ß-Catenin-TCF/LEF signaling promotes steady-state and emergency granulopoiesis via G-CSF receptor upregulation.

The canonical Wnt signaling pathway is mediated by interaction of ß-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of ß-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates ß-catenin-TCF/LEF interaction. Disruption of the ß-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of ß-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of ß-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the ß-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the ß-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.

Magnetic resonance tracking of human CD34+ progenitor cells separated by means of immunomagnetic selection and transplanted into injured rat brain.

Magnetic resonance imaging (MRI) provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied whether superparamagnetic nanoparticles (CD34 microbeads), used clinically for specific magnetic sorting, can be used as a magnetic cell label for in vivo cell visualization. Human cells from peripheral blood were selected by CliniMACS CD34 Selection Technology (Miltenyi). Purified CD34+ cells were implanted into rats with a cortical photochemical lesion, contralaterally to the lesion. Twenty-four hours after grafting, the implanted cells were detected in the contralateral hemisphere as a hypointense spot on T2 weighted images; the hypointensity of the implant decreased during the first week. At the lesion site we observed a hypointensive signal 10 days after grafting that persisted for the next 3 weeks, until the end of the experiment. Prussian blue and anti-human nuclei staining confirmed the presence of magnetically labeled human cells in the corpus callosum and in the lesion 4 weeks after grafting. CD34+ cells were also found in the subventricular zone (SVZ). Human DNA (a human-specific 850 base pair fragment of alpha-satellite DNA from human chromosome 17) was detected in brain tissue sections from the lesion using PCR, confirming the presence of human cells. Our results show that CD34 microbeads superparamagnetic nanoparticles can be used as a magnetic cell label for in vivo cell visualization. The fact that microbeads coated with different commercially available antibodies can bind to specific cell types opens extensive possibilities for cell tracking in vivo.