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

Characterization ofETV2-induced endothelial cells. (A,B) Semi-quantitative real-time PCR on sorted YFP+ VE-CADHERIN+ cells from day 7 of differentiation and infected in day 4 for arterial (A) and venous (B) markers. (C,D) Immunofluorescence against CD31 and VE-CADHERIN on sorted ETV2-mCherry-infected VE-CADHERIN-positive cells cultured for 7 days. Scale bar = 20 μm. Upper panels are anti-CD31 (C) or VE-CADHERIN (D) primary antibody and appropriate secondary. Lower panels, secondary antibody only. (E) Network formation on Matrigel of sorted ETV2-mCherry VE-CADHERIN positive cells grown for 7 days in culture. The cells were imaged 8 h after plating. Scale bar = 1 mm. (F) Flow cytometry on sorted ETV2-mCherry-infected VE-CADHERIN-positive cells cultured for 7 days. The cells were analyzed for YFP expression and VE-CADHERIN expression. Percentages are an average of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Characterization ofETV2-induced endothelial cells. (A,B) Semi-quantitative real-time PCR on sorted YFP+ VE-CADHERIN+ cells from day 7 of differentiation and infected in day 4 for arterial (A) and venous (B) markers. (C,D) Immunofluorescence against CD31 and VE-CADHERIN on sorted ETV2-mCherry-infected VE-CADHERIN-positive cells cultured for 7 days. Scale bar = 20 μm. Upper panels are anti-CD31 (C) or VE-CADHERIN (D) primary antibody and appropriate secondary. Lower panels, secondary antibody only. (E) Network formation on Matrigel of sorted ETV2-mCherry VE-CADHERIN positive cells grown for 7 days in culture. The cells were imaged 8 h after plating. Scale bar = 1 mm. (F) Flow cytometry on sorted ETV2-mCherry-infected VE-CADHERIN-positive cells cultured for 7 days. The cells were analyzed for YFP expression and VE-CADHERIN expression. Percentages are an average of two experiments.

Mentions: We wanted to further characterize the endothelial-like cells that were generated from our viral transductions. Comparable numbers of ETV2-mCherry and control infected cells were sorted for YFP and VE-CADHERIN expression on day 7 of differentiation. RNA was isolated from these cells to determine the venous or arterial identity of these cells. Semi-quantitative real-time PCR performed on cDNA derived from this RNA demonstrated that cells infected with the ETV2-mCherry virus expressed lower levels of both arterial and venous markers (Figure 3A,B). This indicates that cells expressing very high levels of ETV2 did not differentiate into arterial or venous endothelium and likely remain in an angioblast state. In vivo and in vitro data has shown that constitutive expression of ETV2 causes endothelial cells to remain in a more primitive state [12,21].Figure 3


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)

Characterization ofETV2-induced endothelial cells. (A,B) Semi-quantitative real-time PCR on sorted YFP+ VE-CADHERIN+ cells from day 7 of differentiation and infected in day 4 for arterial (A) and venous (B) markers. (C,D) Immunofluorescence against CD31 and VE-CADHERIN on sorted ETV2-mCherry-infected VE-CADHERIN-positive cells cultured for 7 days. Scale bar = 20 μm. Upper panels are anti-CD31 (C) or VE-CADHERIN (D) primary antibody and appropriate secondary. Lower panels, secondary antibody only. (E) Network formation on Matrigel of sorted ETV2-mCherry VE-CADHERIN positive cells grown for 7 days in culture. The cells were imaged 8 h after plating. Scale bar = 1 mm. (F) Flow cytometry on sorted ETV2-mCherry-infected VE-CADHERIN-positive cells cultured for 7 days. The cells were analyzed for YFP expression and VE-CADHERIN expression. Percentages are an average of two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Characterization ofETV2-induced endothelial cells. (A,B) Semi-quantitative real-time PCR on sorted YFP+ VE-CADHERIN+ cells from day 7 of differentiation and infected in day 4 for arterial (A) and venous (B) markers. (C,D) Immunofluorescence against CD31 and VE-CADHERIN on sorted ETV2-mCherry-infected VE-CADHERIN-positive cells cultured for 7 days. Scale bar = 20 μm. Upper panels are anti-CD31 (C) or VE-CADHERIN (D) primary antibody and appropriate secondary. Lower panels, secondary antibody only. (E) Network formation on Matrigel of sorted ETV2-mCherry VE-CADHERIN positive cells grown for 7 days in culture. The cells were imaged 8 h after plating. Scale bar = 1 mm. (F) Flow cytometry on sorted ETV2-mCherry-infected VE-CADHERIN-positive cells cultured for 7 days. The cells were analyzed for YFP expression and VE-CADHERIN expression. Percentages are an average of two experiments.
Mentions: We wanted to further characterize the endothelial-like cells that were generated from our viral transductions. Comparable numbers of ETV2-mCherry and control infected cells were sorted for YFP and VE-CADHERIN expression on day 7 of differentiation. RNA was isolated from these cells to determine the venous or arterial identity of these cells. Semi-quantitative real-time PCR performed on cDNA derived from this RNA demonstrated that cells infected with the ETV2-mCherry virus expressed lower levels of both arterial and venous markers (Figure 3A,B). This indicates that cells expressing very high levels of ETV2 did not differentiate into arterial or venous endothelium and likely remain in an angioblast state. In vivo and in vitro data has shown that constitutive expression of ETV2 causes endothelial cells to remain in a more primitive state [12,21].Figure 3

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