Generation of an RCVRN-eGFP reporter hiPSC line by CRISPR/Cas9 to monitor photoreceptor cell development and facilitate the cell enrichment for transplantation
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ABSTRACT:
PROVIDER: PRJNA804510 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17939975_1.fastq.gz | Fastqsanger.gz | |||
| SRR17939975_2.fastq.gz | Fastqsanger.gz | |||
| SRR17939976_1.fastq.gz | Fastqsanger.gz | |||
| SRR17939976_2.fastq.gz | Fastqsanger.gz | |||
| SRR17939977_1.fastq.gz | Fastqsanger.gz |
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Project description:Stem cell based-cell therapies are considered to be promising treatments for retinal disorders with dysfunction or death of photoreceptors. However, the enrichment of human photoreceptors at specific developmental stage suitable for transplantation is highly challenging so far. This study aimed to generate a specific photoreceptor reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 genome editing, which harbored an enhanced green fluorescent protein (eGFP) sequence at the endogenous locus of the pan photoreceptor marker Recoverin (RCVRN). After confirmation of the successful targeting and gene stability, three-dimensional retinal organoids were induced from this report line. The RCVRN-eGFP reporter faithfully replicated endogenous RCVRN expression and revealed the developmental characteristics of photoreceptors during retinal differentiation. The RCVRN-eGFP specifically and steadily labeled photoreceptor cells from photoreceptor precursors to mature rods and cones. Additionally, abundant eGFP positive photoreceptors could be enriched by fluorescence activated cell sorting and the further RNA sequencing analysis of these purified cells revealed transcriptome signatures and gene networks of photoreceptors. Moreover, potential cluster of differentiation biomarkers were extracted here for enrichment of photoreceptors for clinical applications. Altogether, the RCVRN-eGFP reporter hiPSC line was successfully established and the first global expression database of RCVRN positive photoreceptors was constructed. These achievements will provide a powerful tool for dynamically monitoring photoreceptor cell development and purification of human photoreceptors at specific developmental stage, thus facilitating the photoreceptor cell therapy for the advanced outer retinal disorders.
2022-04-07 | GSE196355 | GEO
Project description:Stem cell-based cell therapies are considered to be promising treatments for retinal disorders with dysfunction or death of photoreceptors. However, the enrichment of human photoreceptors suitable for transplantation has been highly challenging so far. This study aimed to generate a photoreceptor-specific reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 genome editing, which harbored an enhanced green fluorescent protein (eGFP) sequence at the endogenous locus of the pan photoreceptor marker recoverin (RCVRN). After confirmation of successful targeting and gene stability, three-dimensional retinal organoids were induced from this reporter line. The RCVRN-eGFP reporter faithfully replicated endogenous protein expression of recoverin and revealed the developmental characteristics of photoreceptors during retinal differentiation. The RCVRN-eGFP specifically and steadily labeled photoreceptor cells from photoreceptor precursors to mature rods and cones. Additionally, abundant eGFP-positive photoreceptors were enriched by fluorescence-activated cell sorting, and their transcriptome signatures were revealed by RNA sequencing and data analysis. Moreover, potential clusters of differentiation (CD) biomarkers were extracted for the enrichment of photoreceptors for clinical applications, such as CD133 for the positive selection of photoreceptors. Altogether, the RCVRN-eGFP reporter hiPSC line was successfully established and the first global expression database of recoverin-positive photoreceptors was constructed. These achievements will provide a powerful tool for dynamically monitoring photoreceptor cell development and purification of human photoreceptors, thus facilitating photoreceptor cell therapy for advanced retinal disorders.
| S-EPMC9096726 | biostudies-literature
Project description:We generated a transgenic mouse line which express EGFP in the retina driven by the Crx promoter using BAC transgenesis. We sorted EGFP-positive photoreceptor precursors at E17.5 using FACS, and subsequently performed microarray analysis of the FACS-sorted cells. In order to clarify a molecular role of Crx in developing cone photoreceptor precursors, we investigated the expression profile of the BAC-Crx-EGFP-positive cells compared with that of the BAC-Crx-EGFP-negative cells at E17.5.
