Automating iPSC generation to enable autologous photoreceptor cell replacement therapy
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ABSTRACT:
PROVIDER: PRJNA908874 | ENA |
REPOSITORIES: ENA
Dataset's files
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| SRR22537593_1.fastq.gz | Fastqsanger.gz | |||
| SRR22537593_2.fastq.gz | Fastqsanger.gz |
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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
Project description:scRNAseq profiling of retinal organoids derived from iPSCs generated on a robot platform.
2023-02-27 | GSE220154 | GEO
Project description:We generated iPSCs from human urine cells (hUCs) with the aid of small molecules and autologous hUC feeders. A compound cocktail including Cyclic Pifithrin-a, a p53 inhibitor and other compounds known for benefiting reprogramming like A-83-01, CHIR99021, Thiazovivin, NaB and PD0325901 was used to aid hUC reprogramming (Plan B). Aided by this cocktail, we achieved significantly improved efficiency (170 folds more) for hUC reprogramming and iPSC generation. In addition, to enable iPSC generation in some cases that massive cell death occurred during delivering reprogramming factors, we replaced Matrigel with autologous hUCs as feeder for reprogramming and iPSC generation (Plan C). Replacing Matrigel with autologous feeder not only enhanced reprograming, but also avoided concern using animal components for human iPSC generation. These were efficient approaches to enable iPSC generation from hUCs that were otherwise difficult for reprogramming, which would be valuable for banking patient’s specific iPSCs.
2017-02-10 | GSE79134 | GEO
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
Project description:Dozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming. Here we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that pretreatment of iPS-fibro with proinflammatory cytokine IFNγ restored the ligand imbalance, thereby reducing the degranulation and cytotoxicity of NK-cells. In summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies.
2023-03-01 | GSE212796 | GEO
Project description:Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids
| PRJNA1107509 | ENA
Project description:Background:The transposed intestinal segments maintain their absorptive and secretive functions which may lead to metabolic disorders after ileal neobladder construction. The aim of this study was to develop a novel technique to reduce the complications of orthotopic urinary diversion in a porcine model. Methods: An ileal neobladder procedure was performed in eight female pigs. Four pigs (experimental group) received mucosal replacement of the neobladder using autologous peritoneum, while the other four pigs (control group) received a normal U-shaped ileal neobladder. After three months, the neobladder was evaluated using IVU, cystograms and cystoscopy. Blood biochemistries, urine samples and venous gas analyses were obtained preoperatively and postoperatively. The neobladder was then harvested to assess pathological findings. Results: The mean (range) operating time was 248 (224-287) min in the control group and 325 (296-360) min in the experimental group. All animals but one from the experimental group survived to the designated endpoints. Postoperative IVU revealed that the urinary tracts were patent and that there was no hydronephrosis or dilated ureter. Cystograms demonstrated a spherical neobladder. Cystoscopy showed a smooth neobladder wall. Histological analysis revealed that the peritoneum-ileal seromuscular flap surface was lined with the urothelial layers. Transcriptome comparison showed the maximum similarity between mucosa of neobladder from experimental group and mucosa of normal bladder. Conclusions: The novel technique is shown to be feasible and reproducible in a porcine model. The imageological, histological, and transcriptometric analyses all confirmed that autologous peritoneum can be used as a natural biological reconstruction material.
2021-04-07 | GSE145751 | GEO
Project description:The possibility to generate induced oligodendrocyte progenitor cells (iOPCs) from fibroblasts may enable in vitro neurological disease modeling and autologous remyelinating therapies.
2017-05-21 | GSE57890 | GEO
Project description:Transplantation of human iPSC-derived cone photoreceptor sheets in the sub-retinal space of a minipig model of severe photoreceptor degeneration
| PRJNA949697 | ENA