Human embryonic stem cells (hESC) are pluripotent and can thus be used to generate many cell types present in the human body. In addition, the cells are highly expandable, which enables large numbers to be produced prior to differentiation into the cell type required.
A panel of 5 of the 20 hESC lines derived by Roslin Cells were used to make embryoid bodies, which are cell aggregates that are allowed to differentiate in an undirected manner. Our special interest is in the production of hepatocytes, so we selected the hESC line that showed the strongest natural tendency to make these cells (RC10).
After 17 days of directed differentiation, key markers of mature hepatocytes were expressed by RC10 derived hepatocyte-like cells (Figure 2).
In addition, the hepatocyte-like cells were shown to be metabolically active, demonstrating basal CYP3A activity, albumin secretion, urea genesis, and testosterone metabolism.
A feature of hESC lines that has made them more difficult to work with is that, when dissociated into a single-cell suspension and re-seeded into fresh plates, they have a tendency to differentiate spontaneously. For this reason, many laboratories prefer to passage the cells physically without using trypsin; however, this introduces great variance in cell number between wells after re-plating and should be avoided.
Interestingly, the RC10 hESC line is also more resistant to the deleterious effects of single-cell passaging, making it a good choice for producing hepatocytes for cell-based assays. This feature also means that RC10 can be used effectively in cell printing processes to establish artificial stem cell colonies of specific sizes and shapes and to create cell spheroids.