Genome-Edited Human Pluripotent Stem Cell–Derived Macrophages as a Model of Reverse Cholesterol Transport
Objective—To create isogenic human pluripotent stem cell–derived macrophages with and without ABCA1 expression as a model for reverse cholesterol transport.
Approach and Results—The clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9 genome-editing system was used to introduce frameshift mutations into the coding sequence of ATP-binding cassette, subfamily A, member 1. Individual human pluripotent stem cell clones with deleterious mutations were identified, expanded, and differentiated into mature macrophages with a cytokine-based, feeder-free differentiation protocol. Wild-type cells demonstrated effective cholesterol efflux to apoAI acceptor, whereas ABCA1−/− cells displayed significantly reduced efflux ability and increased expression of proinflammatory cytokines.
Conclusions—Human pluripotent stem cell–derived macrophages capable of reverse cholesterol transport can be rapidly generated and genetically edited with clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat–associated 9. Introduction of homozygous frameshift mutations results in loss of ABCA1 expression in differentiated macrophages and subsequent reduction of cholesterol efflux capability. This facile genome-editing approach and differentiation protocol pave the way for future studies of the molecular determinants of reverse cholesterol transport and other macrophage properties.
- Received May 23, 2015.
- Accepted October 22, 2015.
- © 2015 American Heart Association, Inc.