Mesenchymal stromal cells (MSCs) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 h to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 h post irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% dimethyl sulfoxide at −80 °C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) possess pro-regenerative potential in different animal models with renal injury. EVs contain different molecules, including proteins, lipids and nucleic acids. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. The aim of this study was to increase the content of specific miRNAs in EVs that are known to be involved in the pro-regenerative effect of EVs, and to assess the capacity of modified EVs to contribute to renal regeneration in in vivo models with acute kidney injuries. To this purpose, MSCs were transiently transfected with specific miRNA mimics by electroporation. Molecular analyses showed that, after transfection, MSCs and derived EVs were efficiently enriched in the selected miRNAs. In vitro and in vivo experiments indicated that EVs engineered with miRNAs maintained their pro-regenerative effects. Of relevance, engineered EVs were more effective than EVs derived from naïve MSCs when used at suboptimal doses. This suggests the potential use of a low amount of EVs (82.5 × 10⁶) to obtain the renal regenerative effect.

We have previously shown that injury induced by irradiation to murine marrow can be partially or completely reversed by exposure to human or murine mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). Investigation of the biodistribution of EVs in vivo is essential for understanding EV biology. In this study, we evaluated the DiD lipid dye labeled MSC-EV biodistribution in mice under different conditions, including different MSC-EV doses and injection schedules, time post MSC-EV injection, and doses of radiation. DiD-labeled MSC-EVs appeared highest in the liver and spleen; lower in bone marrow of the tibia, femur, and spine; and were undetectable in the heart, kidney and lung, while a predominant EV accumulation was detected in the lung of mice infused with human lung fibroblast cell derived EVs. There was significantly increased MSC-EV accumulation in the spleen and bone marrow (tibia and femur) post radiation appearing with an increase of MSC-EV uptake by CD11b+ and F4/80+ cells, but not by B220 cells, compared to those organs from non-irradiated mice. We further demonstrated that increasing levels of irradiation caused a selective increase in vesicle homing to marrow. This accumulation of MSC-EVs at the site of injured bone marrow could be detected as early as 1 h after MSC- EV injection and was not significantly different between 2 and 24 h post MSC-EV injection. Our study indicates that irradiation damage to hematopoietic tissue in the spleen and marrow targets MSC-EVs to these tissues.

Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. Previously, we found that transcription factor RUNX1-dependent haematopoietic transformation of endothelial progenitor cells may contribute to the pathogenesis of PAH. However, the therapeutic potential of RUNX1 inhibition to reverse established PAH remains unknown. In the current study, we aimed to determine whether RUNX1 inhibition was sufficient to reverse Sugen/hypoxia (SuHx)-induced pulmonary hypertension (PH) in rats. We also aimed to demonstrate possible mechanisms involved. We administered a small molecule specific RUNX1 inhibitor Ro5-3335 before, during, and after the development of SuHx-PH in rats to investigate its therapeutic potential. We quantified lung macrophage recruitment and activation in vivo and in vitro in the presence or absence of the RUNX1 inhibitor. We generated conditional VE-cadherin-CreERT2; ZsGreen mice for labelling adult endothelium and lineage tracing in the SuHx-PH model. We also generated conditional Cdh5-CreERT2; Runx1(flox/flox) mice to delete Runx1 gene in adult endothelium and LysM-Cre; Runx1(flox/flox) mice to delete Runx1 gene in cells of myeloid lineage, and then subjected these mice to SuHx-PH induction. RUNX1 inhibition in vivo effectively prevented the development, blocked the progression, and reversed established SuHx-induced PH in rats. RUNX1 inhibition significantly dampened lung macrophage recruitment and activation. Furthermore, lineage tracing with the inducible VE-cadherin-CreERT2; ZsGreen mice demonstrated that a RUNX1-dependent endothelial to haematopoietic transformation occurred during the development of SuHx-PH. Finally, tissue-specific deletion of Runx1 gene either in adult endothelium or in cells of myeloid lineage prevented the mice from developing SuHx-PH, suggesting that RUNX1 is required for the development of PH. By blocking RUNX1-dependent endothelial to haematopoietic transformation and pulmonary macrophage recruitment and activation, targeting RUNX1 may be as a novel treatment modality for pulmonary arterial hypertension.

Current dogma is that there exists a hematopoietic pluripotent stem cell, resident in the marrow, which is quiescent, but with tremendous proliferative and differentiative potential. Furthermore, the hematopoietic system is essentially hierarchical with progressive differentiation from the pluripotent stem cells to different classes of hematopoietic cells. However, results summarized here indicate that the marrow pluripotent hematopoietic stem cell is actively cycling and thus continually changing phenotype. As it progresses through cell cycle differentiation potential changes as illustrated by sequential changes in surface expression of B220 and GR-1 epitopes. Further data indicated that the potential of purified hematopoietic stem cells extends to multiple other non-hematopoietic cells. It appears that marrow stem cells will give rise to epithelial pulmonary cells at certain points in cell cycle. Thus, it appears that the marrow “hematopoietic” stem cell is also a stem cell for other non-hematopoietic tissues. These observations give rise to the concept of a universal stem cell. The marrow stem cell is not limited to hematopoiesis and its differentiation potential continually changes as it transits cell cycle. Thus, there is a universal stem cell in the marrow which alters its differentiation potential as it progresses through cell cycle. This potential is expressed when it resides in tissues compatible with its differentiation potential, at a particular point in cell cycle transit, or when it interacts with vesicles from that tissue.