Generation of definitive endoderm from human embryonic stem cells cultured in feeder layer-free conditions

The purpose of these studies was twofold: to reduce the level of nonhuman, potentially immunogenic sialic acid N-glycolylneuraminic acid (Neu5Gc) in human embryonic stem cells (hESCs) through culture of the cells in the absence of feeder layers; and to determine whether directed differentiation was preserved under these conditions, that is, using exclusively human-derived products.

Using a technique developed in our laboratory to culture hESCs in the absence of feeder layers, all nonhuman cell culture reagents were replaced with recombinant or human-derived reagents. The level of the nonhuman sialic acid (Neu5Gc) was measured by high-performance liquid chromatography and monitored over many passages. Subsequently, the cells were subjected to in vitro differentiation into definitive endoderm by lowering the serum concentrations and elevating the amount of activin A.

Under standard tissue culture conditions using mouse and other animal products, the basal levels of Neu5Gc were measured between 7 and 10%. After the cell culture reagents were changed to all human products, Neu5Gc levels decreased steadily before leveling below 2%. Upon initiation of the differentiation protocol under these cell culture conditions, we observed robust endoderm formation, as measured by fluorescence-activated cell sorting analysis and the appearance of mRNA for markers of definitive endoderm (Sox17, CXCR4, Goosecoid and FoxA2).

Consistent with other findings, elimination of nonhuman products in cell culture of hESCs decreases the levels of nonhuman and potentially immunogenic sialic acid levels. Furthermore, our studies demonstrate that in this feeder layer-free system, hESCs undergo directed differentiation into definitive endoderm.