Derivation and Characterization of a UCP1 Reporter Human ES Cell Line
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA429821 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6469930.fastq.gz | Fastqsanger.gz | |||
| SRR6469931.fastq.gz | Fastqsanger.gz | |||
| SRR6469928.fastq.gz | Fastqsanger.gz | |||
| SRR6469929.fastq.gz | Fastqsanger.gz |
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Project description:Interest in human brown fat as a novel therapeutic target to tackle the growing obesity and diabetes epidemic has increased dramatically in recent years. While much insight into brown fat biology has been gained from murine cell lines and models, few resources are available to study human brown fat in-vitro. In this study, we detail the derivation and characterization of a novel human ES UCP1 reporter cell line that marks UCP1 positive adipocytes in-vitro. We targeted a mCherry reporter to the UCP1 stop codon via CRISPR-Cas9 and demonstrated that when differentiated to brown adipocytes, reporter cells express UCP1, display high mitochondrial content and multi-locular lipid morphology, and exhibit functional properties such as lipolysis in the presence of isoproterenol and forskolin. Isolation and purification of mCherry positive cells demonstrated elevated expression of brown fat marker genes and a high similarity to isolated human brown fat versus white fat via RNA-seq. This reporter cell line thus presents new opportunities to study human brown fat biology by enabling future work to understand early human brown fat development, performing disease modeling, and enabling drug screening applications.
2020-07-27 | GSE109163 | GEO
Project description:Interest in human brown fat as a novel therapeutic target to tackle the growing obesity and diabetes epidemic has increased dramatically in recent years. While much insight into brown fat biology has been gained from murine cell lines and models, few resources are available to study human brown fat in vitro, which makes the need for new ways to derive and study human brown adipocytes imperative. Human ES cell based reporter systems present an excellent tool to identify, mark, and purify cell populations of choice. In this study, we detail the derivation and characterization of a novel human ES UCP1 reporter cell line that marks UCP1 positive adipocytes in vitro. We targeted a mCherry reporter to the UCP1 stop codon via CRISPR-Cas9 based gene targeting. The brown adipocytes derived from reporter cells express UCP1, display high mitochondrial content, multi-locular lipid morphology, and exhibit functional properties such as lipolysis. The mCherry positive cells purified after cell sorting show elevated expression of brown fat marker genes and a high similarity to isolated human brown fat via RNA-seq analysis. Finally, we demonstrate the utility of this reporter to real time monitor UCP1 expression upon stimulation. This reporter cell line thus presents new opportunities to study human brown fat biology by enabling future work to understand early human brown fat development, perform disease modeling, and facilitate drug screening.
| S-EPMC7376882 | biostudies-literature
Project description:A cell line designated as HFB-ES was established from blastula stage embryos of H. fossilis (Singhi). The embryonic cells were harvested and maintained in Leibovitz's medium supplemented with 15% fetal bovine serum. The cell line had been subcultured for more than 90 passages in a period of 24 months. HFB-ES cells were able to grow at temperatures between 25 and 35°C with an optimum temperature of 28°C. The growth rate of HFB-ES was proportional to FBS concentration, with optimum growth seen at 15% FBS concentration. The originality of the cell line was confirmed by sequencing of cytochrome oxidase c subunit I (COI), cytochrome b gene, and microsatellite DNA profile. Results of chromosome complements of HFB showed normal karyo-morphology with 56 (2n) diploid number of chromosomes after 40 passages which indicated that the developed cell line is chromosomally stable. The pluripotency of HFB was demonstrated by alkaline phosphatase activity and Oct-4 gene expression. Expression of GFP reporter gene was successful in HFB-ES. These results indicated that HFB-ES could be utilized for future gene expression studies.
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Project description:Pluripotency is the differentiation capacity of particular cells exhibited in the early embryo in vivo and embryonic stem (ES) cells have been shown to originate from the inner cell mass (ICM) of an E3.5 blastocyst. Although the potential for ES cells to differentiate into the three germ layers is equated to ICM cells, they differ in the ability to maintain the capacity for self-renewal. Despite several studies on the maintenance of ES cells in the ground state of pluripotency, the precise mechanism of conversion from the ICM to the ES cell remains unclear. Here , we have examined the cell characteristics and expression profile within the intermediate stages of ES cell derivation from the ICM. Gene clustering and ontology (GO) analyses showed a significant change in the expression of epigenetic modifiers and DNA methylation-related genes in the intermediate stages. We have proposed that an epithelial-to-mesenchymal transition (EMT) blockage is required during derivation of mouse ES cells from E3.5 blastocysts. This study suggests a novel mechanistic insight into ES cell derivation and provides a time-course transcriptome profiling resource for the dissection of gene regulatory networks that underlie the transition from ICM to ES cells.
