Abstract
Embryonic stem cells (ES cells), pluripotent and self-renewal, rised from blastocysts, are able to propagate their population and differentiate into various adult cells in vitro. In recent year, ES cells are demonstrated that they can be induced to differentiate into hepatocytes in vitro. Further, the ES cell-derived hepatocytes show therapeutical efficiency to remedy the mice with liver disease. Due to the success of using the mouse model, we expect that people might need a process to produce ES cell-derived hepatocytes in large-scale. The ES cell-derived hepatocytes would be used for treatment of liver disease. They would also be used for drug screening to improve the study of drug development. Base on the requirement of large quantity of hepatocytes in the future, we study the mass production of mouse embryoid bodies (EBs) with hepatic differentiation ability, and induction to differentiate the hepatocytes with high liver function. Initially, we established the cultivation method of mouse ES cells in preliminary study. The ES cell line D3, used in this study, was co-cultured with inactivated primary mouse embryonic fibroblasts. For hepatic differentiation, ES cells were formed EBs, and the hepatic differentiation was appeared in further attached culture. To demonstrate hepatic differentiation, we measured albumin production of EBs by ELISA. Staining by indocyanine green (ICG) was also used to demonstrate that ES cells could differentiate into mature hepatocytes. EB formation is an important step for hepatic differentiation of ES cells. However, production of EBs is a bottleneck of the production of ES cell-derived hepatocytes, because traditional EB formation methods are just used for small-scale EB production. To this end, we attempted to produce mouse EBs with hepatic differentiation ability by a stirred tank bioreactor, “spinner flask.” Our study showed that ES cells could form EBs in spinner flask. Compare to EBs formed by hanging drop method, EBs formed by spinner flask showed in smaller size and size variation. The EBs could also proceed to hepatic differentiation in an attached culture, and promoted by growth factors and inducers. We also observed that EBs formed by spinner flask were similar to that formed by hanging drops on albumin production and hepatic gene expression. Hence, spinner flask has the potential to be an EB formation system for further mass production of ES cell-derived hepatocytes In the production of ES cell-derived hepatocytes with high liver function, we refined the culture method via a series of experimental design studies. We observed that hepatocyte differentiation medium was suitable for EBs to produce more albumin at day 7 of cultivation. Further, we demonstrated that dexamethasone was the most important factor to improve hepatic differentiation. Our refined method could obtain the ES cell-derived hepatocytes with high albumin production reached to 1.90 ± 0.20 pg/h•cell. In addition, the ES cell-derived hepatocytes showed ICG uptake ability and mature hepatic gene expression. In conclusion, the present study examined production method of EBs with hepatic differentiation ability by a stirred tank bioreactor. A more economical hepatic differentiation method was also examined. We could produce ES cell-derived hepatocytes with high liver function without growth factor induction. The present study will benefit for the progress of regenerative medicine and drug development.