GSK-3β Inhibition Promotes Engraftment of Ex Vivo-Expanded Hematopoietic Stem Cells and Modulates Gene Expression

Abstract

Glycogen synthase kinase-3b (GSK-3b) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK-3b, 6-bromoindirubin 3 0 -oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non-obese diabetic/ severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo-expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine-induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK-3b provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation.

Identification of in vitro HSC fate regulators by differential lipid raft clustering

“…52 Consistent with this, FOXO3a, mTOR and GSK3β, downstream targets of the PI3K/Akt pathway, have been shown to be crucial modulators of HSC metabolism and quiescence. 19,[53][54][55] Intriguingly, niche signals such as TGFβ, Wnts or OPN could act on HSC fate by blocking this activation process, which would manifest itself by LRC inhibition. 17,23,24 Our experiments also show that other factors affecting LRC had a direct effect on c-Kit (and Sca-1) distribution in the membrane.…”

GSK3β regulates physiological migration of stem/progenitor cells via cytoskeletal rearrangement

“…34,58), and consequently reduced their numbers in the PB. This is supported by the observation that both treatment with FGF-2 or IGF-1 and direct GSK3β inhibition trigger HSPC proliferation in vitro (6,59,60). Obviously, many signaling molecules are essential for HSPC motility and development, however, the functional linkage between HSC retention (i.e., restriction of motility) and self-renewal could be an important foundation for future studies.…”

“…In a steady state, low levels of HSPCs are constantly circulating as part of homeostasis, whereas in stress situations, HSPCs are recruited in high numbers from the bone marrow (BM) into the circulation as part of host defense and repair mechanisms (2). Glycogen synthase kinase-3β (GSK3β) is a well-established negative regulator of the Wnt/β-catenin pathway, implicated in self-renewal and development of human and murine HSCs (3)(4)(5)(6)(7)(8)(9)(10)(11). In addition to proliferation, GSK3β has been shown to be involved in the motility of various cells, including microglia, epidermal stem cells, human mast cells, and breast cancer cells (12)(13)(14)(15)(16), but its effect on the motility of immature hematopoietic cells has not been addressed.…”

Ex vivo expansion of hematopoietic stem cells

“…It was also reported that ex vivo Wnt5a-treated young LT-HSCs decreased HSC repopulation ability [126]. Pretreatment with a GSK-3 inhibitor, which activates the canonical Wnt downstream effector -catenin, promotes engraftment of ex vivo-expanded human HSCs in xenografted mice [127,128].…”