2010-12-25 | E-GEOD-25607 | biostudies-arrayexpress
Project description:The loss of cone photoreceptor cells, which are critical for optimal daylight vision, have the great impact on vision during retinal degenerations. Retinal differentiation of human induced pluripotent stem cell (hiPSC) sources could provide a renewable source of cone photoreceptors towards developing a cone cell replacement therapy to treat blindness. Demonstration of comparable gene expression profiles between human foetal and stem cell-derived cones at equivalent stages is required to progress the cell transplantation approach into the patient, as it is hypothesised stem cell-derived cones are required to show high levels of developmental recapitulation of the in vivo generated cones. In this study, the AAV2/9.pR2.1.GFP reporter was used to specifically label L/M-opsin cone photoreceptors in human foetal retinal samples, obtained from the MRC-Wellcome Trust Human Developmental Biology Resource, at a range of developmental stages. The L/M-opsin cone population represent the majority cone cell types in the adult human retina and are the only photoreceptors present within the fovea. Using fluorescence activated cell sorting, L/M-opsin GFP+ cones and GFP- retinal populations, alongside total foetal retinal samples containing all retinal cell tytpes, were isolated and processed for bulk RNA sequencing and downstream comparative analysis. Using DESeq2 differential gene expression analyses, statistically significant genes enriched within the GFP+ human foetal LM-opsin cone populations were determined which led to the identification of a cone enriched gene signature of human L/M-opsin cone photoreceptors. The AAV2/9.pR2.1.GFP reporter was applied to hiPSC-derived retinal cultures to isolate and process cone-like cell populations for RNA sequencing using the same strategy developed within the human foetal retina. Applying the cone enriched gene signature to the transcriptome of hiPSC-derived GFP+ samples at equivalent developmental stages revealed some expression similarities in genes found to be enriched within the late foetal L/M-opsin cone photoreceptors. This analysis overall revealed an intermediate stage of cone differentiation was achieved within the hiPSC-derived samples and the comparison to human foetal L/M-opsin gene express profiles suggesting further differentiation of hiPSC-derived sample is required.
2018-04-20 | E-MTAB-6057 | biostudies-arrayexpress
Project description:We generated a transgenic mouse line which express EGFP in the retina driven by the Crx promoter using BAC transgenesis. We sorted EGFP-positive photoreceptor precursors at E17.5 using FACS, and subsequently performed microarray analysis of the FACS-sorted cells.
2010-12-25 | GSE25607 | GEO
Project description:PurposeEarly in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retinal progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist.MethodsCRISPR/Cas9-mediated homology-directed repair (HDR) in hiPSCs facilitated the replacement of the VSX2 (Progenitor), BRN3b (Ganglion), and RCVRN (Photoreceptor) stop codons with sequences encoding a viral P2A peptide fused to Cerulean, green fluorescent protein, and mCherry reporter genes, respectively, to generate a triple transgenic reporter hiPSC line called PGP1. This was accomplished by co-electroporating HDR templates and sgRNA/Cas9 vectors into hiPSCs followed by antibiotic selection. Functional validation of the PGP1 hiPSC line included the ability to generate retinal organoids, with all major retinal cell types, displaying the expression of the three fluorescent reporters consistent with the onset of target gene expression. Disaggregated organoids were also analyzed by fluorescence-activated cell sorting and fluorescent populations were tested for the expression of the targeted gene.ResultsRetinal organoids formed from the PGP1 line expressed appropriate fluorescent proteins consistent with the differentiation of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types.Conclusions and translational relevanceThe PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.
