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Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions.

Sriram G, Tan JY, Islam I, Rufaihah AJ, Cao T - Stem Cell Res Ther (2015)

Bottom Line: Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized.Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse.This could offer a human platform to study arterial-venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.

View Article: PubMed Central - PubMed

Affiliation: Oral Sciences Disciplines, Faculty of Dentistry, National University of Singapore, Singapore, 119083, Singapore. sriramgopu@u.nus.edu.

ABSTRACT

Background: Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage, differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally, current differentiation strategies are hampered by inefficiency, lack of reproducibility, and use of animal-derived products.

Methods: To direct the differentiation of hESCs to endothelial subtypes, H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3), basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation, the endothelial progenitor cells (CD34(+)CD31(+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized.

Results: Sequential modulation of hESCs with GSK-3 inhibitor, bFGF, BMP4 and VEGF resulted in stages reminiscent of primitive streak, early mesoderm/lateral plate mesoderm, and endothelial progenitors under feeder- and serum-free conditions. Furthermore, these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically, the endothelial progenitors differentiated to venous ECs in the absence of VEGF, and to arterial phenotype under low concentrations of VEGF. Additionally, these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse.

Conclusions: We report a simple, rapid, and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.

No MeSH data available.


Related in: MedlinePlus

Assessment of cell migration and survey of angiocrine secretome profiles of hESC-derived arterial and venous ECs. a Graphical representation of kinetics of wound coverage among H1-Ven-ECs and H1-Art-ECs (left panel), H9-Ven-ECs and H9-Art-ECs (middle panel), HUVECs, and HCAECs (right panel). Error bars show standard deviation; n = 3. *p < 0.05, **p < 0.01. b Representative scans of angiogenesis antibody arrays demonstrating the secretion of various angiocrine factors by H1-Ven-ECs and H1-Art-ECs. Array images are obtained from 10-minute exposure of X-ray film. (Refer to Additional file 1 for co-ordinates of the antibody array). c Graphical representation of the relative amounts of selected angiocrine factors that show significant differences among the H1-hESC derived arterial and venous ECs. The bars represent relative amounts of factors secreted based on densitometric analysis of relative pixel density of the blots. Error bars show the standard deviation of two independent experiments. *p < 0.05, **p < 0.01. Art-EC Arterial endothelial cells, HCAEC Human coronary artery endothelial cells, hESC Human embryonic stem cells, HUVEC Human umbilical vein endothelial cells, IGF Insulin-like growth factor, IL Interleukin, MCP monocyte chemoattractant protein, MMP Matrix metalloproteinase, PDGF Platelet-derived growth factor, TIMP Tissue inhibitor of matrix metalloproteinase, Ven-EC Venous endothelial cells
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Fig6: Assessment of cell migration and survey of angiocrine secretome profiles of hESC-derived arterial and venous ECs. a Graphical representation of kinetics of wound coverage among H1-Ven-ECs and H1-Art-ECs (left panel), H9-Ven-ECs and H9-Art-ECs (middle panel), HUVECs, and HCAECs (right panel). Error bars show standard deviation; n = 3. *p < 0.05, **p < 0.01. b Representative scans of angiogenesis antibody arrays demonstrating the secretion of various angiocrine factors by H1-Ven-ECs and H1-Art-ECs. Array images are obtained from 10-minute exposure of X-ray film. (Refer to Additional file 1 for co-ordinates of the antibody array). c Graphical representation of the relative amounts of selected angiocrine factors that show significant differences among the H1-hESC derived arterial and venous ECs. The bars represent relative amounts of factors secreted based on densitometric analysis of relative pixel density of the blots. Error bars show the standard deviation of two independent experiments. *p < 0.05, **p < 0.01. Art-EC Arterial endothelial cells, HCAEC Human coronary artery endothelial cells, hESC Human embryonic stem cells, HUVEC Human umbilical vein endothelial cells, IGF Insulin-like growth factor, IL Interleukin, MCP monocyte chemoattractant protein, MMP Matrix metalloproteinase, PDGF Platelet-derived growth factor, TIMP Tissue inhibitor of matrix metalloproteinase, Ven-EC Venous endothelial cells

