Equine marrow-derived mesenchymal stem cells: isolation, differentiation and culture optimization

Most studies regarding the marrow-derived equine mesenchymal stem cells [MSCs] have mainly focused on the cell transplantation without considering the capacity of differentiation and in vitro requirements of the cells. These concerns were investigated in the present study. Equine MSCs were isolated from the sternal marrow aspirates and expanded through two successive subcultures. Passage-2 equine MSC cultures were then treated with appropriate supplements in order to examine the cell osteogenic, chondrogenic and adipogenic differentiation potential. Furthermore, the culture of the cells was investigated in terms of the optimal concentration of fetal bovine serum [FBS] and the initial cell-seeding density. Additionally, a growth curve was plotted for the cells to study their growth characteristics. According to our findings, equine MSCs were easily generated specialized bone, cartilage and adipose cell lineages as confirmed by specific staining and RT-PCR analysis. Moreover, the cells exhibited rapid expansion when being cultivated in the medium with 15% FBS at 100 cells/cm[2]. Growth curves indicated that these cells rapidly entered the log phase after a brief lag [adaptation] period. In summary, marrow-derived equine MSCs possess tripotent differentiation capacity and rapid growth rate in the appropriate culture conditions

[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

[ comparative study between the structure of cartilage tissue produced from murine MSC differentiation and hyaline costal cartilage]

Vitro cartilage differentiation of mesenchymal stem cells [MSCs] has been noticed in several investigations. In this regard, almost always molecular differentiation of the cells has been examined, while structural and morphological differentiation of them has been ignored. Therefore, the present study examines the structure and ultrastructure of the cartilage differentiated from murine MSCs compared with that of costal cartilage. 2x 10[5] MSCs isolated from the bone marrow of NMRI mice were pleted by centrifugation and cultured for 21 days in chondrogenic medium. At the end of cultivation period, occurrence of chondrogenic differentiation was confirmed by reverse transcriptase-polymerase chain reaction [RT-PCR] analysis for some cartilage-specific genes. To compare the structure of differentiated tissue with that of natural cartilage, the cartilage was differentiated from MSCs and the cartilage obtained from the same murine rib was prepared for transmission electron microscopy [TEM]. Structural studies indicated that similar to the costal cartilage, the cartilage produced from differentiation of perichondrium-like layer was formed. According to the microscopic images, in contrast to costal chondrocytes, the differentiated cells had euchromatic nucleus and their cytoplasm contained plenty of the organelles involved in active cell secretion. Furthermore, intercellular matrix in differentiated cartilage had a fibrillar appearance. Our results indicated that the structure of cartilage produced in micro mass culture system is somewhat different from that of costal cartilage. The cells from differentiated tissue seemed to be more active than those from costal cartilage