The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood. (Blood. 2011;117(8):2476-2483) IntroductionHemopoietic stem cells reside in bone marrow-specialized niches that dictate how they differentiate, proliferate, mature, and enter the peripheral circulation. [1][2][3][4] Megakaryocyte (MK) maturation and platelet generation are consequent to MK migration from the osteoblastic to the vascular niche, where MKs extend pro-platelets and newly generated platelets are released into the bloodstream. 5,6 The characteristics of the microenvironment surrounding MKs play an important role in the regulation of platelet production within the bone marrow. 3,7 In particular, the interaction of MKs with different extracellular matrices (ECMs) that fill the bone marrow spaces seems to orchestrate their maturation in specific sites. 8 It has been demonstrated that interactions of primary human MKs with matrices thought to fill the vascular niche, such as fibrinogen or von Willebrand factor, are able to sustain MK maturation and pro-platelets, whereas type I collagen totally suppresses these events and prevents premature platelet release in the osteoblastic niche. 7,9 The negative regulation of pro-platelets by type I collagen is mediated by the interaction with the integrin ␣2␤1 and involves the Rho/ROCK pathway. 10,11 However, the exact sequence of events that determines the interaction of MKs with the ECM, and therefore their regulation, is not completely understood. 12 Recent studies 13 have demonstrated that the encounter between a cell and an adhesive substrate involves an initial passive interaction characterized by cell adhesion and spreading, followed by an active stage that involves actin polymerization and...

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It is common knowledge that mouse embryonic stem cell (mESC) lines accumulate chromosomal changes during culture. Despite the wide use of mESCs as a model of early mammalian development and cell differentiation, there is a lack of systematic studies aimed at characterizing their karyological changes during culture. We cultured an mESC line, derived in our laboratory, for a period of 3 months investigating its chromosome complement at different times. About 60% of the metaphases analysed were euploid throughout the culture period but, from passage 13, only 50% of the euploid metaphases had a proper chromosome complement. The remaining 50% showed chromosome abnormalities, mainly gain or loss of entire chromosomes, both within the same passage and among different passages analysed. The very heterogeneous spectrum of abnormalities indicates a high frequency of chromosome mutations that arise continuously during culture. The heterogeneity of the aberrant chromosome constitution of 2n = 40 metaphases, observed at different passages of culture, might be due either to their elimination or to a shift towards the hypoeu- or hypereuploid population of those metaphases that accumulate further chromosome abnormalities. The stability of the frequency of eu-, hypoeu- and hypereuploid populations during culture might, however, be due to the elimination of those cells that carry a high mutational burden. Based on our results, we suggest that karyotype analysis of the euploid cell population of mESC lines is necessary when such lines are used in the production of chimeric mice, for their contribution to the germ line, or when they are differentiated into specific cell types.

We studied the role of adenosine diphosphate and P2Y receptors on proplatelet formation by human megakaryocytes in culture. ResultsMegakaryocytes expressed all the known eight subtypes of P2Y receptors, and constitutively released adenosine diphosphate. Proplatelet formation was inhibited by the adenosine diphosphate scavengers apyrase and CP/CPK by 60-70% and by the P2Y 12 inhibitors cangrelor and 2-MeSAMP by 50-60%, but was not inhibited by the P2Y 1 inhibitor MRS 2179. However, the active metabolites of the anti-P2Y 12 drugs, clopidogrel and prasugrel, did not inhibit proplatelet formation. Since cangrelor and 2-MeSAMP also interact with P2Y 13 , we hypothesized that P2Y 13 , rather than P2Y 12 is involved in adenosine diphosphate-regulated proplatelet formation. The specific P2Y 13 inhibitor MRS 2211 inhibited proplatelet formation in a concentrationdependent manner. Megakaryocytes from a patient with severe congenital P2Y 12 deficiency showed normal proplatelet formation, which was inhibited by apyrase, cangrelor or MRS 2211 by 50-60%. The platelet count of patients with congenital delta-storage pool deficiency, who lack secretable adenosine diphosphate, was significantly lower than that of patients with other platelet function disorders, confirming the important role of secretable adenosine diphosphate in platelet formation. ConclusionsThis is the first demonstration that adenosine diphosphate released by megakaryocytes regulates their function by interacting with P2Y 13 . The clinical relevance of this not previously described physiological role of adenosine diphosphate and P2Y 13 requires further exploration.

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