Mentions: ECs interact with the local microenvironment and support tissue regeneration after injury through revascularization of the newly healed tissue and expression of various trophic growth factors, known as angiocrine factors, which lead to proliferation and migration of ECs from pre-existing blood vessels. To assess cell migration, in vitro wound closing assays were performed over a period of 24 hours. Compared to H1-Ven-ECs, the H1-Art-ECs displayed faster closure of the wound area resulting in a complete wound closure by H1-Art-ECs in ~15 hours in contrast to ~27 hours taken by H1-Ven-ECs (Fig. 6a). Similarly, the H9-Art-ECs and HCAECs displayed faster closure of the scratch wound compared to respective venous phenotypes (Fig. 6a). ECs pertaining to distinct vascular beds have recently been reported to display significant differences in expression profiles of various transcriptomes, angiocrine factors and surface markers [39]. However, the distinct expression of various angiocrine factors by different endothelial phenotypes of hESC origin still needs to be explored. To probe into the profile of various angiocrine factors secreted by hESC-derived ECs, we used angiogenesis antibody array of 55 different proteins related to angiogenesis, growth factors and related proteins, cytokines, proteases and their inhibitors, neurotrophic factors, and anti-angiogenic factors. Both H1-Art-ECs and H1-Ven-ECs displayed marked induction of endothelin-1, insulin-like growth factor-binding protein-2 (IGFBP-2), monocyte chemoattractant protein-1 (MCP-1), pentraxin-3, serpin-E1 and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) (Fig. 6; Additional file 1: Figure S7 and Table S1). However, the conditioned media of H1-Ven-ECs had significantly higher levels of Activin-A, IGFBP-3, and interleukin-1β compared to H1-Art-ECs (Fig. 6c). On the contrary, the media conditioned by H1-Art-ECs had significantly higher levels of IGFBP-1, interleukin-8, matrix metalloproteinase-9 (MMP-9), PDGF-AA and thrombospondin-1 (TSP-1) compared to that of H1-Ven-ECs (Fig. 6c). These results emphasize the existence of intrinsic differences between arterial and venous endothelial phenotypes. However, further studies on the differences in secretome profiles among the two endothelial phenotypes and their in vivo relevance are needed to understand the role of endothelial heterogeneity.Fig. 6


Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions.

Sriram G, Tan JY, Islam I, Rufaihah AJ, Cao T - Stem Cell Res Ther (2015)

Assessment of cell migration and survey of angiocrine secretome profiles of hESC-derived arterial and venous ECs. a Graphical representation of kinetics of wound coverage among H1-Ven-ECs and H1-Art-ECs (left panel), H9-Ven-ECs and H9-Art-ECs (middle panel), HUVECs, and HCAECs (right panel). Error bars show standard deviation; n = 3. *p < 0.05, **p < 0.01. b Representative scans of angiogenesis antibody arrays demonstrating the secretion of various angiocrine factors by H1-Ven-ECs and H1-Art-ECs. Array images are obtained from 10-minute exposure of X-ray film. (Refer to Additional file 1 for co-ordinates of the antibody array). c Graphical representation of the relative amounts of selected angiocrine factors that show significant differences among the H1-hESC derived arterial and venous ECs. The bars represent relative amounts of factors secreted based on densitometric analysis of relative pixel density of the blots. Error bars show the standard deviation of two independent experiments. *p < 0.05, **p < 0.01. Art-EC Arterial endothelial cells, HCAEC Human coronary artery endothelial cells, hESC Human embryonic stem cells, HUVEC Human umbilical vein endothelial cells, IGF Insulin-like growth factor, IL Interleukin, MCP monocyte chemoattractant protein, MMP Matrix metalloproteinase, PDGF Platelet-derived growth factor, TIMP Tissue inhibitor of matrix metalloproteinase, Ven-EC Venous endothelial cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697311&req=5

