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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

Flow cytometry analysis of induction of H1-hESCs towards endothelial lineage. Representative flow cytometry overlays display the kinetics of co-expression of vascular endothelial growth factor receptor-2 (VEGFR2) and CD34 (a), and CD31 and CD34 (b) upon induction of H1-hESCs with BMP4 (Gi.F.B), VEGF (Gi.F.V) and BMP4 + VEGF (Gi.F.BV) over a differentiation period of 5 days
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Fig3: Flow cytometry analysis of induction of H1-hESCs towards endothelial lineage. Representative flow cytometry overlays display the kinetics of co-expression of vascular endothelial growth factor receptor-2 (VEGFR2) and CD34 (a), and CD31 and CD34 (b) upon induction of H1-hESCs with BMP4 (Gi.F.B), VEGF (Gi.F.V) and BMP4 + VEGF (Gi.F.BV) over a differentiation period of 5 days

Mentions: Inhibition of GSK-3 followed by bFGF exposure drives the hESCs towards lateral plate mesoderm as evidenced by the upregulation of VEGFR2 and downregulation of PS and endoderm-related genes. We next sought to investigate the potential of these lateral plate mesoderm cells to commit towards endothelial lineage. Differentiation was performed using BMP4 and VEGF after initial treatment with CHIR99021 and bFGF as illustrated in Fig. 2a. The kinetics of differentiation towards endothelial lineage was monitored using VEGFR2 (an early marker for lateral plate mesoderm-derived progenitors), CD34 (early marker for progenitors with potential to differentiate towards hemato-endothelial lineage), CD31 (pan-endothelial lineage marker) and platelet-derived growth factor (PDGF)Rβ (early marker for paraxial mesoderm). Real-time PCR analysis (Fig. 2b) revealed BMP4 supplementation resulted in upregulation of VEGFR2, but had minimal effect on the expression levels of CD34 and CD31. On the contrary, treatment with VEGF resulted in a modest increase in the transcript levels of CD34, CD31 and VEGFR2 and downregulation of PDGFRβ, while combined modulation with BMP4 and VEGF resulted in marked upregulation of CD34, CD31 and VEGFR2 and marked downregulation of PDGFRβ. Furthermore, the pluripotency markers were markedly downregulated under all the three differentiation conditions (Fig. 2c). In accordance with the real-time PCR data, time-course flow cytometry analysis (Fig. 3) revealed the gradual emergence of a VEGFR2+ population with BMP4 supplementation, but only a small subset of this population co-express CD34 (~6.5 % on day 5), while the addition of VEGF resulted in the gradual appearance of CD34+ cells that co-express VEGFR2 and CD31 and account for ~54 % of the differentiated cells by the fifth day of differentiation. Furthermore, combined treatment with BMP4 and VEGF resulted in a robust emergence of CD34+VEGFR2+/ CD34+CD31+ cells which accounted for ~95 % of the differentiated cells by the fifth day of differentiation. In all three differentiation conditions, time-course flow cytometry plots also reveal the temporal emergence of a VEGFR2+ population that gradually attains CD34 and CD31 positivity. These findings suggest the role of VEGF in induction to lateral plate mesodermal progenitors (VEGFR2+ cells) and further to endothelial progenitors (CD34+CD31+VEGFR2+ cells) and a synergistic role of BMP4 resulting in robust commitment to the endothelial lineage. We verified the robustness of the protocol using H9-hESCs which also yielded ~90 % of cells positive for VEGFR2, CD34 and CD31 (Additional file 1: Figure S5). In conclusion, step-wise treatment of hESCs with GSK-3 inhibitor, bFGF followed by BMP4 and VEGF results in sequential emergence of Brachyury+ PS , VEGFR2+ early mesoderm/lateral mesoderm cells followed by robust commitment to endothelial lineage yielding ~90–95 % of CD34+CD31+(VEGFR2+) endothelial progenitor cells within a differentiation span of 5 days under feeder-free, and chemically defined conditions.Fig. 3


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)

