RhoA regulation of cardiomyocyte differentiation.

Earlier findings from our laboratory implicated RhoA in heart developmental processes. To investigate factors that potentially regulate RhoA expression, RhoA gene organisation and promoter activity were analysed. Comparative analysis indicated strict conservation of both gene organisation and coding sequence of the chick, mouse, and human RhoA genes. Bioinformatics analysis of the derived promoter region of mouse RhoA identified putative consensus sequence binding sites for several transcription factors involved in heart formation and organogenesis generally. Using luciferase reporter assays, RhoA promoter activity was shown to increase in mouse-derived P19CL6 cells that were induced to differentiate into cardiomyocytes. Overexpression of a dominant negative mutant of mouse RhoA (mRhoAN19) blocked this cardiomyocyte differentiation of P19CL6 cells and led to the accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac α -actin. Taken together, these findings indicate a fundamental role for RhoA in the differentiation of cardiomyocytes.

A novel cell transplantation protocol and its application to an ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, which selectively affects motor neurons throughout the central nervous system. The extensive distribution of motor neurons is an obstacle to applying cell transplantation therapy for the treatment of ALS. To overcome this problem, we developed a cell transplantation method via the fourth cerebral ventricle in mice. We used mouse olfactory ensheathing cells (OECs) and rat mesenchymal stem cells (MSCs) as donor cells. OECs are reported to promote regeneration and remyelination in the spinal cord, while MSCs have a capability to differentiate into several types of specific cells including neural cells. Furthermore both types of cells can be relatively easily obtained by biopsy in human. Initially, we confirmed the safety of the operative procedure and broad distribution of grafted cells in the spinal cord using wild-type mice. After transplantation, OECs distributed widely and survived as long as 100 days after transplantation, with a time-dependent depletion of cell number. In ALS model mice, OEC transplantation revealed no adverse effects but no significant differences in clinical evaluation were found between OEC-treated and non-transplanted animals. After MSC transplantation into the ALS model mice, females, but not males, showed a statistically longer disease duration than the non-transplanted controls. We conclude that intrathecal transplantation could be a promising way to deliver donor cells to the central nervous system. Further experiments to elucidate relevant conditions for optimal outcomes are required.

RhoA is highly up-regulated in the process of early heart development of the chick and important for normal embryogenesis.

We have used molecular techniques, combined with classic embryological methods, to identify up-regulated genes associated with early heart development. One of the cDNAs identified and isolated by screening a chick lambda cDNA library was the small guanosine triphosphatase RhoA. RhoA has at least three different length mRNA species, each varying in the length of the 3' untranslated region. In situ hybridisation and immunocytochemistry analysis of RhoA expression show marked up-regulation in the heart-forming region. In other systems, RhoA signalling has been shown to be important for both gene expression and morphology. To investigate the function of RhoA in early heart development, we used small interfering RNAs (siRNA) in early chick embryos. Disruption of RhoA expression by siRNA treatment resulted in lack of heart tube fusion and abnormal head development. These data indicate that RhoA is important for normal embryogenesis.