Document Detail

A P19Cl6 GFP reporter line to quantify cardiomyocyte differentiation of stem cells.
MedLine Citation:
PMID:  15005574     Owner:  NLM     Status:  MEDLINE    
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.
Jennifer C Moore; Rene Spijker; Anton C Martens; Teun de Boer; Martin B Rook; Marcel A G van der Heyden; Leon G Tertoolen; Christine L Mummery
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The International journal of developmental biology     Volume:  48     ISSN:  0214-6282     ISO Abbreviation:  Int. J. Dev. Biol.     Publication Date:  2004 Feb 
Date Detail:
Created Date:  2004-03-09     Completed Date:  2004-09-24     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  8917470     Medline TA:  Int J Dev Biol     Country:  Spain    
Other Details:
Languages:  eng     Pagination:  47-55     Citation Subset:  IM    
Hubrecht Laboratory, University Medical Center, Utrecht, The Netherlands.
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MeSH Terms
Biological Markers / analysis
Cell Differentiation*
Cell Line
Clone Cells / metabolism
Genes, Reporter / genetics*
Green Fluorescent Proteins
Luminescent Proteins / genetics,  metabolism*
Molecular Sequence Data
Myocytes, Cardiac / cytology*,  metabolism
Promoter Regions, Genetic / genetics
Stem Cells / cytology*,  metabolism
Tropomyosin / analysis
Reg. No./Substance:
0/Biological Markers; 0/Luminescent Proteins; 0/Tropomyosin; 147336-22-9/Green Fluorescent Proteins

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