Human 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. Human 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. EV 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. We 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.

Oxidative stress is related to ageing and degenerative diseases, including cancer. However, a moderate amount of reactive oxygen species (ROS) is required for the regulation of cellular signalling and gene expression. A low level of ROS is important for maintaining quiescence and the differentiation potential of haematopoietic stem cells (HSCs), whereas the level of ROS increases during haematopoietic differentiation; thus, suggesting the importance of redox signalling in haematopoiesis. Here, we will analyse the importance of ROS for haematopoiesis and include evidence showing that cells from leukaemia patients live under oxidative stress. The potential sources of ROS will be described. Finally, the level of oxidative stress in leukaemic cells can also be harnessed for therapeutic purposes. In this regard, the reliance of front-line anti-leukaemia chemotherapeutics on increased levels of ROS for their mechanism of action, as well as the active search for novel compounds that modulate the redox state of leukaemic cells, will be analysed.

Regulation of transcription requires mechanisms to both activate and terminate transcription factor activity. GATA‐1 is a key haemopoietic transcription factor whose activity is increased by acetylation. We show here that acetylated GATA‐1 is targeted for degradation via the ubiquitin/proteasome pathway. Acetylation positively signals ubiquitination, suggesting that activation by acetylation simultaneously marks GATA‐1 for degradation. Promoter‐specific MAPK phosphorylation then cooperates with acetylation to execute protein loss. The requirement for both modifications is novel and suggests a way by which degradation of the active protein can be specifically regulated in response to external phosphorylation‐mediated signalling. As many transcription factors are activated by acetylation, we suggest that this might be a general mechanism to control transcription factor activity.