| Part I. Comparison of CM-DIL and DAPI labeled the bone marrow derived mesenchymal stem cellsBACKGROUND:Facilitated by the ease of isolation and immunologic privilege, bone marrow-derived mesenchymal stem cells (BMSCs) are emerging as a promising therapeutic agent for cell therapy and tissue engineering. Studies have shown that BMSCs transplantation is a promising therapy for ischemic diseases. However, the destiny of implanted cells in the donor body is one of important issues. Fluorescence staining is used to trace cells via fluorescent chemical compound that can re-emit light upon light excitation bonding to a macromolecule of cells. Because of the ease of its technology, fluorescence staining plays an important role in stem cell transplantation. As fluorescent dyes, CM-DIL and DAPI are commonly used for labeling cells. To our knowledge, there are few reports on comparing the two fluorescent dyes.OBJECTIVE:To compare the efficiency, duration, and visualization of BMSCs labeled by CM-DIL or DAPI in vitro and in vivo.METHODS:Isolation and expansion of BMSCs were performed according to attachment culture. Cell viability was assessed via Trypan blue dye exclusion assay. Growth curves of BMSCs were depicted using MTS assay. The reduction of fluorescent intensity was observed under the inverted fluorescent contrast phase microscope from passage1to passage3after CM-DIL and DAPI labeling BMSCs respectively. Myocardial infarction was induced by left anterior artery ligation in SD rats. One weeks later, BMSCs labeled by CM-DIL or DAPI were injected randomly into the border area of infarct myocardium. After3days, transplanted cell distribution was examined under fluorescent microscope through paraffin sections and frozen sections respectively.RESULTS:In vitro, both BMSCs labeled by CM-DIL and by DAPI decreased cell proliferation during the early period; the percentage of fluorescent-positive cells was approximately100%in two groups; however, fluorescent intensity was significantly reduced from passage1to passage3in BMSCs labeled by DAPI. In vivo, the transplanted BMSCs were detected in a concentrated way both on the paraffin sections and frozen ones; the background colour of frozen sections was lower in CM-DIL group than in DAPI group; false positive results of fluorescent expression can be eliminated in CM-DIL group by using fluorescent mounting medium with the fluorescence of DNA staining.CONCLUSION:Our data indicates that CM-DIL is more appropriate to label cells for visualizing and distinguishing them than DAPI. Part II. Combination of hyaluronic acid and bone marrow-derived mesenchymal stem cells promotes myocardial repair after infarctionBACKGROUND:Bone marrow-derived mesenchymal stem cells (BMSCs) are emerging as an extremely promising therapeutic agent for myocardial infarction. The poor retention and survival of donor cells implanted into the myocardium limit the efficacy of cell therapy for myocardial infarction. Embedding cells in natural or synthetic biomaterials is a strategy to address this issue. Hyaluronic acid (HA), a non-sulfated glycosaminoglycan and a major component of the ECM, is typically chemically modified to make an injectable, in situ polymerizable hydrogel. Due to its biodegradability and high biocompatibility, HA hydrogel is attracting interests as a biomaterial for tissue engineering.OBJECTIVES:Therefore, in the present study, we planned to evaluate:(i) the retention and survival rate of BMSCs delivered in HA hydrogel in infarcted hearts;(ii) the effects of BMSCs encapsulated in HA hydrogel on cardiac function after myocardial infarction;(iii) the mechanism of BMSCs and HA hydrogel in promoting damaged heart repair.METHODS:A biodegradable and in situ polymerizable hydrogel based on hyaluronic acid (HA) was used as the carrier for bone marrow-derived mesenchymal stem cells (BMSC) obtained from male rats. Infarcted hearts of female rats (LAD ligation) were randomized to injection of PBS, HA, BMSCs, or HA+BMSC complex. The survival rate and apoptosis of implanted cells and the expression of MMP2, VEGF, T β4and c-Kit were examined at post-injection day1, week1and week2by RT-PCR, TUNEL staining and western blot, respectively. At post-injection week4, changes of cardiac microstructure and function were evaluated by histological examination and echocardiography.RESULTS:HA hydrogel significantly enhanced the cell retention and survival rate at post injection day1(HA+BMSC vs BMSC:55.72±27.49%vs7.66±5.6%; p=0.041) and week2(HA+BMSC vs BMSC:0.27±0.07%vs0.08±0.06%; p=0.025). The expression of VEGF and MMP2in the infarct zone treated with BMSCs alone were significantly up-regulated at week1. At week2, HA+BMSC group has a higher expression of VEGF and MMP2than HA group; the levels of T β4and c-Kit in hearts injected with HA+BMSC complex or either component alone were increased. Histological analysis revealed that thicker infarct wall in HA group, smaller cardiomyocyte size and higher vessel density in BMSC group, the largest benefits in combined group as indicated by reversing cardiac remodeling, angiogenesis, and more viable myocardium tissue in the infarct zone. At post-injection week4, left ventricular ejection fraction (PBS:47.51%±9.03%; HA:51.26%±8.32%; BMSC:49.3%±10.09%; HA+BMSC:60.26%±8.27%) further reflected that the HA+BMSCs combined treatment yielded the best recoveries of cardiac function.CONCLUSION:HA hydrogel can act as BMSCs delivery vehicles and add to the beneficial effects of implanted BMSCs in repairing damaged heart via enhancing cell retention and survival, and prolonging paracrine effects of engrafted cells. In addition, HA hydrogel alone supplies injured heart with mechanical support and endogenous stem cells recruitment. It should be further explored to use the HA hydrogel and BMSCs for treating the myocardium infarction. |
PDF Full Text Request