Comparison of proliferation, senescence and differentiation into skeletal cell lineages of murine bone marrow-derived and amniotic fluid mesenchymal stem cells

While mesenchymal stem cells [MSCs] have been Isolated from multiple tissue sources, the differences existed between the cells from different tissues have still remained to be clarified. This study compares MSCs from murine amniotic fluid [AF] with those from bone marrow [BM] tissues. AF and BM cells were collected from 10 NMRI mice at second weeks of their pregnancy and the culture was expanded. The isolated MSCs were then compared in terms of in vitro differentiation capacity, expansion rate and the percentages of senescence-associated beta-galactosidase [SA- beta-gal] positive-cells in their cultures. Either cell appeared to be able to differentiate into bone, cartilage and adipose cell lineage. AF-cells were observed to be more proliferative than BM-cells. The population doubling time [PDT] of AF-cells was 92.6 +/- 13.9 hours compared to 168 +/- 40 hours that was recorded for BM-cells. The percentage of SA- beta-gal positive-cells in AF-cell culture appeared to be significantly lower than that in BM-cell culture. Collectively, it seems that murine AF housed MSCs with a relatively higher proliferation property than BM-derived MSCs and a typical tripotent differentiation potential comparable with marrow MSCs, hence it would be as an appropriate source of MSCs for use in regenerative medicine related studies

[Study of differentiation potential of the dedifferentiated chondrocytes from rat articular cartilage into skeletal cell lineages]

Dedifferentiation of the chondrocyte from rat articular cartilage with multiple subcultures and study of the redifferentiation potential of the cells into bone, cartilage and fat cell lineages. In this experimental study, chondrocytes from rat articular cartilage were isolated and expanded through several successive subcultures during which the expression levels of cartilage-specific genes including aggreacan and type II collagen were measured by using real-time PCR to determine the cell dedifferentiation [the time in which cartilage genes ceased their expression]. Furthermore, during the culture period, the chondrocyte was examined morphologically by scanning electron microscopy [SEM]. At the end, the dedifferentiated cells were subjected to osteogenic, adipogenic and chondrogenic culture condition to investigate whether or not they are able to redifferentaite into specialized progenies. Differentiation state was examined by specific staining and RT-PCR analysis. Based on the findings by real time PCR, the expression levels of the both studied genes were high at passage 2 and dramatically decreased at passage 4. Aggreacan expression ceased at passage 10 and collagen II stopped expressing at passage 6. SEM images indicated the flattened morphology of the cells at early passages and the fibroblastic appearance at late passages. Differentiation examination revealed that the dedifferentiated cells were readily differentiated into bone, adipose and cartilage cell lineages. Considering all aspects together, this concluded that articular chondrocyte gradually lost their differentiated state during the long-term culture and changed into multipotent cells capable of differentiating into skeletal cell lineages

Subcutaneous transplantation of marrow-derived murine mesenchymal stem cells cultivated in alginate and their chondrogenesis

Mesenchymal stem cells are appropriate candidates to treat diseases including articular cartilage defects. There are plenty of researches being conducted to make the application of these cells possible. The purpose of this study was to cultivate murine mesenchymal stem cells [MSCs] in alginate gel and transplant them subcutaneously to immuno-suppressed rats to examine their chondrogenic potential in vivo. 4-6 week old NMRI mice were sacrificed and their bone marrow cells were cultivated in 6-cell plates at the density of 500 cell/cm[2]. The pure fibroblastic cells appeared after two passages. 2X10[6] flbroblastic cells were mixed with 1 ml of alginate solution and converted into gel by being exposed to calcium chloride solution. MSCs-embedded alginate gel were then transplanted subcutaneously to 6 rats that had received an immunosuppressive drug [cyclosporine] for transplant rejection to be avoided. 5 weeks after transplantation, the alginate gels were removed and evaluated by histochemistry, RT-PCR for certain cartilage markers, and transmission electron microscopy. 5 weeks after transplantation, the skin was incised and the alginate gel with its surrounding vascular connective tissue were removed. Tuloidine blue staining indicates that the cells within the gel assumed oval morphology and occupied lacuna-like cavities. RT-PCR analysis revealed that in these cells the mRNA of some cartilage markers such as collagen II [the marker of hyaline cartilage], collagen X [hypertrophied chondrocyte marker in osteogenesis], and aggreacan were largely produced. Ultra-thin sections analysis showed that the cells within the lacuna-like cavity of alginate gel contain a large amount of expanded rough endoplasmic reticulum and secret fibrillar extra cellular matrix. Transplanted murine MSCs cultivated in alginate gel can differentiate into hyaline cartilage with the sign of osteogenic initiation