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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 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE206341 | GEO | 2022/09/22

REPOSITORIES: GEO

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Project description:Adipocytes are dynamic cells that have critical functions to maintain body energy homeostasis. Function of adipocyte is affected by adipogenic differentiation, and exogenous stimulation of biochemical factors such as serotonin and TNF-. Serotonin (5-hydroxytriptamine, 5-HT) is a biogenic monoamine, which known to be involved in lipid metabolism in adipocytes. The TNF- is a proinflammatory cytokines involved in adipocyte biology. Here we investigated the global transcriptome alterations of porcine intramuscular adipocytes at undifferentiated preadipocyte, differentiated adipocytes state, and adipocytes after stimulation with serotonin and TNF- for 12 h by using Agilent’s Porcine (V2) one-color Gene Expression Microarray 4 × 44. Transcriptome analysis revealed that the expression of 443, 261, and 249 genes were altered after differentiation, and after serotonin and TNF- stimulation, respectively. Differentiation process of adipocytes are found to be involved with expression changes of APP, HNF4A, ESR1, EGR1, SRC, HNF1A, FN1, ALB, STAT3, CBL, CEBPB, AR, FOS, CFTR, PAN2, PTPN6, VDR, PPARG, STAT5A and NCOA3 genes which are enriched in the ‘PPAR signaling pathway’ and ‘insulin resistance pathway’. Dose-dependent serotonin stimulation resulted in a decreased fat accumulation in the adipocytes as well as decreased adipocyte proliferation. Serotonin-induced differentially expressed genes in adipocytes were participated in the significant enrichment of GPCR ligand-binding, Cell chemotaxis, blood coagulation and complement pathways, metabolism of lipid and lipoproteins, regulation of lipid metabolism by PPARA; and lipid digestion, mobilization and transport pathways. TNF-α-induced transcriptional modification follows the similar trend, and alteration of many genes shared between both serotonin- and TNF-α stimulations. Our results provide new insights of transcriptome modifications associated with adipogenesis, and exogenous stimulation of serotonin- and TNF- to the porcine intramuscular adipocytes.

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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

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