Stem cells hold considerable promise for the therapy of degenerative disease. However, when thinking about stem cells, most people think about cell-replacement approaches, which are costly and present with considerable challenges in relation to engraftment, tissue integration, and function. Small molecules could be an attractive alternative to amplify the stem cells pool or direct their differentiation by targeting specific signaling pathways. Moreover, they can be used to understand basic mechanisms regulating stem cell self-renewal and differentiation. Elucidating how Wnts act as instructive cues for the recruitment, maintenance, and maturation of MSCs and their differentiated progenies is of primary interest given the potential use of these cells in regenerative medicine. Prior studies have implicated canonical Wnt signaling in the regulation of bone metabolism at several levels, in amplifying the undifferentiated MSC pool,7, 8 commitment of undifferentiated MSCs to the osteoblast lineage,13, 14 and stimulation of their differentiation.14 However, differences in cell preparations, use of models that did not take into account the cellular context, or use of nonphysiologic loss- and gain-of-function approaches are some of the reasons for the emergence of contrasting data. Moreover, the presence of large numbers of Wnts, Wnt receptors, coreceptors, and soluble inhibitors with loosely defined actions has made it difficult to define the function of Wnt signaling in MSCs. Here we have addressed some of these issues. Since the bone marrow is a complex and highly organized structure and represents a difficult organ to reconstruct in vitro, we designed experiments that considered the cellular context of MSCs in their in vivo environment, minimizing the loss of functionality that has been associated with the long-term expansion of MSCs. First, in in vitro experiments where Wnt signaling was activated with the GSK-3α/β inhibitor AR28, we have used bone marrow–derived cells that were cultured directly without further fractionation onto tissue culture plastic. Second, we have studied the effect of AR28 following direct injection in BALB/c mice and counted the number of progenitors and their differentiated mature cells. Moreover, to reduce the complexity of the system, we have used a selective inhibitor of GSK-3, leading to enhanced translocation of β-catenin to the nucleus to activate canonical Wnt signaling. Both in vitro and in vivo studies showed an increase in the number of mesenchymal progenitors with osteogenic and adipogenic potential. Of interest is the difference in response in the number of CFU-F between in vitro and in vivo studies. While an increase in the number of CFU-F was seen in vitro, this was not observed in vivo, where a significant decrease was observed after prolonged exposure to AR28. Even more interesting is the temporal regulation of Wnt signaling on MSCs in vivo. An increase in the number of mesenchymal progenitors is seen only at the earlier time point of 3 days. This effect seems to be lost after 14 days of treatment, when the number of osteoblasts is increased significantly. Our in vitro data are in agreement with what has been shown by Baksh and colleagues21 following addition of Wnt3a, a factor that also activates canonical Wnt signaling, to suspension-grown cells. This leads to an increase in the number of CFU-F, CFU-O, and CFU-A in vitro. There are a number of reasons for the lack of increase in the number of CFU-F in vivo seen at early time points followed by a significant decrease with time. One possibility is that mesenchymal progenitors contained in the more primitive CFU-F population have been recruited to commit to progenitors with osteogenic and/or adipogenic potential earlier than 3 days and are driven to exhaustion as a result of the continuous commitment and differentiation process. An alternative or additional explanation holds that activation of canonical Wnt signaling amplifies a more committed subpopulation of mesenchymal progenitors with osteogenic and/or adipogenic potential that are driven to differentiate to the osteogenic lineage at the expense of adipogenic differentiation. The increase in osteoblast numbers and in bone mass would provide a negative-feedback signal to the progenitors, including the CFU-F, causing their inhibition to proliferate. In support of this, differentiated osteoblasts or osteocytes have been found to produce Wnt inhibitors such as DKK1, which is strongly upregulated during the late phases of osteoblast differentiation22 and has been shown to inhibit activation of canonical Wnt signaling and osteoblast differentiation.23 The presence of a feedback mechanism in vivo would explain the discrepancy between our in vivo and in vitro results. In vitro during the CFU-F assay, cells are seeded at low density, and only those with high proliferative capacity are selected for survival. Signals required to provide a negative feedback would not be present owing to the lack of differentiating osteoblasts and to the clonogenic nature of the assay. In vivo, the proliferative effect on the CFU-F compartment is possibly short-lived and masked by the process of commitment, differentiation, and negative feedback. The presence of a negative-feedback mechanism would reconcile some of the contrasting findings on whether activation of canonical Wnt signal had a positive or negative effect on proliferation and differentiation to the osteogenic lineage because this would depend on the subpopulation of mesenchymal progenitors considered, the length of time the cells were exposed to the signal, and the cellular context the target cells were in at the time point analyzed.
While there is agreement that inhibition of GSK-3 leads to enhanced bone mass,14, 16 the mechanism by which this occurs is controversial—whether this is due to enhanced drive to differentiation to osteoblasts and/or inhibition of osteoclast differentiation. We have shown that inhibition of GSK-3 blocks preadipocyte differentiation and enhances osteoblast differentiation in vivo despite progenitors with both potentials being amplified. This is in agreement with in vitro studies on the preadipocyte cell line ST215 and is in line with studies on sustained expression of β-catenin by expression of Wnt10b in preadipocytes or following stimulation by mechanical loading, where adipogenesis is blocked in favor of osteogenesis.13, 24, 25 Of interest is our data on osteoclast maturation and activation. In contrast to what was reported by Glass and colleagues,20 where it was shown that Wnt signaling promoted the ability of differentiated osteoblasts to inhibit osteoclast differentiation, we have shown a transient increase in the number of TRACP+ osteoclasts, most likely owing to an increase in the number of hematopoietic progenitors, from which osteoclasts are derived. Our findings are consistent with hematopoietic stem and progenitor cells described previously to increase in the presence of BIO, an inhibitor of GSK-3.5
Of interest is the multilayer of osteoblasts observed in some areas at the endocortical surface in mice treated with AR28. Although it is difficult to completely exclude endosteal fibrosis, similar to that seen in humans following stimulation with parathyroid hormone,26 this is more likely to reflect a feature of osteoblast formation in young mice. It is not unusual to see areas, at the endocortical surface, with multilayer of osteoblasts in untreated mice at a young age, although this is usually limited to two or three layers. Moreover, a parallel study with AR28 in a model of myeloma bone disease carried out in mice of older age showed an increase in osteoblast numbers, but they were not disposed in multilayers.27
In conclusion, our data are compatible with inhibition of GSK-3 acting on proliferation and commitment of MSCs with osteo- and adipogenic potential, which is driven to osteogenic differentiation at the expense of adipogenic differentiation. It also highlights the complex network of responses taking place with time and occurring in different cellular types at specific stages of commitment. It draws attention to the powerful effect that amplifying stem and progenitor cells in vivo may have on bone mass. Future work is required to exclude the possibility of a reduction in stem cell numbers following prolonged treatment. This may require a more detailed study on the type of stem/progenitors affected and whether those are the more primitive one defined by their ability to engraft following transplantation assay. Moreover, it will be important to determine the best dose and schedule of administration and to dissect the molecular events mediating the feedback mechanism to guarantee a prolonged and sustained effect of this GSK-3 inhibitor as a bone anabolic treatment.