Abstract
Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes.
Highlights
Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells (PSCs), offer promise as in vitro models of human liver development, function, and toxicity [1,2]
The low serum caused significant cell death, which was prevented by Wnt3A (25 ng/ml) for the first two days of culture [7,8,9,14,15], leading to robust detection of the three nuclear transcription factors by day 4 (Fig. 2B)
The downregulation of SOX17 and GATA4 expression between definitive endoderm (DE)-like cells and HLCs complies with the exclusion of these transcription factors, which we recently showed in human liver bud as it develops from the
Summary
Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells (PSCs), offer promise as in vitro models of human liver development, function, and toxicity [1,2]. Thawed cells taken into culture are challenging to maintain [17]; in a well-controlled example, over 90% of the cytochrome P450 (CYP) 3A activity was lost in cryopreserved cells compared to freshly plated cells [18]. This illustrates the risk of over-interpreting the HLC phenotype if compared against dedifferentiated controls. Fresh fetal hepatocyte controls have been lacking when assessing HLC function This risks misunderstanding as we have recently shown human fetal hepatocytes possess proteins, such as CYP3A4, commonly interpreted as adult markers [19]
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