| Objectives:Very small embryonic-like stem cells (VSELs), among sca-1(+) lin(-) CD45(-) cells in mice and CD133(+) CXCR4(+) CD34(+) Lin(-) CD45(-) cells in human, are a novel population of adult stem cells with the ability to differentiate into all three germ-layer lineages, thus could potentially be a promising therapeutic option for myocardial regeneration after acute myocardial infarctioa However, VSELs are scarce in adult tissues and could not satisfy transplantation therapy as well as in vitro studies. Therefore, we propose a new methods to enrich bone marrow drived VSELs without their biological changes.Methods:Murine bone marrow was harvested from young (4-6 weeks old) male C57Black/6 mice by flushing the cavities of femurs and tibias. In group A, the whole bone marrow cells were resuspended in normal culture medium and the non-adherent cells were removed by gently changing the culture medium at 24 h and every 48h thereafter. At about 6-7 days after culture, clone-formed cells were detached and counted. In group B, the whole bone marrow cells were mixed with NH4C1 based BD Pharm Lyse lysing buffer for 15 min at room temperature for hypotonic removal of ery throcytes and the mo no nuclear cells were got.Cells from group A and group B were resuspended in washing buffer. The following primary antibodies were added simultaneously for staining:phycoerythrin (PE)-conjugated monoclonal rat anti-mouse Sca-1, FITC-conjugated monoclonal rat anti-mouse CD45, and APC-conjugated monoclonal rat anti-mouse lineage markers (anti-CD45R/B220, anti-Gr-1, anti-TCR?Chain anti-CDllb and anti-Ter119). After staining, sca-1(+)/lineage(-)/CD45(-) VSELs were sorted by fluorescence-activated cell sorting. For transmission electron microscopy, the sorted VSELs were fixed, embedded, sectioned, stained and subsequently viewed on a Nihon electron microscope. The Oct-4, Nanog, Gata4, Nkx2.5 and Mef-2c mRNA levels were performed by Real time-PCR using an ABI 7300 Sequence Detection SystemResults:In group A, the short-time culture procedure could effectively enrich the sca-l(+) subgroup (89.6±1.99%), lineage(-) subgroup (51.1±3.6%) and CD45(-) subgroup (22.9±1.84%), while in group B, bone marrow mononuclear cells only contains very small amount of this three subgroups (16.53±0.54%,34.7±3.0%and 0.91±0.11%, respectively). As a results, the sca-l(+)/lineage(-)/CD45(-) mouse VSELs were significantly enriched to 9.4±0.62%in group A as compared to 0.04±0.01%in group B (P<0.001). Transmission electronic microscope studies indicated that the ultrastructure of VSELs was same in group A and group B, demonstrated that VSELs could retain their typical characteristic like embryonic stem cells after short-time culture. Real time-PCR studies indicated that the mRNA level of Oct-4, Nanog, Gata4 and Nkx2.5 were very low in group A but nearly no expression in group B.Conclusions:The short-time culture procedure could significantly enrich bone marrow-derived VSELs as compare to sort them directly from bone marrow mononuclear cells. Objectives:Mesenchymal stem cells (MSCs) are the optimal candidate for stem cell therapy in acute myocardial infarction (AMI); however, widespread death of implanted cells hampers its development. The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway participates in regulation of survival of various cells. Although statins are known to have pleiotropic effects, it is unclear if statins could protect MSCs. This study aimed to detect whether rosuvastatin could protect MSCs against hypoxia and serum-deprivation (H/SD) conditions in vitro and their influence on the JAK-STAT signaling pathway.Methods:Rat bone marrow derived-MSCs were exposed to H/SD conditions for 6h without (control) or with different concentrations of rosuvastatin (0.001?M,0.01?M, O.l?M, l?M and lO?M). For inhibitory studies, MSCs were incubated with an JAK2 inhibitor AG490 (1?M) before the addition of rosuvastatin (1?M). Cell apoptosis was assessed using Annexin V-FITC/PI staining. The expression and phosphorylation level of JAK2, STAT3 and eNOS proteins were evaluated by Western blot. Target signals were normalized to the P-actin signal and analyzed semiquantitatively with Quantity One systemResults:We found that rosuvastatin (O.Ol?M-lO?M) remarkably reduced apoptosis of rat bone marrow-derived MSCs