BackgroundHuman mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer.MethodsHuman MSC from bone marrow of healthy donors, mesenchymal cell lines (HS-5 and hTERT) and a leukemic cell line (K562 cells) were used to obtain EV for FCM characterization. EV released from the different cell lines were isolated by ultracentrifugation and were characterized, using a multi-parametric analysis, in a conventional flow cytometer. EV characterization by transmission electron microscopy (TEM), western blot (WB) and Nano-particle tracking analysis (NTA) was also performed.ResultsEV membranes are constituted by the combination of specific cell surface molecules depending on their cell of origin, together with specific proteins like tetraspanins (e.g. CD63). We have characterized by FCM the EV released from BM-hMSC, that were defined as particles less than 0.9 μm, positive for the hMSC markers (CD90, CD44 and CD73) and negative for CD34 and CD45 (hematopoietic markers). In addition, hMSC-derived EV were also positive for CD63 and CD81, the two characteristic markers of EV. To validate our characterization strategy, EV from mesenchymal cell lines (hTERT/HS-5) were also studied, using the leukemia cell line (K562) as a negative control. EV released from mesenchymal cell lines displayed the same immunophenotypic profile as the EV from primary BM-hMSC, while the EV derived from K562 cells did not show hMSC markers. We further validated the panel using EV from hMSC transduced with GFP.Finally, EV derived from the different sources (hMSC, hTERT/HS-5 and K562) were also characterized by WB, TEM and NTA, demonstrating the expression by WB of the exosomal markers CD63 and CD81, as well as CD73 in those from MSC origin. EV morphology and size/concentration was confirmed by TEM and NTA, respectively.ConclusionWe described a strategy that allows the identification and characterization by flow cytometry of hMSC-derived EV that can be routinely used in most laboratories with a standard flow cytometry facility.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0124-8) contains supplementary material, which is available to authorized users.

Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34 þ cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22 phoxdependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.

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PTPN13 is a high-molecular weight intracellular phosphatase with several isoforms that exhibits a highly modular structure. Although in recent years different roles have been described for PTPN13, we are still far from understanding its function in cell biology. Here we show that PTPN13 expression is activated during megakaryocytic differentiation at the protein and mRNA level. Our results show that the upregulation of PTPN13 inhibits megakaryocytic differentiation, while PTPN13 silencing triggers differentiation. The ability of PTPN13 to alter megakaryocytic differentiation can be explained by its capacity to regulate ERK and STAT signalling. Interestingly, the silencing of β-catenin produced the same effect as PTPN13 downregulation. We demonstrate that both proteins coimmunoprecipitate and colocalise. Moreover, we provide evidence showing that PTPN13 can regulate β-catenin phosphorylation, stability and transcriptional activity. Therefore, the ability of PTPN13 to control megakaryocytic differentiation must be intimately linked to the regulation of β-catenin function. Moreover, our results show for the first time that PTPN13 is stabilised upon Wnt signalling, which makes PTPN13 an important player in canonical Wnt signalling. Our results show that PTPN13 behaves as an important regulator of megakaryocytic differentiation in cell lines and also in murine haematopoietic progenitors. This importance can be explained by the ability of PTPN13 to regulate cellular signalling, and especially through the regulation of β-catenin stability and function. Our results hold true for different megakaryocytic cell lines and also for haematopoietic progenitors, suggesting that these two proteins may play a relevant role during in vivo megakaryopoiesis.

