FGF2, TGFβ1, and Activin A cooperatively sustain self-renewal in hESCs.

<p>(<b>A</b>) Colonies in N2B27+ FGF2 with or without DM typically exhibit weak NANOG staining in their centres. (<b>B</b>) RT-qPCR analysis showing that FGF2 and TGFβ1 sustain <i>NANOG</i> in a cooperative or synergistic manner (passage 1: n = 2, passage 3: n = 3, all in presence of DM). (<b>C</b>) Typical morphology at passage 2 showing that FGF2 or TGFβ1 alone fail to robustly sustain self-renewal. When applied in combination, however, larger colonies with reduced rates of differentiation are obtained. (<b>D</b>) Titration of FGF2 and TGFβ1 dosage using the <i>NANOG</i> expression level as a readout, in presence of DM. Arrows mark concentrations chosen for further investigation. (<b>E</b>) RT-qPCR analysis showing that additional supplementation with Activin A further enhances the expression of self-renewal genes in hESCs (left chart, n = 5). Note that a dosage of 2.5 to 5 ng/ml was sufficient to achieve near-saturating expression levels. In contrast, the induction of mesendodermal differentiation genes at this concentration of Activin A was still moderate (right chart, n = 5, in presence of FGF2, TGFβ1, and DM over three passages). (<b>F</b>) Analysis of the mesendodermal gene induction signature by Activin A (n = 3, in presence of FGF2, TGFβ1, and DM). Arrows mark the supplementation with Activin A. Note that the Activin-induced upregulation of mesendodermal markers <i>EOMES, GSC,</i> and <i>MIXL1</i> is fully reversible. Data in this figure: Line HuES6.