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Generation of an RCVRN-eGFP reporter hiPSC line by CRISPR/Cas9 to monitor photoreceptor cell development and facilitate the cell enrichment for transplantation

ABSTRACT: Generation of an RCVRN-eGFP reporter hiPSC line by CRISPR/Cas9 to monitor photoreceptor cell development and facilitate the cell enrichment for transplantation

PROVIDER: PRJNA804510 | ENA |

REPOSITORIES: ENA

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			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: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.

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.

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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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