IPSC-Derived Human Hepatocytes as a Novel Model to Investigate Thyroid Hormone Action and Signaling

Abstract

Thyroid hormone (TH) actions are essential to normal metabolism and neurologic function. Abnormal TH levels can lead to significant morbidity including metabolic abnormalities, cardiac disease, and obesity. TH action is mediated by the thyroid hormone receptor (TR) isoforms and their coregulators. In the disease models of Resistance to Thyroid Hormone (RTH) it is clear that the TR isoforms play tissue-specific roles in humans, however a platform for drug discovery to potentially treat the diseases does not exist. We hypothesize that human hepatocytes (iHeps) derived from induced pluripotent stem cells (iPSCs) will serve as an ideal model to study TH signaling and to delineate mechanisms of TH diseases in humans. To investigate the TH action in humans, we have developed a serum free human iPSC hepatic differentiation protocol that utilizes sequential exposure to growth factors to mimic actual human development. iHeps are robustly responsive to TH with over 1000 regulated genes. Importantly, they allow us to use CRISPR/Cas9 to determine molecular physiology and mechanism of disease. To demonstrate the role of TRs in TH signaling in human liver cells we have focused on TRβ1 the main TR isoform expressed in liver. we knocked out TRβ1 (THRBKO) using guide RNAs (gRNAs) we engineered to specific genomic loci to truncate the expression of this isoform at the DNA binding domain. Also, to define mechanisms of TR action in the context of RTH, we used this same approach to introduce the Δ337T mutation found in human TRβ1 that causes RTH. Both THRBKO and Δ337T lines are karyotypically normal and flow cytometry analysis in both lines demonstrated that these lines differentiated normally into hepatocytes after gene editing with CRISPR/Cas9. Once clones were identified the edited iHeps were treated with PBS (vehicle) or T3 (10nM) for 24 hours. Real-time quantitative PCR (RT qPCR) was done to assess mRNA expression of T3 target genes. RT qPCR analysis confirmed the success of TRβ1 deletion as the response to T3 was lost on both positive and negative THRB targets. In the Δ337T iHeps, the response to TH was diminshed indicating the successful generation of this line. These preliminary results confirm our ability to edit IPSCs and then to differentiate into hepatocytes, allowing us to further study the action of TH and the mutations involved in RTH.