Molecular cloning and characterization of differentiation-inducing factors into the cardiac myocyte lineage using an expression cloning approach

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Since embryonic stem (ES) cells are capable of self-renew and differentiation into specialized cells in response to appropriate signals, ES cells are proposed as a promising source of functional cardiac myocytes for cardiac diseases. However, to date the in vitro differentiation of ES cells into cardiac myocytes remains inefficient and its molecular mechanisms are not fully elucidated. Therefore, the identification of differentiation-inducing factors into the cardiac myocyte lineage has become critical for understanding the molecular mechanisms of differentiation into cardiac myocyte and heart development, and facilitates therapeutic applications of ES cells in cardiac diseases. In the present study, we sought to identify differentiation-inducing factors into the cardiac myocyte lineage using an expression cloning approach. We induced differentiation of the mouse embryonic carcinoma cell line, P19CL cells, into cardac myocytes using DMSO. We made cDNA libraries using mRNA isolated from differentiating cells. We then generated recombinant retroviruses expressing the cDNA libraries. ES cell clones that express EGFP under the transcriptional control of a cardiac-specific α-MHC promoter were infected with recombinant retroviruses expressing the cDNA libraries. Gemonic DNA was extracted from differentiated cells into cardiac myocytes and performed PCR to isolate the integrated cDNAs into the genomic DNA. We obtained G protein beta polypeptide 2 like 1 (Gnb2I1, Rack1), oxidase assembly 1-like (Oxa1l), glutaredoxin 5 homolog (Glrx5), and aurora kinase A interacting protein 1(Aurkaip1) as candidate genes for differentiation-inducing factors into the cardiac myocyte lineage. We are studying their functions in differentiation of ES cells into cardiac myocyte. Our study will provide novel insights into the molecular mechanisms of cardiac myocyte differentiation and the development of novel approaches for the directed differentiation of ES cells into cardiac myocytes.