NKX6.1 induced pluripotent stem cell reporter lines for isolation and analysis of functionally relevant neuronal and pancreas populations - ScienceDirect

Stem Cell Research

Shailesh Kumar Gupta¹ 

关键词: Human induced pluripotent stem cells;    NKX6.1;    Reporter cell line;    Directed differentiation;    hiPSC-derived beta cells;    EBiSC;    European Bank for Induced Pluripotent Stem Cells;    FACS;    Fluorescence activated cell sorting;    GFP;    Green fluorescence protein;    GSIS;    Glucose stimulated insulin secretion;    HBSS;    Hank';    s balanced salt solution;    HDR;    Homology-directed repair;    hESC;    Human embryonic stem cells;    hiPSC;    Human induced pluripotent stem cells;    hPSC;    Human pluripotent stem cells;    IBMX;    3-isobutyl-1-methylxanthine;    IMI;    Innovative Medicine Initiative;    KCl;    Potassium chloride;    KRBH;    Krebs-Ringer bicarbonate hepes buffer;    NEO;    Neomycin-resistance gene;    NKX6.1;    NK6 homeobox 1;    PBS;    Phosphate-buffered saline;    PDX1;    Pancreatic and duodenal homeobox 1;   

DOI  :  10.1016/j.scr.2018.04.010

学科分类：生物科学（综合）

来源: Elsevier

【 摘 要 】

Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies.

【 授权许可】

Unknown