Effect of mild hyperthermia on Mk differentiation

Physical factors can have major influences on the proliferation and differentiation fate of hematopoietic stem cells in culture. Recently, we demonstrated that cord blood CD34+ cells undergo accelerated and increased megakaryocyte differentiation when incubated at 39°C. In this study, we investigated in detail the impacts of mild hyperthermia on the kinetics of megakaryocyte differentiation, maturation, polyploidization and cell viability. The qualitative and quantitative effects on Mk differentiation were found to be rapidly induced, and optimization of the culture length at 39°C led to greater Mk yields. Mild hyperthermia did not promote endomitosis of cord blood-derived Mk, but rather led to a small reduction in the proportion of polyploid Mk. Moreover, it had little impact on viability but induced a significant shift in the proportion of cells undergoing apoptosis at 37°C to necrosis at 39°C. Finally, our results suggest that the effects on megakaryocyte differentiation could be the consequences of aberrant expression of key megakaryocyte transcription factors induced by mild hyperthermia. Keywords: Effect of mild hyperthermia on Mk differentiation To define potential differences between megakaryocytes (Mk) derived at 37°C or 39°C, we compared their gene expression repertoire by micro-gene chip technology (Affymetrix GeneChip HG133A). Two independent experiments were carried out on Mk at 37 or 39°C. CD41a+ Mk issued from CB CD34+ cells were cultured for 7-days with TPO at 100 ng/ml and purified by positive magnetic selection using a CD41a-FITC monoclonal antibody (Immunotech, Marseille, France) and MACS columns according to manufacturer’s instructions (Miltenyi, Auburn, CA, USA) with a purity of >95%.