Medical Research Council Centre for Regenerative Medicine, University of Edinburgh
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Medine, C. N., Lucendo-Villarin, B., Zhou, W., West, C. C., Hay, D. C. Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations. J. Vis. Exp. (56), e2969, doi:10.3791/2969 (2011).
Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.
1. Initial preparation of all chemical stocks and coating of culture plasticware
All steps to be carried out in a tissue culture hood under aseptic conditions.
|Plate / Flask||Volume / Well or Flask|
|12-well plate||0.5 mL per well|
|6-well plate||1 mL per well|
|25cm2 flask||2 mL per flask|
Table 1. Recommended volumes of matrigel for coating typical plasticware for hESC culture.
2. Routine maintenance of hESC cultures and characterisation
3. Differentiation of hESCs to hepatic endoderm
4. Characterisation of hESC derived hepatic endoderm
For RNA isolation and extraction please refer to section 3.3.2
5. Representative Results:
Characterisation of hESCs maintained prior to hepatic differentiation
In order to characterise the stem cell status of the H9 hESCs used in the study we studied a number of parameters. The cells exhibited hESC morphology, small, tightly packed cells growing in defined colonies (Figure 1A) and expressed the pluripotent stem cell gene markers, Oct-3/4 and Nanog (Figure 1B). We did not find significant differences in the expression of these genes compared to a H7 hESC line positive control. Additionally, 90.1% of the hESCs population was positive for the stem cell marker SSEA-4 (Figure 1C).
hESC differentiation to hepatic endoderm
Human embryonic stem cells can be efficiently differentiated to hepatic endoderm in vitro (Hay et al 2008). At day 9 of differentiation, cells were harvested and differentiation of hESC to HLCs was assessed. As previously reported the hESCs exhibited a series of profound morphological changes, and by day 9, exhibited early hepatocyte morphology developing a polygonal appearance (Figure 2A). Moreover, downregulation of Oct-3/4 over the 9 day time course was observed (Figure 2B). In contrast, liver transcripts AFP and albumin were up-regulated (Figure 2C) from day 7 onwards.
In vitro maturation of Hepatic Endoderm
Hepatic endoderm was matured in vitro using the established procedure (Hay et al 2008). On the final day of differentiation cultures were immunostained for human liver markers albumin, AFP and E-cadherin. The yield of HLCs using our procedure is typically 90% (Hay et al 2008 ; Hanoun et al 2010 ; Payne et al 2011). HLCs stained positive for Albumin, Alpha-fetoprotein and E-Cadherin (Figure 3).
Figure 1. Characterisation of hESCs used in this study. (A) Phase contrast microscopy images representative of hESC morphology observed in culture at 4x and 10x magnification. The images were captured using a Nikon TE3000/U inverted microscope (B) hESCs cultured are Octamer (Oct-3/4) positive and Nanog positive. H7 hESCs were use as a control for pluripotency gene expression levels. Relative expression refers to folds of induction compared with the endogenous gene control, β-2-microglobulin. (C) FACS plots show hESC surface marker expression levels, including stage specific embryonic antigens SSEA 4.
Figure 2. (A) Phase contrast microscopy representative images of hESC-derived hepatic endoderm morphology at day 9 of the differentiation observed in culture, at x4 and x10 magnification. (B) Characterization of changes in the gene expression. RNA was extracted and the cDNA was analysed by quantitative polymerase chain reaction, showing progressive downregulation of undifferentiated cell gene expression (Oct-3/4) and (C) Upregulation of hepatocyte gene expression (albumin and α-fetoprotein). Relative expression refers to folds of induction compared with the endogenous gene control, β-2-microglobulin at day 0 of differentiation. P<0.05 is denoted * and P<0.001 is denoted *** measured by students t-test in comparison to hESCs at day 0. Error bars represent 1 standard deviation.
Figure 3. Characterisation of hESC-derived hepatic endoderm
Immunocytochemistry showing expression of hepatocyte markers, albumin, AFP and E-Cadherin in hESC (H9) derived hepatic endoderm. Negative controls were performed with corresponding immunoglobulin G (IgG) and representatives images are shown.
Figure 4. hESC (H9) derived HLCs exhibit metabolic activity. At Day 17, H9 differentiated to HLCs exhibited CYP3A metabolic activity (n = 6).
We have developed a simple, homogeneous and highly reproducible in vitro model to generate scalable levels of human HLCs. Our model has been validated by a number of external collaborating laboratories. We routinely characterise stem cell derived HLCs using our in house tool box of developmental markers and liver specific functional assays (most of which are commercially available). The critical stages in our process are: the maintenance of stem cell pluripotency; the ability to direct stem cell differentiation to definitive endoderm; the specification of homogeneous cultures of hepatic endoderm and the ability to derive mature hepatic endoderm exhibiting broad range function in vitro.
One limitation to the large scale deployment of the stem cell derived HLC technology has been the short term differentiated function of mature hepatic endoderm in culture (˜4 days on matrigel). As such we have screened a polymer library for bio-active and novel cellular supports. This has led to the identification of a novel support which maintains hepatic function for at least 15 days. The future directions for this technology are initially industrial application in the drug discovery process (Hay et al 2011). Mid term gains form this technology are likely to be humanised extra-corporeal liver support devices for bridging or treating patients with liver disease. Long term gains from this technology could in cell based transplantation therapy for liver disease, however the current data demonstrates that this strategy requires significant effort before it can be used safely in the clinic (Payne et al 2011).
In conclusion, the significance of our in vitro technology is the ability to generate homogeneous and limitless cultures of high fidelity human HLCs for applied biology.
No conflicts of interest declared.
Dr Hay was supported by a RCUK Fellowship, Dr West was supported by the Department of Surgery, Dr Medine was supported by a grant from the BHF Core Fund, Mr. Baltasar Lucendo-Villarin was supported by a MRC PhD Studenship. Dr Zhou was supported by a scholarship from the Chinese Government.
Matrigel coating plates and flasks
Passaging hESCs with collagenase
Differentiation of hESCs to hepatic endoderm
Characterisation of hESC derived Hepatic Endoderm
Table 2. The antibodies used for hESC derived hepatic endoderm immunostaining, the concentrations used, the species developed in and the companies they are purchased from.
Functional Analysis of Hepatic Endoderm and Normalisation (per mg protein)
Cytochrome P450 Assays