Multi-Cellular Human Liver Organoids Enable Complete Maturation of Induced Pluripotent Hepatocyte-like Cells Through Purely Endogenous Signals

Abstract

Induced pluripotent stem cells (iPSCs) require further maturation before they can substitute primary human cells. Induced pluripotent stem cell hepatocyte-like cells (iHLCs) exhibit significantly lower hepatic functions than primary human hepatocytes (PHHs). Maturation of iHLCs has relied unsuccessfully on administering chemical cocktails in widely differing temporal patterns that are 106-fold higher in concentration than in vivo. Hence, there is no reproducible approach for iHLC maturation. We report the assembly of a multicellular 3D human liver organoid that recapitulates the in vivo hepatic microenvironment. Intra-and intercellular signaling between human hepatic cells and iHLCs results in their maturation. Within seven-days, iHLCs in organoids expressed markers of hepatocyte maturation that were statistically similar to PHHs including alphafetoprotein (AFP), hepatic nuclear factor (HNF)-4, and albumin. Ki67+ iHLCs decreased by 2-fold from Days 1 to 14. Expression of two cytochrome P450 (CYP) enzymes, CYP3A4 and CYP2E1, in iHLCs were statistically similar to PHHs by Days 7 and 14, respectively. Biotransformation of acetaminophen and ethanol were statistically similar to PHHs by Day 14. On Day 1, the concentration of endogenously secreted prostaglandin E2 (PGE2) was identical to values reported in adult humans. Over the 14-day culture, the concentrations of endogenously secreted hepatocyte growth factor (HGF) and Oncostatin M (OSM) increased until they were 26-36% of in vivo values. The organoids are secreting critically important maturation molecules that are similar to levels reported in healthy humans. These trends demonstrate how closely the multi-cellular organoids are emulating in vivo-like behavior while undergoing further maturation.