Introduction Cellular transplantation is an alternative to liver transplantation, however provision of primary human hepatocytes is limited. Human embryonic stem cell-derived hepatocyte-like cells (ES-HLCs) represent a renewable source of functional hepatocytes. However, engraftment levels of these cells in the liver is low. The mechanisms regulating interactions between transplanted hepatocytes and the host liver remain unclear. Elucidation of these mechanisms may provide a means to enhance recruitment and engraftment in vivo.
Methods ES-HLCs were generated using our previously published protocol.1 Functionality of cells was demonstrated biochemically and by transplantation into immunocompromised fumaryl acetoacetate hydrolase knock-out (FAH−/−) mice. Microarray analysis of adhesion molecule expression was undertaken and compared with primary human hepatocytes. Adhesion molecule expression of ES-HLCs was assessed by flow cytometry. Adhesion of ES-HLCs to extracellular matrices and human hepatic sinusoidal endothelium (HSEC) was quantified using static and physiologically relevant flow assays, respectively. To define the mechanisms underpinning ES-HLCs interactions adhesion molecule neutralising antibodies and a recombinant human BCAM protein (BCAM-fc) were utilised.
Results ES-HLCs displayed markers of functional hepatocytes and resulted in prolonged survival in FAH−/−mice after intra-splenic transplantation. A range of genes for novel adhesion molecules were identified on ES-HLCs including BCAM. ES-HLCs expressed high levels of β1-integrin (84.3±2.3%), as well as high levels of BCAM (84.3±2%). In static adhesion assays, ES-HLCs bound preferentially to laminin, fibronectin and osteopontin. Binding to fibronectin and osteopontin was reduced when β1-integrin was blocked (28.6%±6.5 p=0.022 and 34%±6.5 p=0.008, respectively). Furthermore, adhesion to laminin was reduced by 48.6±3% (p=0.03) when cells were treated with an anti-BCAM blocking antibody or 48±5.1% (p<0.0001) when laminin-binding sites were blocked by BCAM-fc. Blockade of β1-integrins on ES-HLCs or BCAM binding sites on HSEC led to significant (15%±3 and 24%±5, p=0.029 and 0.035, respectively) decreases in adhesion of ES-HLCs to HSEC during flow assays.
Conclusion Using microarrays we have identified novel adhesion molecules on ES-HLCs such as BCAM along with more established adhesion molecules such as β1-integrins. These molecules critically regulate the adhesion of ES-HLCs to specific ECM molecules and HSEC in physiologically relevant flow assays. BCAM and β1-integrins are thus potential targets to manipulate to improve the engraftment of transplanted ES-HLCs.
Competing interests None declared.
Reference 1. Hay, et al. PNAS 2008;105:12301–6.