Flow cytometry analysis of induction of H1-hESCs towards endothelial lineage. Representative flow cytometry overlays display the kinetics of co-expression of vascular endothelial growth factor receptor-2 (VEGFR2) and CD34 (a), and CD31 and CD34 (b) upon induction of H1-hESCs with BMP4 (Gi.F.B), VEGF (Gi.F.V) and BMP4 + VEGF (Gi.F.BV) over a differentiation period of 5 days
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4697311&req=5

Fig3: Flow cytometry analysis of induction of H1-hESCs towards endothelial lineage. Representative flow cytometry overlays display the kinetics of co-expression of vascular endothelial growth factor receptor-2 (VEGFR2) and CD34 (a), and CD31 and CD34 (b) upon induction of H1-hESCs with BMP4 (Gi.F.B), VEGF (Gi.F.V) and BMP4 + VEGF (Gi.F.BV) over a differentiation period of 5 days
Mentions: Inhibition of GSK-3 followed by bFGF exposure drives the hESCs towards lateral plate mesoderm as evidenced by the upregulation of VEGFR2 and downregulation of PS and endoderm-related genes. We next sought to investigate the potential of these lateral plate mesoderm cells to commit towards endothelial lineage. Differentiation was performed using BMP4 and VEGF after initial treatment with CHIR99021 and bFGF as illustrated in Fig. 2a. The kinetics of differentiation towards endothelial lineage was monitored using VEGFR2 (an early marker for lateral plate mesoderm-derived progenitors), CD34 (early marker for progenitors with potential to differentiate towards hemato-endothelial lineage), CD31 (pan-endothelial lineage marker) and platelet-derived growth factor (PDGF)Rβ (early marker for paraxial mesoderm). Real-time PCR analysis (Fig. 2b) revealed BMP4 supplementation resulted in upregulation of VEGFR2, but had minimal effect on the expression levels of CD34 and CD31. On the contrary, treatment with VEGF resulted in a modest increase in the transcript levels of CD34, CD31 and VEGFR2 and downregulation of PDGFRβ, while combined modulation with BMP4 and VEGF resulted in marked upregulation of CD34, CD31 and VEGFR2 and marked downregulation of PDGFRβ. Furthermore, the pluripotency markers were markedly downregulated under all the three differentiation conditions (Fig. 2c). In accordance with the real-time PCR data, time-course flow cytometry analysis (Fig. 3) revealed the gradual emergence of a VEGFR2+ population with BMP4 supplementation, but only a small subset of this population co-express CD34 (~6.5 % on day 5), while the addition of VEGF resulted in the gradual appearance of CD34+ cells that co-express VEGFR2 and CD31 and account for ~54 % of the differentiated cells by the fifth day of differentiation. Furthermore, combined treatment with BMP4 and VEGF resulted in a robust emergence of CD34+VEGFR2+/ CD34+CD31+ cells which accounted for ~95 % of the differentiated cells by the fifth day of differentiation. In all three differentiation conditions, time-course flow cytometry plots also reveal the temporal emergence of a VEGFR2+ population that gradually attains CD34 and CD31 positivity. These findings suggest the role of VEGF in induction to lateral plate mesodermal progenitors (VEGFR2+ cells) and further to endothelial progenitors (CD34+CD31+VEGFR2+ cells) and a synergistic role of BMP4 resulting in robust commitment to the endothelial lineage. We verified the robustness of the protocol using H9-hESCs which also yielded ~90 % of cells positive for VEGFR2, CD34 and CD31 (Additional file 1: Figure S5). In conclusion, step-wise treatment of hESCs with GSK-3 inhibitor, bFGF followed by BMP4 and VEGF results in sequential emergence of Brachyury+ PS , VEGFR2+ early mesoderm/lateral mesoderm cells followed by robust commitment to endothelial lineage yielding ~90–95 % of CD34+CD31+(VEGFR2+) endothelial progenitor cells within a differentiation span of 5 days under feeder-free, and chemically defined conditions.Fig. 3

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