Decision-making can be a difficult process and often a little help is needed. Embryonic stem (ES) cells can choose to differentiate along multiple cell lineages or undergo self-renewal, so how do they decide what to do? Ex vivo stem-cell proliferation is regulated by leukaemia inhibitory factor (LIF), but when ES cells are grown in serum-free cultures, other unknown factors are required. Now, Austin Smith and colleagues report in Cell that inhibitor of differentiation (ID) proteins — induced by the bone morphogenic protein (BMP)–SMAD pathway — collaborate with LIF to ensure that ES cells opt for self-renewal.

Mouse ES cells grown in serum-free media undergo neural differentiation and, although addition of Lif to the media initially reduces the number of differentiating cells, the undifferentiated stem-cell population declines with successive passaging in the presence of Lif alone. BMPs are known to antagonize neural differentiation, so the authors added Bmp2 or Bmp4 to Lif-containing ES cultures. Lif plus Bmp maintained pure populations of undifferentiated, diploid ES cells even after extended passage, and, withdrawal of both factors allowed neural differentiation to resume. Importantly, Bmp alone produced epithelial-like cells, which indicated that the self-renewal response to Bmp is Lif dependent. The Bmp-related growth and differentiation factor-6 (Gdf-6), but not activin or transforming growth factor-β1 (Tgf-β1), also supports self-renewal in the presence of Lif, indicating that this effect is not a general feature of the TGF-β superfamily.

SMAD transcription factors are the principal downstream regulators of BMPs, so Smith and colleagues investigated Smad activation in the ES cells by immunoblotting and found increased phosphorylation of Smad1 in the presence of Bmp4. But does Smad1 activation by BMP support self-renewal? The inhibitory SMAD family members Smad6 and Smad7 were introduced into the ES cells and — in the presence of Lif — produced fewer and smaller ES cell colonies, which expanded poorly after passage compared with wild-type cells. ID genes are induced by the BMP–SMAD pathway, and Id1, Id2 and Id3 expression were strongly induced by Bmp and Gdf — but not by Lif — in the ES cells. Neither activin or Tgf-β1 induced Id expression, confirming that the proliferative response is specific to the BMP–SMAD pathway.

Smith and co-workers then introduced Id1, Id2 or Id3 into the ES cells to determine if overexpression restricted neural differentiation. The resulting Id-transfectants remained Lif dependent under serum-free conditions, no longer required Bmp for self-renewal and had identical properties to parental ES cells cultured in Lif plus Bmp. Removal of Lif caused the Id-transfectants to differentiate into epithelial-like cells, similar to the parental ES cells exposed to Bmp alone. Using revertible Id expression constructs, they confirmed that when Id was expressed it prevents neural differentiation in the absence of Lif although the cells can differentiate along alternate lineages. This ability to control stem-cell decisions ex vivo has important implications for the field of stem-cell therapy, which has been hindered by the inability to maintain stem-cell self-renewal in serum-free cultures.