A P19Cl6 GFP reporter line to quantify cardiomyocyte differentiation of stem cells.
The clinical application of stem cell therapies is still limited by the ability to produce defined, differentiated cell populations in large numbers in culture. High throughput screens to identify factors which enhance differentiation to particular lineages and promote expansion of precursors in culture are dependent on the development of sensitive and reproducible assays for screening. Here we describe a bioassay to identify factors with cardiomyogenic activity which enhance the yield of cardiomyocytes from undifferentiated stem cells. The assay is based on a Green Fluorescent Protein (GFP) reporter under the transcriptional control of the 250 bp MLC-2v promoter expressed in pluripotent P19 embryonal carcinoma cells. We show that reporter expression is limited to developing cardiomyocytes and can be used to determine quantitatively the number of ventricular cardiomyocytes formed in cultures under inducing or non-inducing conditions. This assay differs from all others described previously in that it has an easily quantifiable readout, there is negligible background differentiation in the absence of exogenous cardiogenic factors and it is carried out feeder cell-free. Thus, it is entirely independent of competing differentiation inhibitory factors, such as leukemia inhibitory factor. Patch clamp electrophysiology of the GFP-positive cells confirmed their functional ventricular phenotype and indicated that selection on the basis of GFP would provide cells suitable for transplantation.
Hubrecht Laboratory, University Medical Center, Utrecht, The Netherlands.
Jennifer C
JC
Moore
Rene
R
Spijker
Anton C
AC
Martens
Teun
T
de Boer
Martin B
MB
Rook
Marcel A G
MA
van der Heyden
Leon G
LG
Tertoolen
Christine L
CL
Mummery
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation; D002460:Cell Line; D002999:Clone Cells / Q000378:metabolism; D004594:Electrophysiology; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014335:Tropomyosin / Q000032:analysis
D015415:Biomarkers; D008164:Luminescent Proteins; D014335:Tropomyosin; D049452:Green Fluorescent Proteins