under H/SD conditions. This effect was confirmed by decreased expression of bax, a pro-apoptosis protein; whereas the bcl-2, an anti-apoptosis protein, increased in rosuvastatin treated groups. Meanwhile, rosuvastatin (0.01-10?M) treatment enhanced phosphorylation of JAK2 and STAT3; and rosuvastatin (0.001 and 1O?M) treatment enhanced phosphorylation of eNOS in MSCs. The anti-apoptotic effect of rosuvastatin, as well as the phosphorylation of JAK2, STAT3 and eNOS, was reversed by 1?M AG490 treatment.Conclusions:Rosuvastatin protects MSCs from H/SD-induced apoptosis via activating JAK2-STAT3-eNOS signaling pathway, which suggesting an useful therapeutic methods and underlying mechanism for MSCs transplantion into damaged heart. Objectives:Widespread death of implanted cells hampers stem cell therapy for acute myocardial infarction (AMI). We have shown previously that a short-time administration of statins improved functional activity of transplanted mesenchymal stem cells (MSCs) after AMI by creating a better environment. However, the exact mechnism ofstatins remains unknown.Methods:Female Sprague-Dawley rats were randomized into five groups:AMI (control), rosuvastatin gavage (group R), MSCs transplantation (group M), MSCs and rosuvastatin (group M+R), or MSCs, rosuvastatin and a JAK2 inhibitor AG-490 (group M+R+AG). AMI was created by ligating the left anterior descending coronary artery; MSCs from male rats were injected into the myocardium one week after AMI. Cardiac function and histology as well as expression of Y-chromosomal genes and JAK-STAT signaling proteins were examined at four weeks after transplantationResults:In baseline echocardiography revealed no significant difference between treated and control groups. However, at four weeks after MSCs transplantation, left ventricular end-diastolic diameters (LVEDd) and end-systolic diameters (LVESd) in group M+R (p<0.00l) were significantly different from the control group. But there was no difference between groups R, M and M+R+AG as compared with control. Measurements of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) showed significant improvement in group M (p<0.05) and group M+R (p<0.001) as compared with control The improvement was more striking in group M+R as compared with group M (p<0.001).The LV fibrotic area was reduced markedly in group M+R (8.4%±1.8%) as compared with control group (18.3%±2.8%, p<0.001), while no remarkable changes were observed in group R (17.7%±2.1%, p>0.05), group M (16.1%±2.0%, p>0.05) and group M+R+AG (16.5%±1.9%, p>0.05). The percent of fibrotic area in group M+R also significantly decreased as compared with group M (p<0.001) and group M+R+AG (p<0.001).Although RT-PCR analysis of SRY-specific genes showed that most of donor cells can't survive at 4 weeks after cell transplantation significantly increased expression of SRY genes occurred in group M+R (8.90±0.74%, p<0.001. respectively) as compared with group M (2.3 l%±0.20%) and group M+R+AG (3.89%±0.33%)Capillary density in peri-infarct regions was significantly increased in group M+R (21.72±1.40; p<0.005 vs. control, R and M; p<0.001 vs. M+R+AG) as compared with control (8.54±0.61), group R (13.90±1.01), group M (9.28±0.80) and group M+R+AG (9.57±0.82). Similarly, the density in infarct regions was also significantly increased in group M+R (19.40±1.12; p<0.005 vs. control, R and M; p<0.001 vs. M+R+AG) compared with control (5.20±0.51), group R (11.13±0.9), group M (7.22±0.63) and group M+R+AG (6.20±0.45). Immunofluorescent analyses showed that DAPI-labeled cells expressed the cardiac-specific protein c-Tnl. The efficiency of DAPI-labeled cell differentiation into cardiac myocytes was higher in group M+R than in group M and group M+R+AG.The phosphorylation ratios of JAK2, STAT1 and STAT3 were significantly augmented in group R and group M+R as compared with control (p<0.001, respectively), while no difference in group M (p>0.05) as compared with control. Treatment with AG-490 resulted in reduced phosphorylation of JAK2 (p-JAK2), STAT1 (p-STATl) and STAT3 (p-STAT3) as indicated in group M+R+AG when compared with group M+R (p<0.001, respectively).Conclusions:Rosuvastatin treatment improves efficacy of stem cell transplantation in infarct hearts by activation of the JAK2-STAT3 signaling pathway. |
PDF Full Text Request