Abstract
Large-scale production of hepatocytes from a variety of genetic backgrounds would be beneficial for drug screening and to provide a source of cells to be used as a substitute for liver transplantation. However, fully functional primary hepatocytes remain difficult to expand in vitro, and circumventing this problem by using an alternative source of cells is desirable. Here we describe a 25-d protocol to direct the differentiation of human pluripotent stem cells into a near-homogenous population of hepatocyte-like cells. As cells progress through this protocol, they express genes in a chronological manner similar to that described during in vivo hepatic development. The protocol relies on culture systems devoid of serum, feeders or complex extracellular matrices, which enable molecular analyses without interference from unknown factors. This approach works efficiently with human embryonic stem cells and human induced pluripotent stem cells and was recently used to model liver diseases in vitro.
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Acknowledgements
This work was funded by a Medical Research Council (MRC) Senior Non-clinical Fellowship (T.T. and L.V.); the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V.); the EU grant LivES and the Evelyn Trust (N.R.F.H.); and the Children's Liver Disease Foundation (C.-P.S.).
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Contributions
N.R.F.H. and L.V. contributed to the protocol concept, design, interpretation, validation and optimization; C.-P.S. and T.T. contributed to protocol optimization and validation.
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Supplementary information
Supplementary Table 1
Primer sequences for qPCR analyses of genes expressed during differentiation of artificial hepatocytes. (PDF 220 kb)
Supplementary Figure 1
Characterization of hESC-derived hepatocyte-like cells and comparison with primary hepatocytes. (A) Expression of hepatic markers during differentiation of hESCs. qPCR analyses showing the progressive decrease in AFP expression as cells start to mature and at the same time increase expression of the hepatic makers (Albumin and A1AT) during maturation. Primary Hepatocytes (Biopredicts) were used as positive control. Data are presented as the average of 3 biological replicates and error bars indicate standard deviation. (B) ELISA analyses showing increase in Albumin and A1AT secretion during maturation of hepatocytes like cells. Primary Hepatocytes (Biopredicts) were used as positive control. Data are presented as the average of 3 biological replicates and error bars indicate standard deviation. Medium only was used as negative control (C) hPSCs derived hepatocytes cells also display detoxifying activity associated with cytochrome P450 family members such as CyP3A4. This activity can be further induced by chemical such as dexamethasone. (D) Cells also display the ability to take up and metabolise indocyanine green as described by Yamada et el 200214. Scale bar: 200 μm (PDF 433 kb)
Supplementary Figure 2
Comparison of hPSC-derived hepatocytes to human primary fetal hepatocytes. Foetal hepatocytes differentiated from hPSC's share an expression profile very similar to in-vivo derived primary human foetal hepatocytes. AFP, ALB, APOF and CYP3A4 show very similar expression levels when compared between the two cell types, while A1AT does appear to be generally higher along with several other mature metabolic genes such as such as tyrosine amino-transferase (TAT), tryptophan 2,3-dioxygenase (TDO2) and transthyretin (TTR). (PDF 319 kb)
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Hannan, N., Segeritz, CP., Touboul, T. et al. Production of hepatocyte-like cells from human pluripotent stem cells. Nat Protoc 8, 430–437 (2013). https://doi.org/10.1038/nprot.2012.153
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DOI: https://doi.org/10.1038/nprot.2012.153
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