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Generation of a TPH2-EGFP reporter cell line for Purification and Monitoring of Human Serotonin neurons in vitro and in vivo

ABSTRACT: Generation of a TPH2-EGFP reporter cell line for Purification and Monitoring of Human Serotonin neurons in vitro and in vivo

PROVIDER: PRJNA850356 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19733700_1.fastq.gz	Fastqsanger.gz
	SRR19733700_2.fastq.gz	Fastqsanger.gz
	SRR19733701_1.fastq.gz	Fastqsanger.gz
	SRR19733701_2.fastq.gz	Fastqsanger.gz
	SRR19733702_1.fastq.gz	Fastqsanger.gz

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Generation of a TPH2-EGFP reporter cell line for Purification and Monitoring of Human Serotonin neurons in vitro and in vivo

Project description:Generation of serotonin neurons (SNs) from human pluripotent stem cells (hPSCs) provides a promising platform to explore the mechanisms of serotonin-associated neuropsychiatric disorders. However, neural differentiation always yields heterogeneous cell populations, making it difficult to identify and purify SNs in vitro or track them in vivo following transplantation. Herein, we generated a TPH2-EGFP reporter hPSC line with insertion of EGFP into the endogenous tryptophan hydroxylase 2 (TPH2) locus using CRISPR-Cas9-mediated gene editing technology. This TPH2-reporter, which faithfully indicated TPH2 expression during differentiation, enabled us to obtain purified SNs for subsequent transcriptional analysis and study of pharmacological responses to antidepressants. In addition, the reporter system showed strong EGFP expression to indicate SNs, which enabled us to explore in vitro and ex vivo electrophysiological properties of SNs. In conclusion, this TPH2-EGFP reporter cell line might be of great significance for studies on human SN-related development and differentiation, drug screening, disease modeling, and cell replacement therapies.

2022-09-22 | GSE206341 | GEO

Generation of a TPH2-EGFP reporter cell line for purification and monitoring of human serotonin neurons in vitro and in vivo.

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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

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

scRNA-seq of lung CD140a+ mesenchymal cells (fibroblasts) from CD140a-EGFP reporter mice

Project description:To investigate the heterogeneity of lung CD140a+ mesenchymal cells (fibroblasts) and identify the specific lung fibroblast subset, we purified CD140a+ lung cells using CD140a-EGFP reporter mice for a higher-resolution scRNA-seq.

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Gene expression in haematopoietic stem and progenitor cells from Gata1-EGFP reporter mouse bone marrow

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.

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Project description:Transcriptomic Profiling across Human Serotonin Neuron Differentiation via the FEV Reporter System

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scRNA-seq of lung CD140a+ mesenchymal cells (fibroblasts) from CD140a-EGFP reporter mice

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2023-10-03 | GSE230135 | GEO

Bulk RNA-Seq analysis of TCF1 Reporter eGFP+ and TCF1 Reporter eGFP- CD8+ T cells generated with different metabolic manipulations and isolated from in vitro co-culture with HKP1-ova-GFP mouse lung adenocarcinoma cells

Project description:To investigate how metabolic manipulation alters differentiation and the transcriptional landscape in tumor-specific CD8+ T cells, we isolated OT-I+ CD8+ T cells from spleens of transgenic OT-I+ Tcf7GFP+ mice expressing a TCF1 eGFP Reporter, and activated them for one day on plates coated with 2μg/ml of both anti-CD3 and anti-CD28 in the presence of 50U/mL mouse IL-2. We then treated them with equal volumes of DMSO vehicle control, 3μM glucose 6-phosphate dehydrogenase agonist AG1, 25μM glucose 6-phosphate dehydrogenase inhibitor G6PDi-1, or 2mM hexokinase inhibitor 2-DG, and expanded them for 1 more day on anti-CD3/anti-CD28 coated plates in the presence of 50U/mL mouse IL-2. We subsequently co-cultured these cells with HKP1-ova-GFP tumor cells at a 5:1 T cell:tumor cell ratio for an additional 4 days with continuing drug treatment in the presence of 50U/mL mouse IL-2, passing cultures every other day. At day 6 post-initial-stimulation, CD8+ T cells were harvested, filtered by passing through a 70uM cell strainer, washed, and stained for CD8β and Thy1.2. Samples were dyed with DAPI at 0.2μg/mL, and sorted on a Becton-Dickinson FACS Aria II sorter for DAPI- CD8β + Thy1.2+ eGFP+ or DAPI- CD8β + Thy1.2+ eGFP- cells. We then performed gene expression profiling analysis using data obtained from RNA-seq of sorted cells, comparing across treatment groups and TCF1 eGFP Reporter expression.

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