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There are a few things that I would recommend considering in your trouble shooting:
(1)Stability of Culture Medium-basic fibroblast growth factor is a common component in growth medium that is essential for maintaining the pluripotency of PSCs. This component has been shown to quickly degrade at 37 ºC (Stem Cells (2006) 24:568-574). Therefore, ensuring that, if you are using culture medium which contains FGF as a component, the medium is not repetitively warmed at 37 ºC. Additionally, ensuring that culture medium is not used past the time denoted for complete medium stability is essential (e.g., Essential 8® Medium, upon addition of the supplement to the basal medium, has been shown to remain stable at 4 ºC for 2 weeks). Furthermore, as FGF is shown to rapidly degrade at 37 ºC, daily feeding with culture medium is recommended.
(2)Split Ratios Used-Too high or too low of split ratios resulting in sparse or overcrowded cultures, respectively, can result in higher than normal differentiation of cultures.
(3)Ensuring health of cultures-Ensure that cultures are not overgrown prior to passaging, as this can result in poor cell health and survival following passaging and thus result in too sparse of a culture, which can result in substantial aberrant differentiation of cultures.