Role of the osteoblastic niche in megakaryocyte differentiation and proplatelet formation.

<p>(A) HSCs were plated in direct co-culture with hMSCs, or hOSTs or without any feeder layers (control). Adherent cells were counted as described in methods and normalized to relative control suspension cultures. (B) At day 12 of a direct co-culture cells were stained and the percentage of CD61-positive cells was determined upon conventional fluorescence microscopy analysis. Mks were assigned to distinct stages of differentiation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008359#s4" target="_blank">Materials and Methods</a>. (C) Representative picture of CD61⁺ cells at different stage of maturation. (D) Proplatelet-bearing Mks were then evaluated by fluorescence microscopy, after staining with anti-alpha tubulin. Results are expressed as percentage of adherent megakaryocytes forming proplatelets and normalized to relative controls at different oxygen tensions. Data are the mean ± SD of the results obtained in three different experiments for each sample. Statistical analysis by one-way ANOVA followed by Bonferroni's <i>t-</i>test was performed for unpaired observations between hMSCs and hOSTs values. Student <i>t-</i>test was performed for paired observations to relative control. *p<0.05.