2017-10-02 | GSE87793 | GEO
Project description:Since brown adipose tissue (BAT) dissipates energy through UCP1, BAT has garnered attention as a therapeutic intervention for obesity and metabolic diseases including type 2 diabetes. As we better understand the roles of classical brown and beige adipocytes, increased beige fat mass in response to a variety of external/internal cues is associated with significant improvements in glucose and lipid homeostasis that may not be entirely mediated by UCP1. We aim to analyze transcriptome of wild type and UCP1-null beige adipocyte to identify the UCP1-independent function.
2017-10-28 | E-MTAB-4085 | biostudies-arrayexpress
Project description:Non-shivering thermogenesis in adipocytes is mediated by brown adipose tissue, purportedly through the sole action of uncoupling protein 1 (UCP1). The physiological relevance of UCP1-dependent thermogenesis has primarily been inferred from the attenuation of thermogenic output of mice genetically lacking Ucp1 from birth (germline Ucp1-/-). However, germline Ucp1-/- mice harbor secondary changes within brown adipose tissue beyond UCP1, such as reduced electron transport chain abundance. We show here that these secondary changes also encompass reduced expression of genes regulating fuel liberation, changes that would attenuate the capacity of any thermogenic pathway. Therefore, the quantitative contribution of UCP1-dependent and -independent thermogenesis is not fully understood. To mitigate the potentially confounding ancillary changes to brown adipose tissue of germline Ucp1-/- mice, we constructed mice with inducible adipocyte-selective disruption of Ucp1. We find that, while germline Ucp1-/- mice succumb to cold-induced hypothermia with complete penetrance, most mice with inducible deletion of Ucp1 maintain homeothermy in the cold. However, inducible adipocyte-selective co-deletion of Ucp1 and creatine kinase B (Ckb, an effector of UCP1-independent thermogenesis) exacerbates cold-intolerance, indicative of a negative genetic interaction and thus a parallel thermogenic function. We find no evidence for impairments in insulation or non-shivering thermogenesis in skeletal muscle that would drive this phenotype. Furthermore, following UCP1 deletion or UCP1/CKB co-deletion from mature adipocytes, moderate cold exposure triggers the regeneration of mature adipocytes that coordinately restore UCP1 and CKB to brown adipose tissue, providing further evidence of their parallel thermogenic relationship. Our findings suggest that thermogenic adipocytes utilize non-paralogous protein redundancy – through UCP1 and CKB – to promote cold-induced energy dissipation.
2024-01-10 | PXD047841 | Pride
Project description:Uncoupling protein 1 (UCP1) is thought to be a major regulator of whole-body energy expenditure and metabolic homeostasis. However, the widely employed UCP1 loss of function model has recently been shown to have destructive effects on the entire electron transport chain of thermogenic fat. As such, the role of UCP1 in metabolic regulation in vivo remains unclear. We recently identified cysteine-253 as an allosteric site on UCP1 that elevates protein activity upon covalent modification. Here we examine the physiological importance of this site through the generation of a UCP1 cysteine-253 null mouse (UCP1 C253A), the first genetic model for selective disruption of UCP1 in vivo. UCP1 C253A mice exhibit significantly compromised thermogenic responses but display no measurable effect on fat accumulation in an obesogenic environment. Unexpectedly, instead we find that lack of cysteine-253 results in substantial immune cell infiltration and inflammatory pathology in adipose tissues of male, but not female mice. Together, our results establish the UCP1 cysteine-253 activation site as a regulator of acute thermogenesis and sex-dependent adipose tissue inflammation.
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Project description:lymphoid Es cell line Keywords: cDNA array, ES cells
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Project description:Standard mouse ES cell derivation protocols use blastocysts at 3.5 dpc, but the efficiency of successful line derivation is low in commonly-used inbred strains, and much lower in other mouse strains. By using 2-cell zona pellucida-free embryos, culturing them on embryonic feeder cells, and passaging the outgrowths in several different media formulations, we have dramatically improved mouse ES cell line derivation efficiency. Mouse ES cell lines derived from blastocyst outgrowths using standard procedures were compared to preblastocyst (2-cell outgrowth)-derived lines, each derived in three different media formulations, using the genome-scale MEEBO gene expression oligo probe set. The overall goal of this microarray study is to determine the overall similarity between cell lines derived using the two methods, in terms of gene expression, as well as to identify important differences. Additionally, we hope to identify effects of the media formulation on gene expression in the cell lines.
2010-08-04 | GSE17560 | GEO
Project description:Single cell level analysis of transcriptional circuits regulating UCP1-indepednent theremogensis through Lgr6 in inguinal Adipose tissue. We previously identified an increase in Lgr6 gene expression in the beige adipocyte population of UCP1-KO mice and hypothesized its involvement in UCP1-independent thermogensis. Consistent with our previous data the scATAC-seq data reveals that chromatin accessibility at the Lgr6 loci is significantly higher in Ucp1- KO beige adipocytes as compared to WT and the promter is also more accessible in Ucp1- KO beige adipocytes. Other DNA-binding motifs involved in UCP-1 indepedent thermogensis were also analyzed and found to be upregulated in the KO model.
2021-05-15 | GSE174456 | GEO