Fig6: Assessment of cell migration and survey of angiocrine secretome profiles of hESC-derived arterial and venous ECs. a Graphical representation of kinetics of wound coverage among H1-Ven-ECs and H1-Art-ECs (left panel), H9-Ven-ECs and H9-Art-ECs (middle panel), HUVECs, and HCAECs (right panel). Error bars show standard deviation; n = 3. *p < 0.05, **p < 0.01. b Representative scans of angiogenesis antibody arrays demonstrating the secretion of various angiocrine factors by H1-Ven-ECs and H1-Art-ECs. Array images are obtained from 10-minute exposure of X-ray film. (Refer to Additional file 1 for co-ordinates of the antibody array). c Graphical representation of the relative amounts of selected angiocrine factors that show significant differences among the H1-hESC derived arterial and venous ECs. The bars represent relative amounts of factors secreted based on densitometric analysis of relative pixel density of the blots. Error bars show the standard deviation of two independent experiments. *p < 0.05, **p < 0.01. Art-EC Arterial endothelial cells, HCAEC Human coronary artery endothelial cells, hESC Human embryonic stem cells, HUVEC Human umbilical vein endothelial cells, IGF Insulin-like growth factor, IL Interleukin, MCP monocyte chemoattractant protein, MMP Matrix metalloproteinase, PDGF Platelet-derived growth factor, TIMP Tissue inhibitor of matrix metalloproteinase, Ven-EC Venous endothelial cells
Mentions: ECs interact with the local microenvironment and support tissue regeneration after injury through revascularization of the newly healed tissue and expression of various trophic growth factors, known as angiocrine factors, which lead to proliferation and migration of ECs from pre-existing blood vessels. To assess cell migration, in vitro wound closing assays were performed over a period of 24 hours. Compared to H1-Ven-ECs, the H1-Art-ECs displayed faster closure of the wound area resulting in a complete wound closure by H1-Art-ECs in ~15 hours in contrast to ~27 hours taken by H1-Ven-ECs (Fig. 6a). Similarly, the H9-Art-ECs and HCAECs displayed faster closure of the scratch wound compared to respective venous phenotypes (Fig. 6a). ECs pertaining to distinct vascular beds have recently been reported to display significant differences in expression profiles of various transcriptomes, angiocrine factors and surface markers [39]. However, the distinct expression of various angiocrine factors by different endothelial phenotypes of hESC origin still needs to be explored. To probe into the profile of various angiocrine factors secreted by hESC-derived ECs, we used angiogenesis antibody array of 55 different proteins related to angiogenesis, growth factors and related proteins, cytokines, proteases and their inhibitors, neurotrophic factors, and anti-angiogenic factors. Both H1-Art-ECs and H1-Ven-ECs displayed marked induction of endothelin-1, insulin-like growth factor-binding protein-2 (IGFBP-2), monocyte chemoattractant protein-1 (MCP-1), pentraxin-3, serpin-E1 and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) (Fig. 6; Additional file 1: Figure S7 and Table S1). However, the conditioned media of H1-Ven-ECs had significantly higher levels of Activin-A, IGFBP-3, and interleukin-1β compared to H1-Art-ECs (Fig. 6c). On the contrary, the media conditioned by H1-Art-ECs had significantly higher levels of IGFBP-1, interleukin-8, matrix metalloproteinase-9 (MMP-9), PDGF-AA and thrombospondin-1 (TSP-1) compared to that of H1-Ven-ECs (Fig. 6c). These results emphasize the existence of intrinsic differences between arterial and venous endothelial phenotypes. However, further studies on the differences in secretome profiles among the two endothelial phenotypes and their in vivo relevance are needed to understand the role of endothelial heterogeneity.Fig. 6

Bottom Line: Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized.Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse.This could offer a human platform to study arterial-venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.

View Article: PubMed Central - PubMed

Affiliation: Oral Sciences Disciplines, Faculty of Dentistry, National University of Singapore, Singapore, 119083, Singapore. sriramgopu@u.nus.edu.

ABSTRACT

Background: Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage, differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally, current differentiation strategies are hampered by inefficiency, lack of reproducibility, and use of animal-derived products.

Methods: To direct the differentiation of hESCs to endothelial subtypes, H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3), basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation, the endothelial progenitor cells (CD34(+)CD31(+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized.

Results: Sequential modulation of hESCs with GSK-3 inhibitor, bFGF, BMP4 and VEGF resulted in stages reminiscent of primitive streak, early mesoderm/lateral plate mesoderm, and endothelial progenitors under feeder- and serum-free conditions. Furthermore, these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically, the endothelial progenitors differentiated to venous ECs in the absence of VEGF, and to arterial phenotype under low concentrations of VEGF. Additionally, these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse.

Conclusions: We report a simple, rapid, and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.

No MeSH data available.


Related in: MedlinePlus