Induction and Maturation of Hepatocyte-Like Cells In Vitro: Focus on Technological Advances and Challenges

Abstract

Limited by the poor proliferation and restricted sources of adult hepatocytes, there is an urgent need to find substitutes for proliferation and cultivation of mature hepatocytes in vitro for use in disease treatment, drug approval, and toxicity testing. Hepatocyte-like cells (HLCs), which originate from undifferentiated stem cells or modified adult cells, are considered good candidates because of their advantages in terms of cell source and in vitro expansion ability. However, the majority of induced HLCs are in an immature state, and their degree of differentiation is heterogeneous, diminishing their usability in basic research and limiting their clinical application. Therefore, various methods have been developed to promote the maturation of HLCs, including chemical approaches, alteration of cell culture systems, and genetic manipulation, to meet the needs of in vivo transplantation and in vitro model establishment. This review proposes different cell types for the induction of HLCs, and provide a comprehensive overview of various techniques to promote the generation and maturation of HLCs in vitro.

Rat liver extracellular matrix and perfusion bioreactor culture promote human amnion epithelial cell differentiation towards hepatocyte-like cells

“…Furthermore, the media perfusion and the shear stress resulting from it promote the maturation of cells and tissues in vitro by enhancing nutrient and waste transportation and by providing flow-mediated mechanical stimuli. [44][45][46] The nature and characteristics of the cells seeded in a 3D scaffold are essential features to be considered when generating a bioengineered liver tissue. The liver cells have been for long the elective choice, due to the swinging results reported so far using different pluripotent and multipotent stem cells to generate functional HLC.…”

Phenotypical, functional and transcriptomic comparison of two modified methods of hepatocyte differentiation from human induced pluripotent stem cells

“…Nevertheless, the improvements on HLC maturation obtained using these methods are very limited. An improved understanding of the molecular and cellular basis of organogenesis of the liver coupled with emerging innovative technologies, such as 3D bioprinting (58), organ-on-chip microfluidics (59) and genome modification (60), may lead to breakthroughs in the field.…”

A general fluorescence off/on strategy for fluorogenic probes: Steric repulsion-induced twisted intramolecular charge transfer (sr-TICT)

“…Further, such probes can also be used for monitoring the maturation of human iPS/Embryonic Stem (ES) cell-derived hepatocytes and intestinal epithelial cells. This is important because the preparation of hepatocytes and intestinal epithelial cells by differentiating iPS/ES cells has recently attracted much attention in regenerative medicine and also in connection with drug discovery (29)(30)(31). For example, human iPSC-derived hepatocytes are expected to be a new cell resource for in cellulo assays in pharmaceutical research, overcoming various issues associated with the use of primary human hepatocytes, such as functional variability between human donors, high cost, and short-term loss of functionality (29,(31)(32)(33).…”

“…This is important because the preparation of hepatocytes and intestinal epithelial cells by differentiating iPS/ES cells has recently attracted much attention in regenerative medicine and also in connection with drug discovery (29)(30)(31). For example, human iPSC-derived hepatocytes are expected to be a new cell resource for in cellulo assays in pharmaceutical research, overcoming various issues associated with the use of primary human hepatocytes, such as functional variability between human donors, high cost, and short-term loss of functionality (29,(31)(32)(33). Since CYP3A4 activity is one of the most important markers for differential maturation of hepatocytes and intestinal epithelial cells (34), fluorogenic probes for CYP3A4 activity would also be useful for noninvasively monitoring maturation.…”

“…Next, we applied 2-Me PeER to hiPSC-derived hepatocyte-like and intestinal epithelial-like cells. Because of the utility of hiPSCs for drug discovery research and regenerative medicine, intensive studies have been conducted to differentiate hiPSCs into various types of cells, including hepatocytes and intestinal epithelial cells (29)(30)(31). In the case of hepatocytes, as differentiation proceeds, the cells come to show expression of hepatic markers, such as albumin, drug-metabolizing enzymes, and transporters, which indicate their maturation (29).…”