Abstract
In recent years, the utilization of stem cell therapy to regenerate cardiac tissue has been proposed as a possible strategy to treat cardiac damage (Gnecchi et al., 2012, Aguirre et al., 2013; Sanganalmath and Bolli, 2013). Although encouraging results have been obtained in experimental models, the efficiency of cardiac regeneration is very poor and one of the major barriers to progress in the area of cell therapy for damaged heart is represented by the limited capacity of cells to differentiate into mature cardiomyocytes (CMC) (Laflamme and Murry, 2011). Cell manipulation and transfection represent versatile tools in this context (Melo et al., 2005; Dzau et al., 2005). Murine P19 embryonal carcinoma cells are a well-established cell line capable of differentiating in vitro into spontaneously beating CMC. This cell system with its limited cell culture requirements, protocol reproducibility and ease in uptake and subsequent expression of ectopic genetic materials render it ideal for the study of the cardiac differentiation process. P19 cells have been successfully used to gain important insights into the early molecular processes of CMC differentiation (van der Heyden and Defize, 2003; van der Heyden et al., 2003). P19 cells can also be maintained in an undifferentiated state in a monolayer culture when grown in adherence; this condition allows the enrichment of large cell numbers useful for cardiac differentiation protocols (McBurney, 1993). On the other hand, when cultured in bacterial dishes, P19 cells will grow in suspension and generate embryoid bodies (EB). When exposed to dimethyl sulfoxide (DMSO), EB differentiate into spontaneously beating cells, which can be defined as CMC. This definition is based on their gene and protein expression and their electrophysiological properties (Wobus et al., 1994; van der Heyden et al., 2003). In our laboratory, we used this in vitro model to verify whether the over-expression of a defined combination of miRNA can synergistically induce effective cardiac differentiation (Pisano et al., 2015). We used miRNA1, miRNA133 and miRNA499 alone or in combination. Here, we describe how we transiently transfect P19 cells to over-express a single or a combination of miRNA precursors (pre-miRNA).
Keywords: MicroRNA, Embryoid bodies, Cardiac differentiation
Materials and Reagents
Equipment
Procedure
Notes
Recipes
Acknowledgments
The development of this protocol was supported by the Fondazione IRCCS Policlinico San Matteo Pavia, Italy; the Fondazione Cariplo (2007-5984) and the Ministero Italiano della Sanità (GR-2008-114278). We want to thank Laurene Kelly for help with editing the manuscript.
References
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Firstly, I am curious about the exact transfection efficiency about this method since you use the EB to be transfected directly if I am right. Yes, you are right. We optimized the standard transfection protocol using EB. To quantify transfection efficiency we performed qPCR on dissociated EB (please, see point 12 of our protocol) and using both a positive control (100% of miRNA expression) and a negative control (P19 naïve cells). RNA was extracted from the isolated cells and RT-PCR and qPCR were performed for miRNA1, miRNA133 and miRNA499. The transfection efficiency ranged 88 to 100%.Secondly, is the high transfection efficiency just limited to this cell line or the precursor miRNA. I am wondering how is it for the other ESC lines and the general plasmid? P19 cells are not easy to transfect with liposomes and plasmid. However, the low molecular weight of miRNA precursors (60-80 nucleotides) probably facilitates the transfection efficiency. We initially tried to transfect these cell line with a 10 Kb plasmid but the transfection efficiency was very low. For these reason, we switched to lentivirus to transform P19 cells obtaining high efficiency (please, see Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf).We did not test ESC lines in our studies so we do not have an answer for this specific question. Thirdly, I am wondering what is the difference between the cardiomyocyte differentiated from this embryonal carcinoma cell line and the normal ESC line? If there is no difference, this cell line is really convenient for cardiac differentiation. And how much percentage of the cardiomyocyte in the EBs differentiaed from this cell line?As aforementioned, we did not perform the transfection protocol on ESC. Anyway, ESC and P19 behave very similarly and we supposed that the cardiac differentiation efficiency could be similar between these two cell types. Please, refer again to our manuscript (Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf).