ABSTRACT
BACKGROUND: Bone marrow mesenchymal stem cells have been induced into islet-like cell mass in vitro. However, little researches reported on the morphological changes of cells, the types of endocrine cells in the islet-like cell mass and their relationships. OBJECTIVE: To investigate the morphological changes of cells in the differentiation of bone marrow mesenchymal stem cells into islet-like structure and to explore the composition and distribution of endocrine cells. METHODS: Passage 3 bone marrow mesenchymal stem cells growing well were cultured and expanded until the cell colonies occupies 80% of the bottom of the culture bottle, and the pancreatic tissue lysate was added for continuous induction. Dithizone staining was used to screen the islet-like cell mass directly differentiated from bone marrow mesenchymal stem cells. The types and distribution of endocrine cells were identified by Mallory staining. Expressions of insulin, C-peptide, glucagon and somatostatin protein were detected by immunofluorescence cytochemical staining. RESULTS AND CONCLUSION: (1) Dithizone staining showed that the number of positive cells was increased over the induction time. (2) Mallory staining showed the red α-like cells were located in the periphery of the islet-like cell mass, the yellow β-like cells located in the center and periphery, and the light blue fibroblasts were distributed around the cell mass. (3) Immunofluorescence staining showed insulin, C-peptide, glucagon and somatostatin positive cells in the islet-like cell mass. To conclude, under certain microenvironment, bone marrow mesenchymal stem cells can differentiate into islet-like structures which containing α, β, δ-like cells, and are surrounded by fibroblast-like cells.
ABSTRACT
BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) have been induced to differentiate into cardiomyocyte-like cells in vitro.OBJECTIVE:To explore the association between GATA-4, Nkx-2.5 and α-myosin heavy chain (α-MHC) expression and cell morphological changes and structure formation in the process of BMSCs differentiation into cardiomyocyte-like cells.METHODS:By using myocardial lysate, BMSCs were induced to differentiate into cardiomyocytes.Immunocytochemistry staining was used to detect cardiac troponin T (cTnT) and connexin43, for the identification of cardiomyocytes. In the process of directional differentiation, RT-PCR was used to detect the expression of GATA-4,Nkx2.5 and α-MHC.RESULTS AND CONCLUSION:During the directional differentiation of BMSCs, the cells were changed from long fusiform to short rod, forming protrusions that were interconnected to form mesh-like, bamboo-like or myotube-like structure. When the cells were interconnected like a bamboo, cTNT and connexin43 positive cells were visible, and then the number of positive cells increased with the presence of myotube-like structure. RT-PCR results showed that during the induced directional differentiation of BMSCs, GATA-4, Nkx2.5 and α-MHC mRNA levels increased continuously. When interconnected cells formed a mesh-like structure, GATA-4 expression reached the peak and then kept a high level. When adjacent cells were fused into a myotube-like structure, α-MHC reached the peak. Additionally, the expression of Nkx2.5presented a time-dependent increase trend. Overall, during the induced differentiation of BMSCs into cardiomyocyte-like cells, the expression of cardiomyocyte specific genes, characterized by temporality and spatiality, is related to the changes of cell morphology and special structure formation.