GSK-3β Inhibition Activates WNT, Delays Division and Preserves the Function in Hematopoietic Stem Cell

Wingless (Wnt) signaling appears to play an integral role in expansion and quiescence of stem and progenitor hematopoietic cell populations. To better understand this dichotomous role and aid in the translation of therapeutic goals including cellular therapies, greater understanding of the molecular mechanisms that regulate Wnt/β-catenin in the bone marrow niche are needed. We explored the impact of Wnt signalling in the context of the human HSC niche by using co-culture of human cord blood or bone marrow derived HSC with mesenchymal stem cells (MSCs) together with pharmacological inhibition of GSK-3β (iGSK-3β). We have previously shown that activation of the Wnt/β-catenin pathway using pharmacological inhibition of GSK-3β (iGSK-3β) in human cord blood derived HSCs acts to preserve their primitive status, often lost during the ex-vivo expansion process (1). Here we show using cell division tracking that iGSK-3β delays stem cell divisions induced by cytokines in co-culture with MSCs. Functional analysis of iGSK-3β-treated cells revealed an increased proportion of primitive cells producing mixed Colony Forming Units (CFU-GEMMs) formed in the methylcellulose cultures and Cobblestone Area Forming Cells identified in long-term co-cultures with the bone marrow stroma MS5 cells. In addition, iGSK-3β-treated cells exhibited a higher frequency of SCID Repopulating Cells. We also show that the iGSK-3β effect, at least in part, is mediated through VLA4 integrin engagement by fibronectin. Importantly, iGSK-3β also induced niche-independent delay in stem cell divisions seen in cytokine supported suspension cultures. Up-regulation of HoxB4 and p21Waf1 and down-regulation of cyclin D1 was observed in HSCs treated with iGSK-3β. These findings confirm that iGSK-3β– regulated Wnt/β-catenin activity is crucial for the maintenance of HSC quiescence, both in the MSC niche and in a niche-independent environment and that iGSK-3β can circumvent the requirement of exogenous Wnt ligand for the induction of Wnt/β-catenin signalling in MSC niche. Our results are consistent recent data showing that blocking of canonical Wnt signaling, by over-expression of dickkopf1 (Dkk1) increases the number of proliferating HSCs in the murine marrow microenvironment but reduces the ability to reconstitute the hematopoietic system of irradiated recipient transgenic mice (2). These findings contribute to a better understanding of the mechanisms regulating the balance between self renewal and quiescence and help to facilitate progress towards clinical application of Wnts as mediators of ex-vivo stem cell expansion or interruption of the Wnt pathway as potential anti-leukemic therapeutic strategy.