t FGF2 undergoes marked degradation in stem cell cultures. Alterations in FGF2 level are known to drive differentiation of some stem 21602423 cell lineages. This could, in part, explain why steady growth factor levels achieved by FGF2 beads both minimized spontaneous differentiation, and increased potency of cultures even when cultures were fed every third day. There may be labile media components other than FGF2 and stem cell cultures may also produce unwanted products that can accumulate over time, dependent on culture density, to 
limit culture stability beyond the 34 day feeding period we studied. These can be determined in future experiments and possibly addressed to further improve culture techniques. The MedChemExpress GW 501516 improvements in stem cell cultures we observed appear to be independent of the specific technique used to stabilize the FGF2 level. Frequent manual addition of FGF2, for example, produced results similar to controlled delivery using PLGA microspheres. We anticipate that other biocompatible controlled release FGF2 media additives such as hydrogel or chitosan, would produce similar effects on stem cell cultures. Our working hypothesis is that any technique which provides stable FGF2 levels that resemble the normal in vivo niche activity more closely than unstable soluble FGF2 does, will also more effectively maintain stem cells in the culture dish. Pluripotent stem cells are routinely grown on feeder cell layers such as mouse fibroblasts and/or in the presence of conditioned media to provide supplemental nutrients. Over the past few years, `feeder-free’ culture techniques have been developed. These techniques utilize combinations of high FGF2 levels, biologically active substrates such as matrigel or laminin and/or manipulation of mitogens. Substrates and matrices have been modified to contain growth factors. Culture dish coatings such as matrigel do not eliminate the need for daily media change of pluripotent stem cell cultures, presumably because FGF2 stabilization is not sufficient. Sustained FGF2 Levels Better Maintain Stem Cells 5 Sustained FGF2 Levels Better Maintain Stem Cells Matrix scaffolds that encapsulate stem cells to increase available surface area have also incorporated growth factor. Stem cells encapsulated within such matrices grow well exposed to the growth factor microenvironment within the matrix scaffold. In contrast, the FGF2 beads are a simple media additive and do not encapsulate cells. Beads added to otherwise standard culture media and culture dish techniques release FGF2 to stabilize levels without a fundamental change such as cell encapsulation. As 22440900 such, FGF2 beads provide a very simple new method to improve standard culture techniques for successfully maintaining undifferentiated stem cells. Directed Endoderm and Mesoderm Differentiation Prior to differentiation, pluripotent cells were feeder-depleted and placed in suspension culture in the following conditions: For endoderm: serum-free media supplemented with 100 ng/ml activin A for 5 days and mesoderm: StemPro34 media supplemented with 20 ng/ml BMP4 and 10 ng/ml FGF2, then 10 ng/ml VEGF and 10 ng/ml FGF2 . Cells were collected and analyzed on a FACS Aria 2 Cell Sorter. Spontaneous Differentiation hESCs were spontaneously differentiated by placing clumps of hESCs in 6-well tissue culture treated plates in hESC medium for 1 day to allow cells to settle on Matrigel. This medium was changed to DMEM plus 10% FBS, with feeding every 24 days. After 2-weeks the cells w
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