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Automating iPSC generation to enable autologous photoreceptor cell replacement therapy

ABSTRACT: scRNAseq profiling of retinal organoids derived from iPSCs generated on a robot platform.

ORGANISM(S): Homo sapiens

PROVIDER: GSE220154 | GEO | 2023/02/27

REPOSITORIES: GEO

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Automating iPSC generation to enable autologous photoreceptor cell replacement therapy.

Project description:BackgroundInherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation.MethodsIn this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed.ResultsPatient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory.ConclusionWe have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

| S-EPMC9976478 | biostudies-literature

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy

Project description:Automating iPSC generation to enable autologous photoreceptor cell replacement therapy

| PRJNA908874 | ENA

Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids

Project description:Purpose: Autologous cell replacement shows great promise for the treatment of inherited retinal degeneration. While mature differentiation protocols exist to produce retinal organoids from patient-derived stem cells, not all cell types present in these organoids are desirable for transplant. To increase the potency of future cell therapies, methods for isolating photoreceptors from dissociated retinal organoids are needed. In this work, we show how partial dissociation can be used to exploit the spatial organization of retinal organoids to produce highly pure photoreceptor populations without the use of specialized sorting devices or reagents such as xenobiotic antibodies. Methods: Retinal organoids were generated as we have described previously. For flow cytometry experiments, organoids were dissociated using papain for 30 minutes or for 90 minutes followed by trituration. For scRNAseq experiments, liberated cells were collected from papain-dissociating organoids at 20-minute intervals. At the conclusion of the experiment, remaining tissue was fully dissociated. A control 60-minute full dissociation was performed in parallel. Photoreceptor purity was assessed via flow cytometry and scRNAseq. Results: Photoreceptors desired for transplant are typically found in the outer layers of retinal organoids, suggesting that partial dissociation could selectively release these cells. Flow cytometry results indicating an increase in purity of CD133+ cells from 21.1% to 91.7% when cells were partially dissociated as compared to full dissociation. A time dependent release of photoreceptors, with a maximum purity of 96% CRX+ cells at 40 minutes as compared to 66% pure for traditional dissociation. Conclusions: By timing the dissociation of retinal organoids, highly pure photoreceptor cell populations suitable for cell replacement therapies can be obtained under cGMP without the use of sorting reagents or equipment.

2024-09-02 | GSE266526 | GEO

scRNAseq comparison of iPSC-derived retinal organoids from Control and LCA7 patients

Project description:To comprehensively capture changes in retinal transcriptome for the LCA7 organoids compared to control, we performed single cell RNA-sequencing (scRNAseq) using the 10X Genomics platform. Retinal organoids at D150 of differentiation were dissociated for scRNAseq analysis. scRNAseq data revealed significant dysregulation of specific photoreceptor genes between control and LCA7 organoids, as well as mutation-specific differences in various genes, including CRX, RCVRN, ARR3, and AIPL1.

2021-09-17 | GSE184080 | GEO

Characterization of transcriptomes of human iPSC-derived retinal lineages

Project description:Retinal ganglion cells (RGCs) and retinal pigment epithelium (RPE) cells are two retinal cell types that are affected by the most prevalent retinal diseases leading to irreversible blindness, such as glaucoma affecting the former and age-related macular degeneration affecting the latter. One of the most promising approaches for the therapy of these diseases is via the autologous transplantation of RGC or RPE cells derived from the induced pluripotent stem cells (iPSCs). This emphasizes the importance of detailed characterization and understanding of the mechanisms of differentiation of iPSCs into retinal lineages on the genome-wide scale. Such information can be used to identify novel crucial regulators of differentiation, optimisation of differentiation protocols to make them more efficient and safe, identification of novel specific biomarker signatures of differentiated cells. In this study, we performed the genome-wide transcriptome analysis of terminally differentiated RGC and RPE lineages, as well as intermediate retinal progenitor cells (RPCs) of optic vesicles (OVs) derived from the human induced pluripotent stem cells (iPSCs). In our analysis we specifically focused on the classes of transcripts that encode regulators of gene expression, such as transcription factors, epigenetic factors, and components of signaling pathways.

2017-03-22 | GSE96853 | GEO

RNA-seq of retinal organoids derived from RPGR patient-, control- and mutation-corrected-iPSC at different developmental stages

Project description:Retinitis pigmentosa (RP) is an irreversible and inherited retinopathy. RPGR mutations are the most common causes of this disease. It remains challenging to decipher the mechanism of RPGR mutation because of the lack of appropriate study models. The substitution of patient-specific diseased retina without ethical restrictions is desired and iPSC-derived 3D retina is the best choice. In our experiment, we generated iPSCs from one RP patient with 2-bp frameshift mutation in the exon14 of RPGR gene, which were differentiated into retinal organoids. Also we generated iPSCs from a normal control and differentiated those control-iPSCs into healthy retinal organoids. Samples of patient- and control-retinal organoids at W0, W7, W13 (two replicates), W18 (two replicates) and W22 (two replicates for patient) were collected for RNA-seq. Corrected-iPSC were derived from CRISPR/Cas9-mediated gene correction. Then we collected the corrected-iPSC derived retinal organoids at W0, W7, W13 (two replicates), W18 (two replicates) and W22 (two replicates) for RNA-seq. Through the RNA-seq data, we demonstrate that patient-specific iPSC-dervied 3D retinae can recapitulate disease progress of Retinitis Pigmentosa through presenting defects in photoreceptors' gene profile. CRISPR/Cas9-mediated gene correction can rescue photoreceptor gene profile. Those transcriptome are consistent with the phenotype and function.

2020-02-01 | E-MTAB-6490 | biostudies-arrayexpress

Transplantation of human iPSC-derived cone photoreceptor sheets in the sub-retinal space of a minipig model of severe photoreceptor degeneration

Project description:We used single cell RNA sequencing to investigate the cell diversity in our in vitro differentiation from iPSC to Retinal sheets culture and the development of our culture.

2024-06-30 | GSE228436 | GEO

Optic nerve hypoplasia iPSC derived retinal ganglion cells exhibit differential gene expression with potential relevance to disease etiology

Project description:To investigate gentic contributions to optic nerve hypoplasia, induced pluripotent stem cells were produced from 3 patients and 3 controls. The iPSCs were differentiated to retinal organoids from which differential gene expression was performed on FACS isolated retinal ganglion cells.

2023-05-01 | GSE227975 | GEO

Pigment Epithelium Derived Factor (PEDF) secreted from iPSC derived-RPE facilitates apoptotic causes cell death, not the differentiation, of iPSCs

Project description:We show that Retinal pigment epithelium (RPE) secreted-factor, pigment epithelium derived factor (PEDF) secreted/derived from primary or iPSC-derived retinal pigment epithelium (RPE)RPE, dramatically inhibitsed the cell growth of iPSCs. PEDF was detected abundantly in culture supernatant media of primary and iPSC-derived RPE. We examined the gene expression in primary RPE and iPS-derived RPE. Two samples: RPE derived from 253G1 iPSC, Primary RPE.

2013-01-03 | E-GEOD-43257 | biostudies-arrayexpress

Deregulation of neuro-developmental genes and primary cilium cytoskeleton anomalies in iPSC retinal sheets from human syndromic ciliopathies

Project description:We generated iPSCs from control and ciliopathy cases and differentiated them into polarized 3D-adherent retinal sheets that could recapitulate normal photoreceptor development and disease state, respectively.

2020-03-01 | GSE133247 | GEO

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