Abstract
Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have been extensively studied as an alternative cellular model for recapitulating phenotypic and pathophysiologic characters of human diseases. Particularly, hiPSCs generated from the genetic disease somatic cells could provide a good cellular model to screen potential drugs for treating human genetic disorders. However, the patient-derived cellular model has a limitation when the patient samples bearing genetic mutations are difficult to obtain due to their rarity. Thus, in this study, we explored the potential use of hPSC-derived Wilson’s disease model generated without a patient sample to provide an alternative approach for modeling human genetic disease by applying gene editing technology. Wilson’s disease hPSCs were generated by introducing a R778L mutation in the ATP7B gene (c.2333G>T) using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system into wildtype hESCs. Established Wilson’s disease hESCs were further differentiated into hepatocyte-like cells (HLCs) and analyzed for disease phenotypes and responses against therapeutic agent treatment. R778L mutation in the ATP7B gene was successfully introduced into wildtype hESCs, and the introduction of the mutation neither altered the self-renewal ability of hESCs nor the differentiation capability into HLCs. However, R778L mutation-introduced HLCs exhibited higher vulnerability against excessive copper supplementation than wildtype HLCs. Finally, the applicability of the R778L mutation introduced HLCs in drug screening was further demonstrated using therapeutic agents against the Wilson’s diseases. Therefore, the established model in this study could effectively mimic the Wilson’s disease without patient’s somatic cells and could provide a reliable alternative model for studying and drug screening of Wilson’s disease.
Highlights
Wilson’s disease (Online Mendelian Inheritance in Man, OMIM #277900) is a genetic disorder in which excessive copper accumulates due to abnormal metabolism of copper [1,2]
In Wilson’s disease patients, copper is not removed properly due to mutations in the ATP7B gene that encodes the copper transporting P-type ATPase, which results in damages in several organs [3,4]
We suggested a promising approach for modeling Wilson’s disease without patient samples by introduction of disease mutation in wildtype human embryonic stem cells (hESCs) using gene-editing technology and demonstrated the effectiveness of the mutation introduced model by comparing with the same mutation bearing Wilson’s patient-derived model
Summary
Wilson’s disease (Online Mendelian Inheritance in Man, OMIM #277900) is a genetic disorder in which excessive copper accumulates due to abnormal metabolism of copper [1,2]. In Wilson’s disease patients, copper is not removed properly due to mutations in the ATP7B gene that encodes the copper transporting P-type ATPase, which results in damages in several organs [3,4]. The most affected organ in Wilson’s disease patient is the liver because it is the primary organ that encounters copper metabolism [5,6]. When the disease is diagnosed in the early phase, the important strategy for curing it is lowering the amount of copper level in the body in order to prevent the accumulation of excessive copper. A reliable disease model that recapitulates Wilson’s disease is strongly required
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