Mesenchymal Stem Cells Stimulated by Growth Factors Release Exosomes with Potent Proangiogenic Activity / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the proangiogenic activity of exsomes released by human umbilical cord mesenchymal stem cells (MSCs) stimulated by erythropoietin and platelet-derived growth factor BB (PDGF-BB).</p><p><b>METHODS</b>Human umbilical cord-derived MSCs were seeded and maintained in culture overnight. The media were then replaced by alpha-MEM containing EPO (1 U/ml) and/or PDGF-BB (50 ng/ml), and the culture was maintained for 72 hours. The exosomes from the culture supernatants were isolated with a routine ultra-catrifagation method. Flow cytometric analysis was performed to identify the origin of the exosomes, and their morphological features were observed by using a transmission electron microscopy. The exosomes were added at a concentration of 10 µg/ml into the culture system of human umbilical cord vein endothelial cells. MTT assay was used to evaluate the proliferative status. The Matrigel assay was used to observe the formation of net-work structures which were calculated after culture for 12 hours.</p><p><b>RESULTS</b>Flow cytometric analysis showed that microparticles released by human umbilical cord MSCs expressed CD9, CD63 and CD81, which was in accordance with the surface molecular features of exosomes. Under an electron microscope, the exosomes took the featured cystic shape. The protein contents of exosomes released by untreated, EPO-stimulated, PDGF-BB-stimulated and EPO plus PDGF-BB stimulated MSCs (10 cells) were 256±124 µg, 1021±392 µg, 830±265 µg and 2207±733 µg, respectively. The results revealed that MSCs treated by EPO and PDGF-BB released significantly higher amounts of exosomes (P<0.01). MTT assay proved that the exosomes from EPO and PDGF-BB treated MSCs had more potent proliferation-promoting activity on human umbilical cord vein endothelial cells than those from untreated MSCs. The Matrigel assay showed that the numbers of capillary-like structures in untreated, EPO-, PDGF-BB and EPO plus PDGF-BB-treated groups were 2.6±0.84, 4.6±1.57, 4.2±0.78 and 6.3±1.34 per high power objective. Treatment with EPO or PDGF-BB dramatically enhanced the numbers of capillary liue structure, compared with that of untreated group (P<0.01) and those in EPO and PDGF-BB combination group was significantly greater than those of EPO or PDGF-BB group (P<0.01).</p><p><b>CONCLUSION</b>EPO and PDGF-BB can stimulate MSCs to release exosomes with more potent proangiogenic activity.</p>

Mesenchymal stem cells release membrane microparticles in the process of apoptosis / 中国实验血液学杂志

Though mesenchymal stem cells (MSC) have been clinically used to repair a variety of damaged tissues, the underlying mechanisms remain elusively as the majority of the ex vivo expanded MSC die shortly after transplantation. To explore the mechanism in which the death cells play tissue repair effect, apoptosis of rat bone marrow MSC was induced by culturing cells in the conditions of hypoxia or/and serum-free medium, and the subcellular structures in the supernatants were analyzed. The results showed that apoptosis occurred in the presence of either hypoxia or serum-free condition as well, and the apoptotic proportion reached up to (17.44 ± 2.15) after the cells were treated by hypoxia plus serum free culture for 72 hours. The flow cytometric analysis of the sub-cellular substances harvested by ultracentrifugation of the supernatants found that the MSC released substantial amount of membrane microparticles into the supernatants, which expressed CD29, CD44A and Annexin-V-binding phosphatidylserine. It is concluded that the MSC can release membrane microparticles after induction, the amount of these membrane microparticles was around 15-fold of the parent cell numbers. The membrane microparticles is the mediators in the cross-talk between the transplanted cells and their surrounding tissues. This study provides some novel information for the mechanisms of MSC therapy.

Mesenchymal stem cells exist in the compact bones from four species of mammals / 中国实验血液学杂志

The biological properties of cultured mesenchymal stem cells (MSC) have been intensively investigated, while there is still a paucity of information about the definite in vivo sites that harbor these stem cells due to the lack of specific surface markers. Previous data have demonstrated that human and murine MSC can be isolated from the compact bones. To investigate if it is the case for other species, the femurs from Wistar rats, Beagles, C57 mice and New Zealand rabbits were collected, minced and digested with collagenase type I. The digested bone fragments were seeded into the medium for human bone marrow culture after removal of the suspended cells in the digestion. The results showed that the fibroblast-like cells were observed to migrate from the bone fragments after several days of culture, and they gradually formed an adherent confluent layer. The adherent cells could be passaged and expressed homogenously the mesenchymal cell marker vimentin. Differentiation assays showed that these cells had the capacity to differentiate into osteoblasts and adipocytes. In conclusion, the results here provide new information for the further investigations on the in vivo biological features of MSC in the context of the simplicity of the compact bone structure.

In vivo tracing of transplanted bone marrow mesenchymal stem cells with bioluminescence imaging / 中国实验血液学杂志

Mesenchymal stem cell (MSC)-based cell therapy has shifted into clinical trials to repair the damage of various tissues. In this setting, the survival of the transplanted cells contributes critically to the therapeutic effectiveness. To investigate the in vivo tracing of MSCs, a recombinant retroviral vector carrying firefly-luciferase reporter gene [pL (FLUC) SN] was constructed and several GPE+86 cell clones that stably expressed fluc were selected. The retroviral supernatants were collected and used to transfect MSC derived from C57 mice. The cells were then screened with G418 and the expression of the exogenous gene was identified by luciferase enzyme activity analysis. Labeled mouse MSCs (2x10(6)) were injected into skeletal muscles, and the in situ expression was noninvasively tracked by in vivo bioluminescence imaging for 1, 3 and 6 days after transplantation. The results showed that the survival rates of the grafted cells dropped sharply with time, they were 57.2+/-11.7%, 8.6+/-2.5% and 5.4+/-3.1% on day 1, 3 and 6 after transplantation, and no fluorescent signals above background were detected on day 10. It is concluded that the method described above could be used for in vivo tracing of grafted cells. Furthermore, MSCs could not survive even transplanted into the none-ischemic skeletal muscles.