Paper

A nuclear receptor facilitates differentiation of human PSCs into more mature hepatocytes

Dall'agnese, Alessandra, De Zwaan, Esmee, Jeppesen, Jacob, Sudeepa Syamala, Abraham, Brian, Young, Richard, Yang, Eric Guo, Fu, Dongdong, Prathapan Thiru, Griffith, Linda, Van De Bunt, Martijn, Long, Henry, Cejas, Paloma, Lee, Tony, Jaenisch, Rudolf, Ma, Haiting, Garrett-Engele, Carrie

2022

Overview

The capacity to generate functional hepatocytes from renewable human pluripotent stem cells (hPSCs) could address limited supplies of primary human hepatocytes. However, hepatocytes differentiated from hPSCs in vitro are functionally immature. To understand mechanisms regulating maturation of in vitro derived hepatocytes, we developed a 3D spheroid differentiation system and compared gene regulatory elements in uncultured human primary hepatocytes with those in hepatocytes that were differentiated in 2D or 3D conditions from human PSCs by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Three-dimensional differentiation improved enhancer activity and expression of transcription factor ONECUT1, but was insufficient to upregulate human-specific mature hepatocytes marker gene CYP3A4 or super-enhancer regulated transcription factor gene NFIC. Regulome comparisons showed reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and in active enhancers without reduced transcription of THRB, suggesting the regulation at the level of THRB ligands in PSC-differentiated hepatocytes. Addition of thyroid hormone T3 to the PSC-differentiated hepatocytes increased CYP3A4 expression. T3 increased binding of THRB to the CYP3A4 proximal enhancer and restored the super-enhancer status and gene expression of NFIC and reduced expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the 3D PSC-hepatocytes into immunocompromised mice resulted in engraftment of human hepatocytes in the mouse liver parenchyma without disrupting normal liver histology at 6 months after transplantation. This work provides insights into the functions of nuclear receptor THRB and highlights the importance of the environmental factors-nuclear receptors axis in regulating maturation of human PSC-differentiated cell types. Competing Interest Statement R.J. is a cofounder of Fate, Fulcrum, and Omega Therapeutics and an advisor to Dewpoint and Camp4 Therapeutics. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. J.F.J. and M.B. are employees of Novo Nordisk A/S. T.L. is a shareholder of Syros Pharmaceuticals and a consultant to Camp4 Therapeutics. The remaining authors have no competing interests.

• Publisher: Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
• Subject: Cell differentiation / Chromatin / Developmental Biology / Enhancers / Environmental factors / Epigenetics / Gene expression / Hepatocytes / Liver diseases / Maturation / Nuclear receptors / Parenchyma / Pluripotency / Regulatory sequences / Single-nucleotide polymorphism / Stem cell transplantation / Stem cells / Stockholders / Thyroid / Thyroid gland / Transcription factors / Triiodothyronine