Hepatic Differentiation of Human Embryonic Stem Cells as Microscaled Multilayered Colonies Leading to Enhanced Homogeneity and Maturation

Directed differentiation of human embryonic stem cells (hESCs) towards hepatocyte‐like cells on planar tissue culture plates has been extensively investigated with great promise to provide alternative cell sources for drug metabolism/toxicity testing. Recently, hepatic differentiation of hESCs in 3D configuration with better mimicry of embryonic liver development represents incremental efforts to improve the differentiation efficiency and cellular maturation. However, most of the present 3D differentiation configurations involved interruptive operations during the multi‐staged differentiation process, which might impose unwanted influence on cellular differentiation. Most of the current researches resulted in generation of hepatocytes with high expression of AFP, which is minimally expressed in primary hepatocytes. Here, off‐the‐shelf micro‐stencil arrays are developed to generate adherent multilayered colonies composed of hESCs‐derived cells. Uninterrupted cellular differentiation and proliferation is achieved to recapitulate the continuous and multi‐stage liver development. Compared with conventional 2D format, the micro‐scaled multilayered colonies with uniform and defined sizes constrained within the microwells are composed of more homogenous and mature hepatocyte‐like cells with significantly lowered AFP expression and elevated hepatic functions. The multilayered colonies as novel 3D configuration for hepatic differentiation of hESCs represent a significant step toward efficient generation of functional hepatocytes for regenerative medicine and drug discovery. 3D hepatic differentiation of pluripotent human embryonic stem cells in multilayered microtissue format is achieved within a versatile and off‐the‐shelf micro‐stencil array. Compared with 2D format, cells in the micro‐scaled platform show more homogenous morphology, maturer hepatic induction, and enhanced hepatic functions, indicating a versatile methodology for the derivation of human hepatocytes with mature phenotype and a potential cell source for hepatocyte‐based applications.