Induced pluripotent stem (iPS) cells are genetically reprogrammed somatic cells that exhibit embryonic stem cell-like characteristics such as self-renewal and pluripotency. These cells have broad differentiation capability to convert into diverse cell types that make up the primary germ layers during embryonic development. iPS cells can spontaneously differentiate and form cell aggregates termed embryoid bodies (EBs) in the absence of differentiation inhibitory factors. Unlike other methods used to generate EBs, "the hanging drop" method offers reproducibility and homogeneity from a set number of iPS cells. As such, we describe the differentiation of rat-induced pluripotent stem cells into cardiac myocytes in vitro using the hanging drop method. Both the confirmation and identification of the cardiac myocytes are done using immunocytochemistry, RT-PCR, Western Blot, and Flow Cytometry. Briefly, a specific number of iPS cells are placed in droplets on the lid of culture dishes and incubated for 2 days, yielding embryoid bodies, which are suspended and plated. Spontaneous beating of cardiomyocytes can be seen 7-14 days after the plating of EBs and specific cardiac markers can be observed through identification assays.
Keywords: Cardiac myocytes; Cell culture and differentiation; Embryoid bodies; Hanging drop method; Induced-pluripotent stem cells.
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