Pluripotent stem cell-derived hepatocyte-like cells

Abstract

Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and function. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for pharmacology and toxicology drug screening. iPS cells can be differentiated in a stepwise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iPS-derived hepatocyte-like cells resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iPS-derived hepatocyte-like cells express fetal markers such as alpha fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (0.1%) of many detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iPS-derived hepatocyte-like cells and adult hepatocytes have limited the use of stem cells as a renewable source of functional adult human hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are poorly understood, which has hampered the field in its efforts to induce further maturation of iPS-derived hepatic lineage cells. This review analyzes recent developments in the derivation of hepatocyte-like cells, and proposes important points to consider and assays to perform during their characterization. In the future, we envision that iPS-derived hepatocyte-like cells will be used as in vitro models of human disease, and in the longer term, provide an alternative cell source for drug testing and clinical therapy.

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

“…The hepatic differentiation protocol used in our study is similar to that in many reports (7,9). Our results of characterization of each differentiation stage by both immunofluorescent staining of key markers and expression levels of important genes are within the ranges of those observed in literature (7,9), suggesting that the differentiation process has been successful. The characterizations of the cells along differentiation stages and before encapsulation by staining ( Fig.…”

“…Major liver functions are tightly linked to the 3D assembly of hepatocytes with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Current approaches to use HLCs for an in vitro liver model, however, are mostly limited to 2D culture or simple 3D spheroid cultures (3,7,(10)(11)(12)(13)(14). The lack of a biomimetic microenvironment provided from the 3D interactions of parenchymal and nonparenchymal cell types along the hepatic differentiation stages may potentially be one of the limiting factors to functional maturation of the hepatic progenitor cells (HPCs), as well as the functional preservation of HLCs in vitro (15,16).…”

“…In addition, the practical difficulties in obtaining liver biopsy samples from every patient further hinder their use in personalized liver models. Consequently, hepatocytes derived from human induced pluripotent stem cells (hiPSCs), with the potential to be patient specific and easily accessible, have been widely acknowledged as the most promising cell source for developing personalized human hepatic models (4,7).…”

“…Many groups have reported monolayer differentiation of hiPSCs into hepatocyte-like cells (HLCs) and their ability to metabolize drugs (7)(8)(9). Nevertheless, hiPSC-derived HLCs are still considered immature in terms of many liver-specific gene expressions, functions, and cytochrome P450 (CYP) enzyme activities (7,9).…”

“…Nevertheless, hiPSC-derived HLCs are still considered immature in terms of many liver-specific gene expressions, functions, and cytochrome P450 (CYP) enzyme activities (7,9). Major liver functions are tightly linked to the 3D assembly of hepatocytes with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit.…”