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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

Differentiation of hESC to endothelial cells. (A) Diagram of the differentiation protocol. (B–D) Semi-quantitative real-time PCR analysis of gene expression in cells from days 0 to 8 of differentiation. Genes examined: (B)BRACHYURY and ETV2. (C)VE-CADHERIN and CD-31. (D)KDR and CD34. (E) Flow cytometry analysis of day 7 differentiation of hESC. Percentages shown represent averages from five experiments with standard deviations. (F) Quantitative analysis of data in panel (E). Immunofluorescence of day 7 of differentiation for CD31 (G) and VE-CADHERIN (H).
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Fig1: Differentiation of hESC to endothelial cells. (A) Diagram of the differentiation protocol. (B–D) Semi-quantitative real-time PCR analysis of gene expression in cells from days 0 to 8 of differentiation. Genes examined: (B)BRACHYURY and ETV2. (C)VE-CADHERIN and CD-31. (D)KDR and CD34. (E) Flow cytometry analysis of day 7 differentiation of hESC. Percentages shown represent averages from five experiments with standard deviations. (F) Quantitative analysis of data in panel (E). Immunofluorescence of day 7 of differentiation for CD31 (G) and VE-CADHERIN (H).

Mentions: We wanted to determine if addition of exogenous ETV2 during differentiation could induce endothelial cells from hESCs more effectively than addition before differentiation. First, we determined the timing of the expression of endogenous ETV2 in a hESC differentiation model. hESCs were differentiated into endothelial cells using a method that utilized both embryoid body (EB) and adherent stages and were similar to those reported previously (Figure 1A) [1,3]. The cells were collagenase IV digested into clusters and allowed to form EBs overnight in mTeSR1 media in low adherence plates for 24 h. The EBs were collected by gravity and the medium was replaced with mTeSR1 supplemented with 10 ng/ml BMP4. Four days later, the EBs were digested to single cells with Accutase and plated on Matrigel-coated plates in DMEM/F12 media supplemented with 15% KSR, 25 ng/ml VEGF and 20 ng/ml bFGF2. To determine the timing of gene expression, we collected RNA samples from days 0 to 8 of hESC differentiation. Semi-quantitative real-time PCR performed on cDNA generated from the extracted RNA showed that BRACHYURY expression, a marker of mesoderm specification, peaked on day 2 while ETV2 expression peaked on day 5 of differentiation (Figure 1B). This is comparable to the timing of the expression of Brachyury and Etv2 in the mesoderm of mice, where the Brachyury expression precedes a wave of Etv2 expression by 2 days [9,13,14]. The endothelial markers VE-CADHERIN, CD31, KDR, and CD34 showed an increase on day 5 that continued for the next 3 days (Figure 1C,D).Figure 1


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)

Differentiation of hESC to endothelial cells. (A) Diagram of the differentiation protocol. (B–D) Semi-quantitative real-time PCR analysis of gene expression in cells from days 0 to 8 of differentiation. Genes examined: (B)BRACHYURY and ETV2. (C)VE-CADHERIN and CD-31. (D)KDR and CD34. (E) Flow cytometry analysis of day 7 differentiation of hESC. Percentages shown represent averages from five experiments with standard deviations. (F) Quantitative analysis of data in panel (E). Immunofluorescence of day 7 of differentiation for CD31 (G) and VE-CADHERIN (H).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Differentiation of hESC to endothelial cells. (A) Diagram of the differentiation protocol. (B–D) Semi-quantitative real-time PCR analysis of gene expression in cells from days 0 to 8 of differentiation. Genes examined: (B)BRACHYURY and ETV2. (C)VE-CADHERIN and CD-31. (D)KDR and CD34. (E) Flow cytometry analysis of day 7 differentiation of hESC. Percentages shown represent averages from five experiments with standard deviations. (F) Quantitative analysis of data in panel (E). Immunofluorescence of day 7 of differentiation for CD31 (G) and VE-CADHERIN (H).
Mentions: We wanted to determine if addition of exogenous ETV2 during differentiation could induce endothelial cells from hESCs more effectively than addition before differentiation. First, we determined the timing of the expression of endogenous ETV2 in a hESC differentiation model. hESCs were differentiated into endothelial cells using a method that utilized both embryoid body (EB) and adherent stages and were similar to those reported previously (Figure 1A) [1,3]. The cells were collagenase IV digested into clusters and allowed to form EBs overnight in mTeSR1 media in low adherence plates for 24 h. The EBs were collected by gravity and the medium was replaced with mTeSR1 supplemented with 10 ng/ml BMP4. Four days later, the EBs were digested to single cells with Accutase and plated on Matrigel-coated plates in DMEM/F12 media supplemented with 15% KSR, 25 ng/ml VEGF and 20 ng/ml bFGF2. To determine the timing of gene expression, we collected RNA samples from days 0 to 8 of hESC differentiation. Semi-quantitative real-time PCR performed on cDNA generated from the extracted RNA showed that BRACHYURY expression, a marker of mesoderm specification, peaked on day 2 while ETV2 expression peaked on day 5 of differentiation (Figure 1B). This is comparable to the timing of the expression of Brachyury and Etv2 in the mesoderm of mice, where the Brachyury expression precedes a wave of Etv2 expression by 2 days [9,13,14]. The endothelial markers VE-CADHERIN, CD31, KDR, and CD34 showed an increase on day 5 that continued for the next 3 days (Figure 1C,D).Figure 1

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