Connexin 40 promoter-based enrichment of embryonic stem cell-derived cardiovascular progenitor cells.
BACKGROUND
Pluripotent embryonic stem (ES) cells that can differentiate into functional cardiomyocytes as well as vascular cells in cell culture may open the door to cardiovascular cell transplantation. However, the percentage of ES cells in embryoid bodies (EBs) which spontaneously undergo cardiovascular differentiation is low (<10%), making strategies for their specific labeling and purification indispensable.


METHODS
The human connexin 40 (Cx40) promoter was isolated and cloned in the vector pEGFP. The specificity of the construct was initially assessed in Xenopus embryos injected with Cx40-EGFP plasmid DNA. Stable Cx40-EGFP ES cell clones were differentiated and fluorescent cells were enriched manually as well as via fluorescence-activated cell sorting. Characterization of these cells was performed with respect to spontaneous beating as well as via RT-PCRs and immunofluorescent stainings.


RESULTS
Cx40-EGFP reporter plasmid injection led to EGFP fluorescence specifically in the abdominal aorta of frog tadpoles. After crude manual enrichment of highly Cx40-EGFP-positive EBs, the appearance of cardiac and vascular structures was increased approximately 3-fold. Immunofluorescent stainings showed EGFP expression exclusively in vascular-like structures simultaneously expressing von Willebrand factor and in formerly beating areas expressing alpha-actinin. Cx40-EGFP-expressing EBs revealed significantly higher numbers of beating cardiomyocytes and vascular-like structures. Semiquantitative RT-PCRs confirmed an enhanced cardiovascular differentiation as shown for the cardiac markers Nkx2.5 and MLC2v, as well as the endothelial marker vascular endothelial cadherin.


CONCLUSIONS
Our work shows the feasibility of specific labeling and purification of cardiovascular progenitor cells from differentiating EBs based on the Cx40 promoter. We provide proof of principle that the deleted CD4 (DeltaCD4) surface marker-based method for magnetic cell sorting developed by our group will be ideally suitable for transference to this promoter.
David
R
R
Medizinische Klinik und Poliklinik I, Klinikum Grosshadern, Ludwig-Maximilians-Universität, München, Germany.
Theiss
H
H
Franz
W-M
WM
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002319:Cardiovascular System / Q000166:cytology*; D002469:Cell Separation; D002999:Clone Cells; D017630:Connexins / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D021381:Protein Transport; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D014981:Xenopus; D014841:von Willebrand Factor / Q000378:metabolism; D000097003:Gap Junction alpha-5 Protein
D015415:Biomarkers; D017630:Connexins; D004247:DNA; D049452:Green Fluorescent Proteins; D014841:von Willebrand Factor; D000097003:Gap Junction alpha-5 Protein; C485184:enhanced green fluorescent protein