Connexin 40 promoter-based enrichment of embryonic stem cell-derived cardiovascular progenitor cells
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vor 16 Jahren
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. Immuno fluorescent
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 (Delta CD4) surface marker-based
method for magnetic cell sorting developed by our group will be
ideally suitable for transference to this promoter. Copyright (c)
2008 S. Karger AG, Basel.
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. Immuno fluorescent
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 (Delta CD4) surface marker-based
method for magnetic cell sorting developed by our group will be
ideally suitable for transference to this promoter. Copyright (c)
2008 S. Karger AG, Basel.
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