A clonal derivative named CL6 has been isolated from pluripotent P19 embryonal carcinoma (EC) cells, after long term culture under conditions for mesodermal differentiation. The CL6 subline has a morphology similar to P19 cells and grows exponentially in standard medium, but unlike P19 cells, it efficiently differentiates into beating cardiac muscle in adherent culture with 1% dimethyl sulfoxide (DMSO). During differentiation, CL6 cells displayed the following general cardiac muscle properties: (1) α- and β-cardiac myosin heavy chain (MHC) transcripts were first detected on day 10 after treatment with DMSO. (2) A sarcomere MHC protein also appeared on day 10, simultaneously with the initiation of contraction. (3) Immunofluorescence analysis showed that vigorously contracting cells have a striated structure. (4) Skeletal muscle specific transcripts, such as myogenin and MyoD, were not detected throughout differentiation.
On the other hand, in suspension culture with 1 μM retinoic acid (RA), the condition for neural differentiation of P19 cells, the CL6 cells developed into neurons with poor outgrowth.
Taken together, the CL6 subline seems not to be committed to a mesodermal lineage but to represent a developmental stage closer to differentiated cardiac muscle than the P19 cell line. Since CL6 cells directly differentiate into cardiac muscle in adherent culture, it is the most useful cell line for studying the differentiation of cardiac muscle in vitro.