Growth of dissociated rat cerebellar cells using serum-free supplemented media and varied transferrin concentrations

Dissociated neonatal rat cerebellar cells were grown on medium supplemented with 10% horse serum (HS) and compared with those grown using a serum-free supplemented (SFS) medium, modified from Bottenstein and Sato (1979). containing insulin, transferrin, progesterone, putrescine, and selenium (after an initial 24 hr in 10% horse serum). Cells survived for several weeks using either medium. Cells grown in SFS had higher levels of GABA uptake than cells grown in HS. Cellular morphology and the proportion of neurons to glial cells were similar under the two conditions. Transferrin concentrations of 0.5, 10, and 100 µg/ml were tested. Neither neuronal nor glial cells were sensitive to this 200-fold variation. The SFS medium supports survival and maturation of both neurons and glial cells from rat cerebellum. However, the medium is not completely defined since (1) one day of serum is still required and (2) the heterogeneous cell population is undoubtedly conditioning the medium to some extent.This work was supported in part by grants from the Scottish Rite Schizophrenia Research Program, N.M.J., USA, and by Biomedical Research Grant S 07-RR5394, from the National Institutes of Health, PHS/DHHS.     

Gata4, Tbx5 and Baf60c induce differentiation of adipose tissue-derived mesenchymal stem cells into beating cardiomyocytes

The adipose tissue-derived mesenchymal stem cells (ADMSCs) are extensively utilized in tissue engineering, regenerative medicine and cell therapy. ADMSCs can differentiate into cardiomyocytes, and it has been shown that over-expression of a cocktail of factors can induce ectopic heart formation and program cardiogenesis in ESCs. However, which genes are responsible for differentiation of ADMSCs into beating cardiomyocyte-like cells remains unknown. In this study we have shown that the combination of Gata4, Tbx5 and Baf60c is sufficient for inducing ADMSCs to form cardiomyocytes. It also appears that, while Gata4 and Baf60c are key inducers of myocardial differentiation, Tbx5 is essential for the ability of cardiac cells to contract. These findings provide additional experimental references for myocardial tissue engineering in the emerging field of cell-based therapy of heart diseases.     

p38/NF-kB-dependent expression of COX-2 during differentiation and inflammatory response of chondrocytes

Studying cartilage differentiation, we observed the emergence of inflammation-related proteins suggesting that a common pathway was activated in cartilage differentiation and inflammation. In the present paper, we investigated the expression pathway of the inflammation-related enzyme Cyclooxygenase-2 (COX-2) during differentiation and inflammatory response of the chondrocytic cell line MC615. Cells were cultured either as (i) proliferating prechondrogenic cells expressing type I collagen or (ii) differentiated hyperconfluent cells expressing Sox9 and type II collagen. The p38 and the NF-kB pathways were investigated in standard conditions and after inflammatory agents treatment. NF-kB was constitutively activated in differentiated cells. The activation level of NF-kB in differentiated cells was comparable to the level in proliferating cells treated with the inflammatory agent LPS. In both cases, p65 was bound to the NF-kB consensus sequence of COX-2 promoter. p38, constitutively activated in differentiated cells, was activated in proliferating cells by treatment with LPS or IL-1alpha. In stimulated proliferating cells the two pathways are connected since addition of the p38-specific inhibitor SB203580 inhibited p38 activation, significantly reduced NF-kB activation and repressed COX-2 synthesis indicating that p38 is upstream NF-kB activation and COX-2 synthesis. In differentiated cells, the treatment with the inflammatory agent neither enhance NF-kB activation, nor synthesis of COX-2 while the addition of SB203580 neither repressed activation of p38, nor COX-2 synthesis, suggesting a constitutive activation of a p38/NF-kB/COX2 pathway. Our data indicate that in chondrocytes, COX-2 is expressed via p38 activation/NF-kB recruitment during both differentiation and inflammatory response.     

TGF-β1 modifications in nuclear matrix proteins of osteoblasts during differentiation

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4–6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β–treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β–treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β–dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291–303, 1998. © 1998 Wiley-Liss, Inc.