| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2004: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Research Abstract |
We previously reported that during in vitro cardiomyocyte differentiation of mouse embryonic stem (ES) cells, Cx40 transcripts significantly increased with the appearance of beating cells and that the time course was similar to that of cardiac-specific genes, whereas the transcripts for Cx43 and Cx45 were detected in undifferentiated ES cells, and before and after the appearance of beating (Exp Cell Res. 229:318, 1996; Cardiac & Vascular Regeneration 1: 54, 2000). We also reported that during early neuronal differentiation stages of ES cells, gap junctional intercellular communication was absent between cell groups that were committed to different cell lineages. In the present study, we hypothesized that different intercellular communication compartments are formed due to distinctive combinations of connexin species when a novel cell lineage arise. To test this hypothesis and to understand at what stages mRNA expression and localization of connexins are initiated during cardiomyocyte d
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ifferentiation, we analyzed connexin expression using an in vitro cardiomyocyte differentiation system of P19CL6 cells, a clonal derivative of P19 mouse embryonal carcinoma cells, that are pluripotent and efficiently differentiate into beating cardiomyocytes under adherent condition when treated with 1% dimethyl sulfoxide (DMSO). For early detection of cells that are committed to cardiomyocyte differentiation, we transfected an EGFP-expression vector driven by cardiac-specific myosin heavy chain alpha (MHC-α) promoter into P19CL6 cells and established a cell line.
After inducing cardiomyocyte differentiation by DMSO, beating cell populations that were EGFP-positive emerged. Immunolabeling for Cx40 revealed that most Cx40-positive spots were localized in MHC-α-positive cardiomyocytes, whereas Cx43-positive spots were present not only in MHC-α-positive cardiomyocytes but also in MHC-α-negative non-cardiomyocytes. Even in small clusters of MHC-α-positive cells, Cx40-positive spots were detected. These results indicate that Cx40 is specifically linked to the early stage of cardiomyocyte differentiation in vitro. Less
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