Proteomics Analysis Between Bone Marrow Mesenchymal Stem Cells And Fibroblasts

Background:Bone Marrow Stem Cells(BMSCs) were considered as the ideal seed cell source for creating tissue engineered heart valves (TEHV). In comparison to blood vessel derived interstitial seed cells,the application of MSC has several advantages of showing characteristics of multipotent progenitor cells which are able to differentiate into a variety of mesenchymal cell types, allowing persistence in an allogeneic setting owing to their unique immunological characteristics, and avoiding the sacrificing of intact cardiovascular structures in comparison to blood vessel derived interstitial cells by easy collection and isolation methods. The interstitial cells play important roles in maintaining the normal structure integrity and functions of the heart valves, which are composed of fibroblasts, myofibroblasts and smooth muscle cells. Fibroblasts are the more important interstitial cell phenotypes of the native heart valves. Recent advances have revealed that TEHV created with BMSCs showed a similar distribution of fibroblasts with native adult heart valves after remolding in vivo. Unfortunately, owing to failing to be aware of the molecular mechanism of fibroblasts differentiation from BMSCs till now, the BMSCs constructed TEHV need to be remolded in vivo.Though a few genes were confirmed to be differently expressed in BMSCs and fibroblasts, HBMSCs resemble to terminally differentiated fibroblasts in cell morphology, cell phenotype and cell surface antigens.Proteome, the entire protein complement of the genome, could provide detailed descriptions of the cell structure and function.AIM:To find out the differences and similarities on the protein expressions between human bone marrow mesenchymal stem cells (hBMSCs) and human fibroblasts, and to further study the two different cells'structure and function.METHODS:1. We adopt the ways of density gradient centrifugation and adherence screening to isolate, purify hBMSCs from the human bone marrow. After culturing and amplifying the cells in DMEM/F12, we observe the cells'growth and morphology through inverted microscope and detect the cell surface antigens CD29, CD34 and CD45. At the last time, we differentiated hBMSCs into bone cells and fat cells. Fibroblasts were human fibroblast cell line cells (purchased from Cell Applications Company).2. The protein extracts were obtained from hBMSCs and fibroblasts, and separated by two-dimensional gel electrophoresis (2-DE). And then the differential protein spots were identified by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The results from simultaneous proteomic profiling were further validated by Western Blot of selected proteins and immunohistochemistry.RESULTS:1. Primary cells grow in the way of adherence and cluster. After passage, most of the cells look like fusiform, few look like polygon, and they grow in the mode of fish of school and radition. hBMSCs exhibited positive expression of CD29, in the absence of CD45, CD34 expression. hBMSCs'multipotential ability, Vacossa and oil red O staining were performed after specific inducing culture for osteogenesis and adipogenesis respectively. As a result, the obtained cells were certainly human bone marrow stem cells.2. Protein extracts from the two samples were separated by 2-DE gel electrophoresis and the gels were visualized by silver staining. Three independent replications were performed to each protein samples to ensure reproducibility of the protein homogenates on the 2-DE gels. Two representative 2-DE maps from BMSCs and fibroblasts have similar distribution character. In the pH range 3–10, 2-DE maps of BMSCs displayed about 782±65 protein spots; tha map of fibroblasts displayed 760±57 spots For MS analysis, spots of interest were excised from the each gel and subsequently digested by trypsin. Among these spots, 29 selected protein spots were successfully identified with high confidence with in-gel trypsin digestion followed by MALDI-TOF MS and subsequent bio-information data mining. Among them, 17 proteins were expressed significantly differently in hBMSCs from that in fibroblasts, and another 12 proteins equally expressed in the 2 types of cells.To further confirm our proteomics results, we performed Western blot analysis and immunocytochemical staining with few selected proteins. In concordance with the proteomics results, Western blot analysis immunocytochemical staining further confirm that these proteins are indeed expressed in two types of cells, as quantified and quantitated by comparative proteomics method. CONCLUSION:We first find the differential protein expressions between hBMSCs and fibroblasts by proteomics, which provides a new insight into the differential cell structures and functions between the 2 types of cells. This might be valuable for further study of fibroblasts differentiation from hBMSCs and the application of hBMSCs for creating tissue engineered heart valves in the future.