Turning hESCs into Functional Hepatocytes
Plasticell has developed the Combinatorial Cell Culture (CombiCult®) platform—a high-throughput technology specifically designed for the discovery of novel stem cell differentiation protocols.
The way it works is that stem cells on beads are exposed to multiple combinations of media, containing active agents such as growth factors or small molecules. A unique tagging system allows the cell culture history of each bead to be determined, and tens of thousands of media combinations can be tested in each screen. The goal is to discover the optimal combinations for effective stem cell differentiation.
A disposable split-pool device that facilitates bead manipulation and a custom developed bioinformatics software program, Ariadne™, for protocol selection have further streamlined the CombiCult system. The platform reportedly has been successfully used to discover novel differentiation protocols for many different starting stem cell types and differentiated progeny, e.g., hepatocytes, neurons, cardiomyocytes, and osteoblasts from human and mouse embryonic and mesenchymal stem cells.
According to Plasticell, since large numbers of conditions can be tested in each screen it is possible to efficiently discover optimal protocols that have advantages over more traditional cell culture methods.
For example, in a poster entitled “Directed differentiation of human embryonic stem (hES) cells into functional hepatocytes using the combinatorial cell culture platform CombiCult,” Jeyakumar et al., from Plasticell describe a method for identifying novel serum-free protocols for generating hepatocytes from human pluripotent stem cells.
In this CombiCult screen, hES cells cultured on microcarrier beads were shuffled through 3,375 combinations of cell culture media, containing growth factors and/or small molecules over a 21-day period. Immunostaining for albumin and CYP3A4 expression was used to identify beads bearing differentiated cells.
These positive “hit” beads were identified and sorted using a large particle flow sorter (COPAS) and the tags attached to the beads analyzed, enabling identification of hepatic differentiation protocols.
The researchers reported the discovery of a number of efficient serum-free differentiation protocols. These protocols were further validated in a monolayer culture system, and the cells characterized at phenotypic and functional levels. Specifically, they demonstrated that hepatic cells generated using CombiCult-discovered protocols: 1) exhibit a polygonal shape resembling hepatic morphology, 2) express hepatic markers such as CYP3A4, albumin, and FoxA2, and 3) illustrate functional characteristics of hepatocytes such as glycogen storage and AcLDL uptake.
Such hepatic-like cells could serve as “a valuable tool for regenerative medicine and drug discovery applications,” wrote the scientists.