Comparative Evaluation of Mouse Bone Marrow Mesenchymal Stromal Cells Characteristics Cultured in Two Different Supplemented Media

@#Introduction: Preclinical studies on mesenchymal stromal cells (MSC) have allowed the cells to be considered as a promising candidate for cellular therapy. In recent years, conflicting data have been reported regarding various aspects of their characteristics, development and differentiation potential, which may be due to arrange of factors. Among the factors worth investigating is the culture medium formulation. Methods: Here we have made a comparative characterization of mouse bone marrow mesenchymal stromal cells (mBM-MSC) that were cultured using two common supplements, MesenCult™ Stimulatory Supplement (MSS) and fetal bovine serum (FBS), under the same experimental conditions at different passages. Clonogenic potential, cumulative population doubling level (CPDL), population doubling time (PDT), immunophenotyping, differentiation, immunosuppression potentials and chromosome analysis of early and late passages mBM-MSC were assessed. Results: Our findings showed that the CPDL, immunophenotype and immunosuppression potential of mBM-MSC were similar. However, variations were seen in their clonogenicity, population doubling time and differentiation efficacy whereby all of these were enhanced in DMSS. These observations suggest that their genetic make-up may be affected by both supplements upon prolonged culture. Interestingly, this conjecture was supported when chromosomal analysis revealed genetic instability of mBM-MSCs cultured in both supplements. Conclusion: In conclusion, culture medium formulation was shown to cause variations and spontaneous transformation in mBM-MSCs raising concerns on the usage of late passages mBMMSCs in fundamental and preclinical downstream experiments.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND: Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMEC US) or Swiss Holstein-Friesian (bMEC CH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA. RESULTS: The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS: The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Isolation and characterization of canine umbilical cord blood-derived mesenchymal stem cells

Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III beta tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.

Comprehensive characterization of rat pulmonary microvascular endothelial cells cultured by tissue-sticking method / 第三军医大学学报

Objective To establish a comprehensive characterization protocol of rat pulmonary microvascular endothelial cells(RPMVECs) cultured by tissue-sticking method.Methods RPMVECss were cultured with peripheral lung tissue-sticking method.Histological sections from peripheral lung tissue pieces used for cell culture were examined.With rat pulmonary artery smooth muscle cells and human umbilical vein endothelial cells as controls,the cultured cells were identified by immunocytochemical staining using CD34 antibody,lectin from Bandeiraea simplicifolia and factor Ⅷ related antigen.The cell morphology and ultrastructure were observed with inverted light microscope and transmission electron microscope respectively.Results Histological sections showed that tissue pieces were scissored from peripheral lung lobes accurately.The cultured cells had characterization of binding lectin from Bandeiraea simplicifolia and positive immunocytochemical staining with CD34 antibody,but negative for factor Ⅷ related antigen.Weibel-Palade bodies were not found in cells.Conclusion Factor Ⅷ related antigen and Weibel-Palade body are not ideal indexes for RPMVECs identification.The combination of peripheral lung tissue section,CD34 immunocytochemical staining and lectin from Bandeiraea simplicifolia binding assay provides a simple and reasonable comprehensive characterization protocol for RPMVECs.