to recognize and predict retinal differentiation in stem cell-derived organoids based on bright-field imaging. The three-dimensional "organoid" approach for the differentiation of pluripotent stem cells (PSC) into retinal and other neural tissues has become a major in vitro strategy to recapitulate development. We decided to develop a universal, robust, and non-invasive method to assess retinal differentiation that would not require chemical probes or reporter gene expression. We hypothesized that basic-contrast bright-field (BF) images contain sufficient information on tissue specification, and it is possible to extract this data using convolutional neural networks (CNNs). Retina-specific Rx-green fluorescent protein mouse embryonic reporter stem cells have been used for all of the differentiation experiments in this work. The BF images of organoids have been taken on day 5 and fluorescent on day 9. To train the CNN, we utilized a transfer learning approach: ImageNet pre-trained ResNet50v2, VGG19, Xception, and DenseNet121 CNNs had been trained on labeled BF images of the organoids, divided into two categories (retina and non-retina), based on the fluorescent reporter gene expression. The best-performing classifier with ResNet50v2 architecture showed a receiver operating characteristic-area under the curve score of 0.91 on a test dataset. A comparison of the best-performing CNN with the human-based classifier showed that the CNN algorithm performs better than the expert in predicting organoid fate (84% vs. 67 ± 6% of correct predictions, respectively), confirming our original hypothesis. Overall, we have demonstrated that the computer algorithm can successfully recognize and predict retinal differentiation in organoids before the onset of reporter gene expression. This is the first demonstration of CNN's ability to classify stem cell-derived tissue in vitro.

| S-EPMC7350982 | biostudies-literature

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