Hepatic differentiation of human embryonic stem cells on growth factor-containing surfaces.

Embryonic stem cells (ESCs) hold considerable promise in tissue engineering and regenerative medicine as a source of tissue-specific cells. Hepatocytes derived from ESCs will be useful for therapies, bioartificial liver assistance devices and drug discovery. In traditional stem cell cultivation/differentiation experiments, growth factors (GFs) are added in soluble form in order to provide signals for tissue-specific differentiation. In contrast, we investigated differentiation of hESCs cultured on top of GFs. In these experiments, glass substrates were imprinted with a mixture of ECM and GF molecules to form 500 µm diameter spots. hESCs were cultured onto these GF-containing ECM spots for up to 12 days to induce differentiation towards the hepatic lineage. The dynamics of differentiation were examined by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and immunocytochemistry. Stem cells cultured on GF-containing surfaces stained positive for the endoderm markers SOX17 and FOXA2, as well as early liver markers such as α-fetoprotein and albumin. qRT-PCR confirmed that pluripotency, endoderm and liver gene expression of hESCs cultured on GF-containing surfaces was consistent with hepatic differentiation. In comparison, hESCs cultured on ECM spots without GFs showed less pronounced loss of pluripotency and lower levels of liver gene expression. In summary, our study demonstrates that hESCs receive differentiation-inducing signals from GF-containing surfaces and may be pushed along the hepatic lineage when cultured on such surfaces. Compared to traditional approaches, the advantages of GF immobilization include reduction in the cost of experiments, stronger and longer stimulation and the possibility of screening GF-stem cell interactions in a multiplexed manner.