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

Abstract

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.

Nanotopographical control of human embryonic stem cell differentiation into definitive endoderm

“…We concluded for the clump culture that the 200 nm substrate improved DE differentiation of hESCs predominantly through effective cell–nanofiber contact rather than providing 3D cell–cell contacts by spheroid formation. A similar conclusion was reached in a recent study where the formation of multilayered colonies by micropatterned substrates was shown to have no significant effect on DE differentiation of hESCs compared to monolayer colonies …”

“…Since nanotopography-mediated spheroid formation has been reported to support maintenance of hESC pluripotency, [39][40][41] the newly formed 3D cell-cell contacts on 200 nm nanofibers could be considered as a favorable event which might prevent non-desirable early differentiation during the three-day expansion and subsequently present a more homogeneous population for endodermal induction. 42 By DE differentiation, hESCs undergo epithelial-to-mesenchymal transition processes 38 where cells assume a mesenchymal, migratory phenotype which might flatten the colony and increase cellsubstrate interactions. 43 In order to investigate whether the multilayered colonies that formed on the 200 nm substrate could switch to a more spread shape upon differentiation, we analyzed SEM images of the differentiated colonies.…”

“…A similar conclusion was reached in a recent study where the formation of multilayered colonies by micropatterned substrates was shown to have no significant effect on DE differentiation of hESCs compared to monolayer colonies. 42…”

Cell sources forin vitrohuman liver cell culture models

“…115 Under this view, hepatic differentiation was performed in differently scaled 3D culture models. Microscale approaches are mainly based on 3D cell aggregation in matrices like collagen, 116 alginate, 117 hydrogel, 118 or Matrigel. 119 Liver-specific parameters, such as albumin production, urea secretion, and CYP expression indicate a higher grade of hepatic differentiation in these models when compared to conventional 2D cultures.…”

Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening

“…However, one of the remaining questions that arises when using HLCs derived from PSCs is the stability of their phenotype and function in long-term cultures, a problem commonly encountered with cultured adult hepatocytes (Schwartz et al 2014). Hence, distinct strategies to improve and stabilize the hepatic phenotype of derived HLCs, such as employing complex 3D culture systems (spheroidal aggregates, perfused bioreactors) (Sivertsson et al 2013;Gieseck et al 2014;Sengupta et al 2014;Subramanian et al 2014;Takayama et al 2014;Yao et al 2014;Kim et al 2015) or using different extracellular matrices (Hay et al 2011), have been promoted.…”