PROTOCOL FOR DIRECTED DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS TOWARD A HEPATOCYTE FATE

Abstract

Hepatocytes contribute ∼70% of hepatic mass and are responsible for the majority of functions associated with the liver including detoxification, carbohydrate metabolism, protein storage, urea synthesis, formation of bile, cholesterol homeostasis and the secretion of a plethora of proteins that are present within serum. Studies in mice and rats have demonstrated that transplant of exogenous hepatocytes can successfully replace dysfunctional host parenchymal cells . Such studies have led to clinical trials to test the feasibility of correcting inborn errors of hepatic metabolism using exogenous cell transplant . Although preliminary results from such studies are encouraging, the availability of donor livers is limited raising the need for an alternative source of human hepatocytes. One possible source of hepatocytes could be provided by human pluripotent stem cells because such cells have a high capacity for self-renewal as well as the potential to differentiate into a wide variety of somatic cell types including hepatocytes . The generation of hepatocytes from human induced pluripotent stem cells (hiPSCs) is particularly appealing because it could be used to model heritable hepatic disorders such as familial hypercholesterolemia and alpha-1-antitrypsin deficiency A large number of groups have described protocols that can induce the differentiation of cells with gene expression and functional profiles that closely resemble that of hepatocytes using both hESCs and hiPSCs . Most of the approaches that have been described have successfully exploited data generated from decades of research into the molecular mechanisms that control hepatocyte differentiation during embryogenesis . In addition, unlike many other cell fates, human pluripotent stem cells seem particularly amenable to following a hepatic fate. While the results have generally been impressive, it is important to keep in mind that no protocol has successfully generated hepatocytes that can fully replace the parenchymal cells of the mouse liver in the manner that primary human hepatocytes are capable of Although such a caveat may impact the use of such cells for cell transplant, hESC– and hiPSC–derived hepatocytes have already proven to be a valuable tool to study human hepatocyte dysfunction and differentiation and are likely to provide platforms for drug discovery in the near future Original protocols, which were adapted from studies using mouse ES cells, generally used suspension culture of stem cell aggregates (embryoid bodies) as a mechanism to initiate differentiation. Although effective in inducing differentiation, the procedure tends to be chaotic and as a consequence generates multiple cell lineages that commonly require subsequent cell sorting or selection to be useful. Moreover, the efficacy of producing high quality hepatocytes through an embryoid body intermediate tends to be variable, which affects reproducibility and complicates interpretation of data. Similar problems can be associated with protocols that include culture of differentiated cells using feeder layers or include ill-defined additives such as fetal bovine serum that typically exhibit substantial lot–to–lot variation. With this in mind we have described a protocol, illustrated in , that relies on highly defined culture conditions that facilitate the reproducible differentiation of hepatocytes from a wide-range of hESCs and hiPSCs . Although the basic protocol described here is efficient and has proven effective in a large number of hESC and hiPSC lines, optimization may be required for specific cell lines