Purpose: Cancer cells show higher levels of reactive oxygen species (ROS) than normal cells and increasing intracellular ROS levels are becoming a recognized strategy against tumor cells. Thus, diminishing ROS levels could be also detrimental to cancer cells. We surmise that avoiding ROS generation would be a better option than quenching ROS with antioxidants. Chronic myelogenous leukemia (CML) is triggered by the expression of BCR-ABL kinase, whose activity leads to increased ROS production, partly through NADPH oxidases. Here, we assessed NADPH oxidases as therapeutic targets in CML.Experimental Design: We have analyzed the effect of different NADPH oxidase inhibitors, either alone or in combination with BCR-ABL inhibitors, in CML cells and in two different animal models for CML.Results: NADPH oxidase inhibition dramatically impaired the proliferation and viability of BCR-ABLexpressing cells due to the attenuation of BCR-ABL signaling and a pronounced cell-cycle arrest. Moreover, the combination of NADPH oxidase inhibitors with BCR-ABL inhibitors was highly synergistic. Two different animal models underscore the effectiveness of NADPH oxidase inhibitors and their combination with BCR-ABL inhibitors for CML targeting in vivo.Conclusion: Our results offer further therapeutic opportunities for CML, by targeting NADPH oxidases. In the future, it would be worthwhile conducting further experiments to ascertain the feasibility of translating such therapies to clinical practice. Clin Cancer Res; 20(15); 4014-25. Ó2014 AACR.

SummaryThe regulation of hematopoietic stem cells (HSCs) depends on the integration of the multiple signals received from the bone marrow niche. We show the relevance of the protein tyrosine phosphatase PTPN13 and β-catenin as intracellular signaling molecules to control HSCs adhesiveness, cell cycling, and quiescence. Lethally irradiated mice transplanted with Lin– bone marrow cells in which PTPN13 or β-catenin had been silenced showed a significant increase of long-term (LT) and short-term (ST) HSCs. A decrease in cycling cells was also found, together with an increase in quiescence. The decreased expression of PTPN13 or β-catenin was linked to the upregulation of several genes coding for integrins and several cadherins, explaining the higher cell adhesiveness. Our data are consistent with the notion that the levels of PTPN13 and β-catenin must be strictly regulated by extracellular signaling to regulate HSC attachment to the niche and the balance between proliferation and quiescence.

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Background The differentiation-based therapy for acute promyelocytic leukemia (APL) is an inspiring example for the search of novel strategies aimed at treatment of other subtypes of acute myeloid leukemia (AML). Thus, the discovery of new molecular players in cell differentiation becomes a paramount research area to achieve this goal. Here, the involvement of the protein tyrosine phosphatases SHP1 and SHP2 on leukemic cells differentiation is shown, along with the therapeutic possibilities of their targeting to enhance the differentiation induction effect of phorbol esters. Methods The oxidation status and enzymatic activity of SHP1 and SHP2 during PMA-induced differentiation of HEL cells was evaluated. Additionally, the effects of RNAi-mediated downregulation of these phosphatases on cell differentiation was studied. Afterwards, the impact of chemical inhibition of SHP1 and SHP2 on differentiation both in the presence and absence of phorbol esters was tested. Finally, the anti-leukemic potential of phorbol esters and chemical inhibitors of SHP1 and SHP2 was addressed in several AML model cell lines, a xenograft mouse model and AML primary cells in vitro . Results An increase of oxidation with a concomitant decrease of activity was observed for both phosphatases at the onset of PMA-induced differentiation. Consistently, silencing of these proteins favored the process, with an enhanced effect upon their simultaneous downregulation. Moreover, the proteins SRC and β-catenin were identified as downstream targets of SHP1 and SHP2 in this context. In agreement with these findings, chemical inhibition of the phosphatases promoted cell differentiation itself and enhanced the effect of phorbol esters. Interestingly, treatment with the phorbol ester prostratin and the dual inhibitor of SHP1 and SHP2 NSC87877 synergistically hampered the proliferation of AML cell lines, prolonged the survival of xenografted mice and reduced the clonogenic potential of AML primary cells. Conclusions SHP1 and SHP2 are relevant mediators of differentiation in AML cells and their inhibition either alone or in combination with prostratin seems a promising differentiation-based therapeutic strategy against different subtypes of AML beyond APL. Electronic supplementary material The online version of this article (10.1186/s13046-019-1097-z) contains supplementary material, which is available to authorized users.