Abstract
Several reports have implicated canonical Wnt/β-catenin pathway in murine and human hematopoietic stem/progenitor cells (HSC/HPCs) ex vivo expansion. In addition, it was demonstrated augmentation of hematopoietic repopulating ability in vivo by post transplantation treatment with an ATP-competitive GSK-3 inhibitor, which leads to activation of intrinsic β-catenin. Conversely, it is also reported that constitutive activation of β-catenin enforced cell cycle entry of murine HSCs, thereby, exhausting the long-term repopulating cell pool and leading to hematopoietic failure associated with loss of multilineage differentiation. In this way, the precise roles of individual molecules concerning in canonical Wnt/β-catenin pathway for normal hematopoiesis have not been elucidated. In this study, we examined the effects of GSK-3 inhibition on stem-cell maintenance, progenitor cell expansion, and lineage decisions of murine and human HSC/HPCs. At first, the expression and localization of β-catenin in human CD34+ HSC/HPCs treated with GSK-3 inhibitor 9 (6-bromoindirubin-3-oxime) (GI9) was observed with confocal microscopy. After the treatment for 24 hrs, expression of β-catenin in vehicle-treated (negative control; NC) cells was scarcely detected except for the membrane-bounded form. On the other hand, in GI9-treated cells, β-catenin accumulated in their nucleus in a dose dependent manner. These results suggested that GI9-treatment activates intrinsic β-catenin in human HSC/HPCs. Next, CD34+ HSC/HPCs were cultured for 7 days in a serum-free medium containing with cytokines (SCF, FL, TPO, IL-6 and sIL-6R) and also with 2μM, 10μM of GI9 or vehicle. After 7 days culture, total viable cells and CD34+ cells were expanded 31.6±4.6 and 17.9±3.8 fold in NC cells, respectively (n=3). However, GI9-treatment could not maintain a proportion of CD34+ cells compared with NC significantly caused the growth inhibition in a dose dependent manner. From the analysis of cumulative distribution of first cell division among the cells treated with GI9 or vehicle, GI9-treatment caused delayed cell cycling especially in fractionated immature CD34+CD38− cells. In addition, GSK-3 inhibition lost SCID repopulating cells (SRCs) as tested in the NOD/SCID mouse model (SRCs was calculated to be 1 in 8,452 NC cells vs. in 45,503 GI9-treated cells using limiting dilution methods). These results suggested that activation of intrinsic β-catenin followed GSK-3 inhibition suppressed self-renewal of immature hematopoietic cells via modulating its cell cycle kinetics. Next, as for the multipotency of HSC/HPCs after the culture, the distribution pattern of immunophenotype and the colony forming ability were evaluated. About 80% of expanded cells expressed myeloid marker, CD33 in our culture system, however, GI9-treatment perturbed myeloid differentiation of CD34+ HSC/HPCs but induced the differentiation toward to megakaryocyte and erythroid lineages. Furthermore, in methylcellulose assay, although expanded cells with GI9-treatment generated all types of progenitors, GI9-treatment was inferior significantly in terms of expansion rate of myeloid progenitor, CFU-GM and superior in formation of erythroid progenitor, BFU/CFU-E compared with NC (No. of CFU-GM/1000 cells 151±65.8 vs. 284±17.0, No. of BFU/CFU-E/1000 cells 132±18.5 vs. 32.7.±7.0, respectively) (p<0.05, n=3). Similarly, in murine model, GI9-treatment tended to convert differentiation potential of common myeloid progenitor (CMP) from granulocyte and macrophage progenitor (GMP) to megakaryocyte and erythroid progenitor (MEP). As for this mechanism, we found that activated β-catenin suppresses the transcriptional activity of C/EBPα, which is essential transcription factor for granulocyte development, while it promotes the function of GATA1, essential transcription factor for megakaryocyte and erythrocyte development during the differentiation of HSC/HPCs. In addition, β-catenin competitively impeded the interaction between C/EBPα and its transcriptional coactivator, CBP/p300 in coimmunoprecipitaion analysis. Together, these results indicated that intrinsic β-catenin was supposed to play an important role in self-renewal and multipotency of HSC/HPCs and control the balance of lineage commitment of HSC/HPCs for normal hematopoiesis, presumably by regulating the interaction with essential transcription factors.
Dose-dependent roles for canonical Wnt signalling in de novo crypt formation and cell cycle properties of the colonic epithelium