| S-EPMC7352077 | biostudies-literature
Project description:The lack of understanding of the molecular and cellular characteristics of human retinal progenitor cells (RPCs) has hindered their application in cell therapy for retinal degenerative diseases. This study aims to employ retinal organoids (ROs) derived from a VSX2-eGFP fluorescence reporter human induced pluripotent stem cell (hiPSC) line for positive selection of human RPCs, investigate their features, and facilitate their applications. hiPSCs were differentiated into three-dimensional ROs following established protocols. The fidelity of the VSX2-eGFP reporter was confirmed through immunostaining. Fluorescence-activated cell sorting was employed to select VSX2-eGFP-positive (+) cells at distinct developmental stages, followed by bulk RNA sequencing (RNA-seq) analysis to assess their transcriptome profile. Immunostaining and flow cytometry were utilized to validate the identity of VSX2-eGFP+ cells and potential cluster of differentiation (CD) biomarkers for identifying human RPCs. hiPSCs were successfully differentiated into ROs containing abundant RPCs. The spatiotemporal activity of the VSX2-eGFP reporter recapitulates the dynamic expression of endogenous VSX2 protein. Compared to VSX2-eGFP- cells, VSX2-eGFP+ cells mainly exhibited characteristics of RPCs at early stages of retinal development and of bipolar cells at late stages. RNA-seq analysis revealed transcriptional heterogeneity within VSX2-eGFP+ cells across four distinct developmental stages. Moreover, the dynamic expression of 394 known CD biomarkers at distinct developmental stages was analyzed herein for the first time. One CD biomarker, TNFRSF1B, which has never been reported to be expressed in RPCs, was found to be highly expressed in RPCs at the early stages and might serve as a candidate CD biomarker for sorting RPCs. This study provides valuable insights into the molecular and cellular characteristics of human RPCs, especially their expression profiles of CD biomarkers, laying a foundation for research on retinal development and the clinical translation of hiPSC-derived RPCs.
2025-09-15 | GSE185475 | GEO
Project description:For isolation of rod, cone, and pineal photoreceptor cells, we used the transgenic zebrafish lines, Tg(rho:egfp)ja2Tg, Tg(gnat2:egfp)ja23Tg, and Tg(exorh:EGFP)ja1Tg, which express EGFP in rods, all cone subtypes, and pineal rod cells, respectively. Retinas were dissected from dark-adapted adult fish under dim red light. The isolated retinas were digested with trypsin in Ca2+-free Ringer’s solution. The reaction was terminated by adding soybean trypsin inhibitor and fetal bovine serum. The dissociated EGFP-positive cells were isolated with a fluorescence activating cell sorter (FACSAria, BD Biosciences) by the following three parameters: forward scatter, side scatter and green fluorescence. Goal was to determine differentially expressed genes among three types of photoreceptor cells.
2021-10-06 | GSE168749 | GEO
Project description:Vertebrate vision is mediated by two kinds of photoreceptors, rods and cones, responsible for dim- and bright-light vision, respectively. Gene expression differences among cone subtypes remain poorly understood compared with rods. We generated single-cell transcriptome data using a droplet-based approach to reveal the extent of gene expression diversity among adult zebrafish photoreceptor subtypes. Populations of photoreceptor cells were enriched by using the transgenic zebrafish lines, Tg(rho:EGFP)ja2Tg and Tg(gnat2:EGFP)ja23Tg, which express GFP in rods and all cone subtypes, respectively. By analyzing the single-cell transcriptomes, we found that in addition to the four canonical zebrafish cone types (ultraviolet, blue, green and red), there exist subpopulations of green and red cones in the ventral retina that express red-shifted opsin paralogs (opn1mw4 and opn1lw1). This work lays a foundation for future studies aimed at understanding how molecular differences among cone subtypes affect photoreceptor function.
2021-08-30 | GSE175929 | GEO
Project description:Haematopoietic stem cells can differentiate into all blood cell types. In this process, cells become progressively restricted to a single cell type. The order in which differentiating cells loose lineage potential, and the prospective isolation of cells with a defined potential remains a long-standing question. We performed gene expression analysis of haematopoietic cells from Gata1-EGFP reporter mice, leading to a model for hematopoiesis where the initial lineage decision consists of a separation of erythroid/megakaryocyte/mast cell/eosinophil potential from lymphopoietic/monocyte/neutrophil potential RNA was isolated from HSC EGFP-, HSC EGFP+, LMPP, CLP, preGM EGFP-, preGM EGFP+, GMP EGFP-, GMP EGFP+ and preMegE cells with the QIAGEN RNeasy Micro Kit.
2016-04-04 | E-GEOD-49241 | biostudies-arrayexpress