Derivation and characterization of a UCP1 reporter human ES cell line.

Stem Cell Research
Suranjit MukherjeeShuibing Chen

Abstract

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 ...Continue Reading

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Citations

Mar 19, 2019·Journal of Enzyme Inhibition and Medicinal Chemistry·Zhanfei ChenNanhong Tang

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Datasets Mentioned

BETA
GSE66686
GSE109163
E-MTAB-26032

Methods Mentioned

BETA
PCR
genotyping
Assay
protein assay
RNA-seq
Fluorescence
FACS

Software Mentioned

R
DAVID
R pheatmap package
DESeq2
Applied
Biosystems Copy Caller
hclust

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