Human embryonic stem cells (hESCs) can self-renew and become all three germ layers. Nodal/Activin signaling specifies developmental status in hESCs: moderate Nodal/Activin signaling maintains pluripotency, while enhancement and inhibition promote definitive endoderm (DE) and neuroectoderm (NE) development, respectively. However, how modulation of Nodal/Activin signaling influences developmental competence and commitment toward specific lineages is still unclear. Here, we showed that enhancement of Nodal/Activin signaling for 4 days was necessary and sufficient to upregulate DE markers, while it diminished the upregulation of NE markers by inhibition of Nodal/Activin signaling. This suggests that after 4 days of enhanced Nodal/Activin signaling, hESCs are committed to the DE lineage and have lost competence toward the NE lineage. In contrast, inhibition of Nodal/Activin signaling using LY364947 for 2 days was sufficient to impair competence toward the DE lineage, although cells were still able to activate LEFTY1 and NODAL, direct targets of Nodal/Activin signaling. Expression analyses indicated that the levels of pluripotency regulators NANOG and POU5F1 were significantly diminished by 2 days of LY364947 treatment, although the expression of NANOG, but not POU5F1, was restored immediately upon Activin A treatment. Thus, downregulation of POU5F1 coincided with the abrogation of DE competence caused by inhibition of Nodal/Activin signaling.

This study investigated the effect of mechanical load on human mesenchymal stem cell (hMSC) differentiation under different exogenous transforming growth factor-beta1 (TGF-beta(1)) concentrations (0, 1 or 10 ng/ml).The role of the TGF-beta signalling pathway in this process was also studied. Human MSCs were seeded into fibrin-biodegradable polyurethane scaffolds at a cell density of 5 x 10(6) cells per scaffold and stimulated using our bioreactor. One hour of surface motion superimposed on cyclic compression was applied once a day over seven consecutive days. Scaffolds were analysed for gene expression, DNA content and glycosaminoglycan amount. Addition of TGF-beta(1) in the culture medium was sufficient to induce chondrogenesis of hMSCs. Depending on the TGF-beta(1) concentration of the culture medium, mechanical load stimulated chondrogenesis of hMSCs compared to the unloaded scaffolds, with a much stronger effect on gene expression at lower TGF-beta(1) concentrations. With TGF-beta(1) absent in the culture medium, mechanical load stimulated gene transcripts and protein synthesis of TGF-beta(1) and TGF-beta(3). TGF-beta type I receptor inhibitor LY364947 blocked the up-regulation on TGF-beta(1) and TGF-beta(3) production stimulated by mechanical load, and also blocked the chondrogenesis of hMSCs. Taken together, these findings suggest that mechanical load promotes chondrogenesis of hMSCs through TGF-beta pathway by up-regulating TGF-beta gene expression and protein synthesis.

Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Here, using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a(+/+) and Foxo3a(-/-) mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, we find that TGF-beta is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-beta inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-beta inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-beta-FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.