Bone marrow derived mesenchymal stem cells (MSCs) can be used to repair articular cartilage defects, these cells should be properly stimulated so that they could differentiate morphologically and hold cellular synthetic features closer to maturely differentiated chondrocytes. It is well known that tissue specific environment plays an important role in cell fate determination. Once improved isolation, proliferation and differentiation protocols have been developed, the likelihood of spontaneous differentiation of MSCs into divergent lineages will be reduced, thus increasing their value for cartilage repair. The purpose of this study was to improve chondrogenic differentiation of equine MSCs using coculture with mature equine articular chondrocytes (ACs), along with the determination of the effect of adding transforming growth factor (TGF) beta1 in the pellet culture system. Following confirmation of multilineage (adipogenic, osteogenic and chondrogenic) differentiation, isolated MSCs, ACs and coculture of both cell types were transferred into pellet culture system in a DMEM-based medium supplemented with or without TGFbetal. Chondrogenic differentiation was evaluated histologically and the relative mRNA expressions of collagen type 1 alpha1 (COL1A1), collagen type 2 alpha1 (COL2A1), aggrecan (ACAN) and SRY-box 9 (SOX9) were estimated by quantitative RT-PCR. Cocultured cells showed diffuse distribution of extracellular matrix (ECM), whereas in chondrocyte pellets it was more localized to central regions. Expression of COL2A1, ACAN and SOX9 genes were higher in cocultured pellets when compared to MSCs and ACs-composed pellets. Addition of TGFbeta1 in chondrogenic differentiating medium did not consistently amplify expression of the above mentioned genes. Differentiation of equine MSCs was enhanced by coculturing in association with mature ACs, improving expression of cartilage-specific genes and producing a more homogeneous production of ECM within the newly formed cocultured cartilage. The use of the coculture system could possibly enhance the capacity of MSC-derived chondrocytes to build up stable articular cartilage-like constructs, which could play an important role in articular cartilage repair and regeneration.

Rabies virus (RV) is highly neurotropic and migrates to the neuronal soma by retrograde axonal transport from nerve terminals, after which it is taken by anterograde axonal transport to be finally released into the central nervous system (CNS) from which it disseminates, resulting in lethal encephalitis. Dorsal root ganglia (DRG) are crucial in the initial events of the infection by RV since they can act as a gate for the viral entrance into the CNS. In the present study, we examined cell tropism of RV and the roles of neuronal cytoskeletal components in the production of viral nucleoprotein (N protein) using cultured nerve cells and non-neuronal cells from DRG of newborn mice. Our in vitro study demonstrated a low propagation rate of RV in nerve cells, susceptibility of non-neuronal cells to RV, and independence of cytoplasmic synthesis of viral N protein from the neuronal cytoskeleton. The present study also suggests that Schwann cells should be considered as another possible candidate supporting RV propagation.

Bovine bone marrow mesenchymal stem cells (MSCs) cultured in condensate culture, spontaneous and independent for any external biostimulants, undergo chondrogenic differentiation. In the present study, the bovine MSC chondrogenesis pathway was studied by analyzing stage-specific gene expression using quantitative 'Real Time' reverse transcriptase polymerase chain reaction (qRT-PCR). Results showed that bovine MSCs underwent complete chondrogenesis; the initial stage was characterized by expression of sox 9 messenger ribonucleic acid (mRNA), followed by high transcription of chondrocyte specific genes, collagen type II and IX, biglycan and cartilage oligomeric matrix protein, and the final prehypertrophic and/or hypertrophic stage was distinguished by increased expression of collagen type X. From day 7 to day 14 of differentiation increased mRNA expression of the transforming growth factors beta1 and beta2, basic fibroblast growth factor (FGF 2), bone morphogenic protein 6 (BMP 6), insulin-like growth factors 1, parathyroid hormone related peptide and indian hedgehog (Ihh) were detected. These results suggest that these well know chondrogenic growth factors may play a role in bovine chondrogenesis in autocrine and/or paracrine manner. On day 21 of the culture, FGF 2, BMP 6 and Ihh were highly expressed, compared to cells cultured in monolayer manner, which suggests a possible function in maintaining the terminal stage of differentiation. This data extends our knowledge about the unusual species-specific bovine MSC chondrogenesis, allowing us to define the phenotype of the differentiated cells. Furthermore, this study contributes to our in understanding of known chondrogenic-growth factors in autocrine and/or paracrine manner playing a role in the spontaneous differentiation.

This study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells (fetal fibroblasts, adult skin and muscle cells, and cumulus cells) after culture under a variety of conditions ; 1 ) growth to 60- 70% confluency (cycling) , 2) serum starvation, 3) culture to confluency. Cell -cycle phases were determined by flow cytometry with propidium iodide staining enabling the calculation of percentages of cells in GO /G1, S and G2 /M. The majority was in GO /GI regardless of cell type and treatment. Serumstarved or confluent cultures contained higher percentages of cells in GO /G1 (89.5-95.4% ; P <0.05) . Percentages of cells in GO /G1 increased as cell size decreased regardless of the cell type and treatment. In the serum-starved and confluent cultures, about 98% of small cells were in GO /G1. Serum-starved cultures contained higher percentages of small cells (38.5-66.9%) than cycling and confluent cultures regardless of cell type (P < 0.05) . After trypsinization of fetal fibroblasts and adult skin cells that were serum-starved and cultured to confluency, the percentages of cells in GO /G1 increased (P < 0.05) on incubation for 1.5 (95.7-99.5%) or 3 hr (95.9-98.6%). These results verify that serum starvation and culture to confluency are efficient means of synchronizing bovine somatic cells in GO /G1, and indicate that a more efficient synchronization of the cells in GO /G1 can be established by incubation for a limited time period after trypsinization of serum-starved or confluent cells.

Endothelial cells are important for maintenance of vascular integrity by producing a variety of bioactive molecules such as nitric oxide (NO) . Recent evidence has suggested that there are some differences in characteristics between endothelial cells from different origins. Here we examined responses of two typical endothelial cells to hepatocyte growth factor (HGF), which induces endothelium-dependent relaxation of microvessels. Stimulation of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) with HGF increased endothelial NO synthase activity, accompanied with an increase of activity-related site-specific phosphorylation of protein kinase B/Akt. However, HGF stimulated phosphorylation of p38 mitogenactivated protein kinase (MAPK) only in HUVEC, but not in BAEC, while it induced phosphorylation of p44 /p42 MAPK in both cells. These results suggest that HGF transduces different intracellular signals between aortic and umbilical venous endothelial cells, and that the differences might represent divergent endothelial responses to growth factors, especially those that activate receptor-tyrosine kinases.