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The physical culture parameters have important influences on the proliferation and differentiation fate of hematopoietic stem cells. Recently, we have demonstrated that CD34cord blood (CB) cells undergo accelerated and increased megakaryocyte (Mk) differentiation when incubated under mild hyperthermic conditions (i.e., 39°C). In this study, we investigated in detail the impacts of mild hyperthermia on Mk differentiation and maturation, and explored potential mechanisms responsible for these phenomena. Our results demonstrate that the qualitative and quantitative effects on Mk differentiation at 39°C appear rapidly within 7 days, and that early transient culture at 39°C led to even greater Mk yields (p< 0.03). Surprisingly, cell viability was only found to be significantly reduced in the early stages of culture, suggesting that CB cells are able with time to acclimatize themselves to 39°C. Although mild hyperthermia accelerated differentiation and maturation of CB-derived Mks, it failed to promote their polyploidization further but rather led to a small reduction in the proportion of polyploid Mks (p 0.01). Conversely, gene arrays analysis demonstrated that Mks derived at 39°C have a normal gene expression program consistent with an advanced maturation state. Finally, two independent mechanisms that could account for the accelerated Mk differentiation were investigated. Our results suggest that the accelerated and increased Mk differentiation induced by mild hyperthermia is not mediated by cell-secreted factors but could perhaps be mediated by the increased expression of Mk transcription factors. [ABSTRACT FROM AUTHOR]