Glycogen Synthase Kinase-3β Regulates Ex-Vivo Expansion and Engraftment of Umbilical Cord Blood Hematopoietic Stem Cells through the Activation of Wnt Signaling.

Rapid availability of an HLA matched marrow donor for allogeneic hematopoietic stem cell transplantation remains a challenge in the clinical setting and has resulted in increased use of alternate stem cell sources, particularly unrelated umbilical cord blood (UCB). Although UCB transplantation has been used to treat a multiplicity of malignant and non-malignant hematological diseases, success has been limited particularly in adult recipients, because of reduced numbers of stem/progenitor cells in a cord unit resulting in higher graft failure rates, delayed hematopoietic reconstitution and increased transplant related mortality when compared to BM or PBSCs. This has prompted investigation of alternate strategies including multi-unit infusions and ex-vivo expansion systems. Clinical trials incorporating cytokine-based HSC ex-vivo expansion have failed to demonstrate significant shortening of hematologic recovery despite substantial increases in cell dose, suggesting loss of HSC function. Activation of Wnt signaling has previously been shown to maintain HSC function; as such we hypothesized that use of a specific inhibitor of glycogen synthase kinase (GSK-3β) to activate Wnt signaling in combination with cytokine (Flt3L, SCF, TPO) based ex-vivo HSC expansion would result in expansion, maintenance of HSC function and ultimately improve hematological recovery. UCB CD34+ cells were treated with a potent GSK-3β inhibitor; 6-bromoindirubin-3′-oxime (BIO), a synthetic cell-permeable derivative of a natural product, indirubin-3′-oxime, isolated from the molusk Tyrian purple. UCB CD 34+ cells treated with BIO exhibited increased expression of β-catenin that re-located from the cytoplasm to the nuclei to activate transcription of Wnt targets including cyclin D1, c-myc, and HoxB4. When compared to control cells (cytokine only); BIO treated cells demonstrated an increase in the absolute number of nucleated, CD34+ and clonogenic cell numbers in 5–7 day expansion cultures with the best expansion observed at 0.1 μM of BIO. Increased cell cycling, reduced apoptosis and up-regulation of the cell cycle inhibitor p21^Waf1 previously shown to maintain HSC function in-vivo was demonstrated in BIO treated cells. Furthermore, BIO significantly improved the hematopoietic supporting ability of murine bone marrow stroma MS5 cells. Increased expansion of myeloid, megakaryocytic and B-cell progenitor cells were all observed. Using a NOD/SCID mouse model we have demonstrated both preservation of function and expansion with increased numbers of engrafting BIO treated human CD34+ UCB (120-fold expansion) compared to control cells (45-fold expansion). The increased expansion of human NOD/SCID repopulating cells suggests improved maintenance of stem cell function during ex-vivo expansion of UCB in the presence of BIO. Activation of the Wnt pathway, through GSK-3β inhibition, is a promising strategy to facilitate expansion and maintain function of UCB HSCs and may overcome current limitations of UCB and allow for greater application of this stem cell source.