Is TGF-β a stemness regulator?

In this issue of Blood, Yamazaki and colleagues show that TGF-β inhibits cytokine-mediated LRC clustering in purified HSCs freshly isolated from the bone marrow and induces hibernation of HSCs ex vivo. Since cytokine stimulation of LRC is important to stimulate proliferation of HSCs, the authors propose that TGF-β may be a hibernation/quiescence regulator in the HSC bone marrow niche.

It has been 2 decades now since Ruscetti and colleagues discovered TGF-β as the first potent negative regulator of hematopoietic stem- and progenitor cells.¹  Through many detailed experiments, it was demonstrated that TGF-β treatment of hematopoietic stem and progenitor cells inhibited their proliferation in vitro (with the exception CFU-GM progenitors). From these initial studies it was not clear whether TGF-β is a physiologic regulator of hematopoietic stem cells (HSCs) in vivo. Once conditional knockout mice were available to study the role of TGF-β signaling in the regulation of HSCs in the bone marrow niche, it became clear that HSCs deficient in activin-like kinase 5 (ALK5, TGF-β type I receptor) have normal stem cell function in vivo, although primitive ALK5 null hematopoietic progenitors exhibit increased proliferation capacity in vitro, consistent with Ruscetti et al's initial findings.²  However, these findings would question a physiologic role for TGF-β as an important regulator of HSCs in the bone marrow niche. Recently, it was shown that HSCs deficient in Smad4 (removal of all Smad signals) exhibit a profound defect in self-renewal,³  demonstrating an important role for Smad signaling in hematopoiesis. But specific removal of Smad2/3 activation due to deficient TGF-β signaling has no impact on HSC function in vivo.² 

Here, Yamazaki et al use elegant techniques to examine cell signaling and behavior of single purified HSCs. The same group has recently demonstrated that cytokines (thrombopoietin and stem cell factor) induce lipid raft clustering (LRC) in purified HSCs leading to augmented cytokine signals to mediate cell-cycle entry and proliferation.⁴  In the present paper, single, pure HSCs (LSK CD34⁻ cells) were examined with respect to lipid raft clustering, activation of signaling pathways, and potential to proliferate, differentiate and repopulate irradiated recipients. In the presence of stem cell factor and thrombopoietin, TGF-β could inhibit lipid raft clustering and activate phosphorylation of Smad2/3, the intracellular transducers downstream of the TGF-β receptor, in freshly isolated purified HSCs. The authors suggest that these findings indicate that TGF-β signaling is active in the bone marrow niche to keep the HSCs in hibernation (quiescent). A potent negative regulator of HSCs, p57, was found to be up-regulated following TGF-β stimulation in vitro, and single purified HSCs grown in SCF, TPO, and TGF-β for 5 days could repopulate irradiated recipients. This was impossible without TGF-β stimulation. This demonstrates that genuine HSCs were being examined.

Is TGF-β an important regulator of HSCs in the bone marrow niche to mediate hibernation/quiescence and to maintain intact stemness properties, as Yamazaki and colleagues would like to suggest? All the signaling studies of the purified HSC, while elegant, are done in vitro and can only indirectly imply what is happening in vivo in the stem cell niche. Earlier studies show that HSC s deficient in ALK5 (and thereby TGF-β signaling) have normal self-renewal, repopulation capacity, and differentiation potential in vivo, indicating that TGF-β is not an important regulator of hibernation/quiescence/stemness in vivo.²  Therefore, the 2 studies appear to present conflicting findings. However, both hypotheses may be correct. It is possible that TGF-β regulates quiescence/hibernation in the bone marrow niche. But this role may be redundant because there are many other regulators and signals to preserve stemness of the HSCs in the niche and the removal of one may not lead to an observable phenotype. Another possible explanation could be that studies with the TGF-β signaling deficient mice were performed after bone marrow transplantation,²  and this experimental setting following the hematopoietic stress of the transplantation procedure may not be optimal for studying the regulation of hibernation/quiescence. It is challenging to look at regulation of individual HSCs in the bone marrow niche, but new techniques and future experimental approaches may be able to reveal whether TGF-β regulates quiescence/hibernation/stemness of HSCs in the niche.