Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

Abstract

Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1.

Adult and iPS-derived non-parenchymal cells regulate liver organoid development through differential modulation of Wnt and TGF-β

“…Also, TGF-β is positively correlated with lower O 2(g) levels and activation of HIF1A during liver organogenesis [31]. In addition, Wnt signaling inhibition is known to control hepatocyte differentiation in 3D culture [32]. The combined inhibition of Wnt and TGF-β significantly increases the expression of albumin (more pronounced by TGF-β inhibition), as opposed of what was observed, when these two pathways were activated (Fig.…”

The importance of transporters and cell polarization for the evaluation of human stem cell-derived hepatic cells

“…There is a wide variety of solutions to enhance the liver-specific character of the stem cell-derived HLCs. These include chemically defined xeno-free media [47,48], different coating substances and 3D culturing compositions [35][36][37][38][39], as well as co-culturing with supporting cells [26], bioprinting of HLCs onto structures mimicking in vivo situation [49]. It is important to note that the criteria for defining stem cell-derived HLCs were restricted in most cases to the expression of AFP and albumin, secretion of urea and albumin, as well as glycogen storage.…”

“…Previous studies demonstrated that 3D and so-called sandwich culturing conditions facilitate polarization of hepatocytes and have a positive effect on the hepatic differentiation [35][36][37][38][39]. It has also been reported that bile acids can elevate the expression of liver specific transporters, and enhance their membrane localization [40][41][42].…”

Vascularized Liver Organoids Generated Using Induced Hepatic Tissue and Dynamic Liver‐Specific Microenvironment as a Drug Testing Platform

“…In the last decades, mounting evidence has indicated that 3D cell culture configuration can lead to phenotypical and functional improvements by providing a permissive context that preserves cellular phenotypes on a molecular and histological level and mimicking the architecture and cell–cell contact of the native tissue. In recent studies, microwell array‐based 3D aggregation methods have been reported to generate multicellular hepatic spheroids . Microwell platforms allow for efficient production of multicellular liver constructs, but cannot create dynamic environment required to maintain viability and functions of large 3D constructs.…”