Minireview: Genome Editing of Human Pluripotent Stem Cells for Modeling Metabolic Disease

Abstract

The pathophysiology of metabolic diseases such as coronary artery disease, diabetes, and obesity is complex and multifactorial. Developing new strategies to prevent or treat these diseases requires in vitro models with which researchers can extensively study the molecular mechanisms that lead to disease. Human pluripotent stem cells and their differentiated derivatives have the potential to provide an unlimited source of disease-relevant cell types and, when combined with recent advances in genome editing, make the goal of generating functional metabolic disease models, for the first time, consistently attainable. However, this approach still has certain limitations including lack of robust differentiation methods and potential off-target effects. This review describes the current progress in human pluripotent stem cell-based metabolic disease research using genome-editing technology.

Figure 1.

Overview of generation of three types of hPSCs: hESCs generated through isolation of inner cell mass from blastocyst; human induced pluripotent stem cells (hiPSCs) generated through reprogramming of adult cells by exogenous expression of transcription factors; and hPSCs generated through SCNT (hPSCs-SCNT).

Figure 2.

Overview of ZFNs, TALENs, and CRISPR/Cas-mediated gene targeting. ZFNs and TALENs, which usually function in pairs, are chimeric proteins consisting of endonuclease FokI catalytic domain and DNA binding motif as depicted at top left. Upon binding to targeting sites, nuclease domains of FokI dimerize and subsequently generate a DSB between the two binding sites. In the CRISPR/Cas system, Cas9 nuclease is recruited to targeting site by a sgRNA that recognizes and binds to a 20-bp DNA sequence in the presence of 5′-NGG motif (PAM, indicated in red). Upon binding, Cas9 mediates a DSB 3 bp upstream of the PAM (red triangle). Endonuclease-generated DSBs can be either repaired by NHEJ, which is error prone and usually leads to the introduction of indels, causing a gene knockout due to frameshift, or repaired by HDR in the presence of a homologous template, which can either be a sister chromosome or a DNA donor template introduced exogenously. HDR-mediated repair is usually used to knock in a site mutation or a tag.

Figure 3.

Experiment design for hPSC-based disease modeling. hPSCs are targeted by genome-editing tools, such as ZFNs, TALENs, and CRISPR/Cas, to generate isogenic cell lines. Then wild-type and mutated hPSCs are differentiated into disease-relevant cell types (HLCs are depicted here as an example) followed by phenotypic comparison and pathophysiological studies.

Figure 4.

Differentiation of hPSCs into various metabolic cell types, including white adipocytes (WAs), HLCs, skeletal muscle cells, pancreatic β-cells, VSMCs, ECs, and hypothalamic neurons.