Limits...
ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells.

Lindgren AG, Veldman MB, Lin S - Cell Regen (Lond) (2015)

Bottom Line: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs.Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification.This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cellular and Developmental Biology, University of California, 615 Charles E. Young Drive South, Los Angeles, CA 90095 USA.

ABSTRACT

Background: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.

Findings: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.

Conclusions: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

No MeSH data available.


Related in: MedlinePlus

Introduction of exogenousETV2increases the percentage of endothelial cells generated during differentiation. (A) Construct used to generate virus for introduction of ETV2 to hESC. (B–D) Flow cytometry for YFP and VE-CADHERIN. Left panels of each subset show YFP expression in infected cells. Right panels of each subset show VE-CADHERIN expression within YFP+ population. Percentages represent an average from three (B, D) or six experiments (C) and cell counts represent and average from three (B,D) or four experiments (C). Cells were infected/analyzed on days −7i/+7a (B), +4i/+7a (C), and +1i2/+15a (D). Cell counts and percentages are calculated from cells gated to be non-debris, alive, and single cells. (E) Graphical summary of flow cytometry for infection efficiency in cells infected and analyzed on days indicated. Error bars indicate standard deviation. (F) Graphical summary of results of flow cytometry for VE-CADHERIN of YFP expressing virally infected cells. Error bars indicate standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4318149&req=5

Fig2: Introduction of exogenousETV2increases the percentage of endothelial cells generated during differentiation. (A) Construct used to generate virus for introduction of ETV2 to hESC. (B–D) Flow cytometry for YFP and VE-CADHERIN. Left panels of each subset show YFP expression in infected cells. Right panels of each subset show VE-CADHERIN expression within YFP+ population. Percentages represent an average from three (B, D) or six experiments (C) and cell counts represent and average from three (B,D) or four experiments (C). Cells were infected/analyzed on days −7i/+7a (B), +4i/+7a (C), and +1i2/+15a (D). Cell counts and percentages are calculated from cells gated to be non-debris, alive, and single cells. (E) Graphical summary of flow cytometry for infection efficiency in cells infected and analyzed on days indicated. Error bars indicate standard deviation. (F) Graphical summary of results of flow cytometry for VE-CADHERIN of YFP expressing virally infected cells. Error bars indicate standard deviation.

Mentions: We constructed two lentiviral vectors to express either mCherry, as a control, or an ETV2-mCherry fusion protein (Figure 2A). Based upon transient transfection experiments, we found that the ETV2-mCherry fusion protein was localized to the nucleus but difficult to visualize by either microscopy or flow cytometry (data not shown). To ensure that we could identify virally infected cells, we co-expressed yellow fluorescent protein (YFP) with the mCherry or ETV2-mCherry proteins (Figure 2A). YFP expression was used as proxy for mCherry and ETV2-mCherry expression for the remainder of the experiments.Figure 2


ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells.

Lindgren AG, Veldman MB, Lin S - Cell Regen (Lond) (2015)

Introduction of exogenousETV2increases the percentage of endothelial cells generated during differentiation. (A) Construct used to generate virus for introduction of ETV2 to hESC. (B–D) Flow cytometry for YFP and VE-CADHERIN. Left panels of each subset show YFP expression in infected cells. Right panels of each subset show VE-CADHERIN expression within YFP+ population. Percentages represent an average from three (B, D) or six experiments (C) and cell counts represent and average from three (B,D) or four experiments (C). Cells were infected/analyzed on days −7i/+7a (B), +4i/+7a (C), and +1i2/+15a (D). Cell counts and percentages are calculated from cells gated to be non-debris, alive, and single cells. (E) Graphical summary of flow cytometry for infection efficiency in cells infected and analyzed on days indicated. Error bars indicate standard deviation. (F) Graphical summary of results of flow cytometry for VE-CADHERIN of YFP expressing virally infected cells. Error bars indicate standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4318149&req=5

Fig2: Introduction of exogenousETV2increases the percentage of endothelial cells generated during differentiation. (A) Construct used to generate virus for introduction of ETV2 to hESC. (B–D) Flow cytometry for YFP and VE-CADHERIN. Left panels of each subset show YFP expression in infected cells. Right panels of each subset show VE-CADHERIN expression within YFP+ population. Percentages represent an average from three (B, D) or six experiments (C) and cell counts represent and average from three (B,D) or four experiments (C). Cells were infected/analyzed on days −7i/+7a (B), +4i/+7a (C), and +1i2/+15a (D). Cell counts and percentages are calculated from cells gated to be non-debris, alive, and single cells. (E) Graphical summary of flow cytometry for infection efficiency in cells infected and analyzed on days indicated. Error bars indicate standard deviation. (F) Graphical summary of results of flow cytometry for VE-CADHERIN of YFP expressing virally infected cells. Error bars indicate standard deviation.
Mentions: We constructed two lentiviral vectors to express either mCherry, as a control, or an ETV2-mCherry fusion protein (Figure 2A). Based upon transient transfection experiments, we found that the ETV2-mCherry fusion protein was localized to the nucleus but difficult to visualize by either microscopy or flow cytometry (data not shown). To ensure that we could identify virally infected cells, we co-expressed yellow fluorescent protein (YFP) with the mCherry or ETV2-mCherry proteins (Figure 2A). YFP expression was used as proxy for mCherry and ETV2-mCherry expression for the remainder of the experiments.Figure 2

Bottom Line: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs.Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification.This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cellular and Developmental Biology, University of California, 615 Charles E. Young Drive South, Los Angeles, CA 90095 USA.

ABSTRACT

Background: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.

Findings: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.

Conclusions: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

No MeSH data available.


Related in: MedlinePlus