Spontaneous chondrogenic differentiation

Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system.

"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 sox9 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[LSJL induces expression of all of these except COMP]. From day 7 to day 14 of differentiation increased mRNA expression of the transforming growth factors beta1{upregulation is predicted} and beta2, basic fibroblast growth factor (FGF 2){up in LSJL}, 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."

"For inducing chondrogenesis in vitro, strong cell to cell interaction, growth factors or cytokines possessing chondrogenic potentiality and structure which support three dimensional cell orientations were reported to be necessary"

"Strong cell to cell interaction mediated by cell adhesion molecules such as N-cadherin and integrins allowed MSC conversion to prechondroblasts at the precartilage mesenchymal condensation stage during limb development.  This condition in vitro can be obtained by pellet or micromass culture systems."

"Bone marrow was aspirated from three calves months old.  The bone marrow

sample was washed twice with phosphate-buffered saline and twice with Dulbecco‘ s Modified Eagle Medium DMEM...Non-adherent cells were removed by changing the culture medium."

"MSCs from the first or second passage were used in the experiments. There were not notable differences on chondrogenic potential among cells from different passages, or reconstructed cells after deep freeze in liquid nitrogen. Within one or two days of samples preparations, cells formed compact pellet, which increased in size during culturing"

"Cells cultured in monolayer manner with serum free-chemically defined medium had bipolar to polygonal fibroblastic cell-shape and grew in uniform monolayer"

"[Cells grown in pellet culture] plump, round cell-shape, located in lacunae and surrounded with notable newly synthesized ECM"

"Cell shape and structure of the newly generated tissue had typical characteristics of chondrocytes and cartilage.  Only cells in the few periphery layer of the pellet had elongated , fibroblast-like shape ."

"Sox9 was firstly detected on day 7 and its expression increased in the following 7 days of culture, when reaches the maximum [expression], and then decreased at the terminal stage"

"over expression of [Sox9] in mouse MSC can enhance chondrogenesis, and that cell mediated sox9 gene therapy could be treatment for articular cartilage regeneration"