Key Points Endothelial cells secrete exosomes containing miR-214, which suppress senescence and stimulates an angiogenic program in target cells. Exosomal miR-214 regulates ataxia telangiectasia mutated expression in recipient endothelial cells.

Regeneration of the myocardium by transplantation of cardiomyocytes is an emerging therapeutic strategy. Human embryonic stem cells (HESC) form cardiomyocytes readily but until recently at low efficiency, so that preclinical studies on transplantation in animals are only just beginning. Here, we show the results of the first long-term (12 weeks) analysis of the fate of HESC-derived cardiomyocytes transplanted intramyocardially into healthy, immunocompromised (NOD-SCID) mice and in NOD-SCID mice that had undergone myocardial infarction (MI). Transplantation of mixed populations of differentiated HESC containing 20-25% cardiomyocytes in control mice resulted in rapid formation of grafts in which the cardiomyocytes became organized and matured over time and the noncardiomyocyte population was lost. Grafts also formed in mice that had undergone MI. Four weeks after transplantation and MI, this resulted in significant improvement in cardiac function measured by magnetic resonance imaging. However, at 12 weeks, this was not sustained despite graft survival. This suggested that graft size was still limiting despite maturation and organization of the transplanted cells. More generally, the results argued for requiring a minimum of 3 months follow-up in studies claiming to observe improved cardiac function, independent of whether HESC or other (adult) cell types are used for transplantation.

Accumulating evidence indicates that programmed necrosis plays a critical role in cell death during ischemia-reperfusion. Necrostatin-1 (Nec-1), a small molecule capable of inhibiting a key regulator of programmed necrosis (RIP1), was shown to prevent necrotic cell death in experimental models including cardiac ischemia. However, no functional follow-up was performed and the action of Nec-1 remains unclear. Here, we studied whether Nec-1 inhibits RIP1-dependent necrosis and leads to long-term improvements after ischemia-reperfusion in vivo. Mice underwent 30 min of ischemia and received, 5 min before reperfusion, 3.3 mg/kg Nec-1 or vehicle treatment, followed by reperfusion. Nec-1 administration reduced infarct size to 26.3 ± 1.3% (P = 0.001) compared to 38.6 ± 1.7% in vehicle-treated animals. Furthermore, Nec-1 inhibited RIP1/RIP3 phosphorylation in vivo and significantly reduced necrotic cell death, while apoptotic cell death remained constant. By using MRI, cardiac dimensions and function were assessed before and 28 days after surgery. Nec-1-treated mice displayed less adverse remodeling (end-diastolic volume 63.5 ± 2.8 vs. 74.9 ± 2.8 μl, P = 0.031) and preserved cardiac performance (ejection fraction 45.81 ± 2.05 vs. 36.03 ± 2.37%, P = 0.016). Nec-1 treatment significantly reduced inflammatory influx, tumor necrosis factor-α mRNA levels and oxidative stress levels. Interestingly, this was accompanied by significant changes in the expression signature of oxidative stress genes. Administration of Nec-1 at the onset of reperfusion inhibits RIP1-dependent necrosis in vivo, leading to infarct size reduction and preservation of cardiac function. The cardioprotective effect of Nec-1 highlights the importance of necrotic cell death in the ischemic heart, thereby opening a new direction for therapy in patients with myocardial infarction.

The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2-4 wk at 32 degrees C or 37 degrees C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37 degrees C than at 32 degrees C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.