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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

Characterization of hESC-derived venous and arterial endothelial cells. a Profiles of transcripts related to endothelial, arterial and venous phenotypes among Ven-ECs and Art-ECs derived from H1-hESCs, H9-hESCs and primary cells (PC: HUVECs and HCAECs). Gene expression levels were normalized to corresponding β-ACTIN values and are represented as relative to undifferentiated hESCs. b Representative photomicrographs of H1-Ven-ECs, H1-Art-ECs, H9-Ven-ECs, H9-Art-ECs, HUVECs, and HCAECs show the cobblestone morphology of ECs under phase contrast microscopy, and immunofluorescence images demonstrate the expression of pan-endothelial markers CD31, VE-Cadherin, and von Willebrand factor (vWF), uptake of Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) and formation of cord-like structures over Matrigel (Green-CalceinAM). Scale bars = 150 μm. Error bars show standard deviation; n ≥ 3. *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, Ven-EC Venous endothelial cells
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Fig5: Characterization of hESC-derived venous and arterial endothelial cells. a Profiles of transcripts related to endothelial, arterial and venous phenotypes among Ven-ECs and Art-ECs derived from H1-hESCs, H9-hESCs and primary cells (PC: HUVECs and HCAECs). Gene expression levels were normalized to corresponding β-ACTIN values and are represented as relative to undifferentiated hESCs. b Representative photomicrographs of H1-Ven-ECs, H1-Art-ECs, H9-Ven-ECs, H9-Art-ECs, HUVECs, and HCAECs show the cobblestone morphology of ECs under phase contrast microscopy, and immunofluorescence images demonstrate the expression of pan-endothelial markers CD31, VE-Cadherin, and von Willebrand factor (vWF), uptake of Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) and formation of cord-like structures over Matrigel (Green-CalceinAM). Scale bars = 150 μm. Error bars show standard deviation; n ≥ 3. *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, Ven-EC Venous endothelial cells

Mentions: Under serum-containing conditions, high concentrations (50 ng/ml) of VEGF have been reported to aid arterial differentiation, while lower concentrations (10 ng/ml) aid venous commitment of mouse ESCs and human iPSCs [27–29]. However, differentiation of hESCs to arterial and venous ECs and, specifically, differentiation under serum-free conditions has not been reported so far. The CD34+CD31+ endothelial progenitors were sorted and further differentiated towards endothelial subtypes in serum-free conditions using commercially available ESFM. Serum-containing endothelial medium typically requires supplementation with FBS (2–5 %), insulin, heparin, ascorbic acid, hydrocortisone, insulin-like growth factor, bFGF, EGF and VEGF, but the serum-free endothelial medium as per manufacturer’s instructions requires supplementation with bFGF (20 ng/ml) and EGF (10 ng/ml) only. Hence we initially carried out the differentiation of the CD34+CD31+ cells in ESFM supplemented with bFGF and EGF for 3–6 passages (Fig. 4a). Differentiation under these conditions yielded 98–99 % CD34+/CD31+/VE-CAD+ ECs (Fig. 4b). Real time RT-PCR analysis demonstrated upregulation of all transcripts associated with endothelial lineage (Fig. 5a). Additionally, immunocytochemistry revealed the expression of CD31, VE-CAD and vWF (Fig. 5b). Further analysis into the arterial and venous phenotype markers showed almost 80–90 % of the cells to be positive for venous markers (NRP2, EPH-B4) while only ~2–10 % of the cells differentiated from H1/H9-hESCs expressed NRP1 and DLL4, and ~13–17 % expressed CXCR4 (Fig. 4b). The endothelial, arterial and venous marker expression profiles were similar to those expressed by human umbilical vein endothelial cells (HUVECs) (Figs. 4 and 5).These observations suggest the commitment of CD34+CD31+ cells towards venous endothelial phenotype and these would be referred to as hESC-Ven-ECs (H1/H9).Fig. 4


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)

Characterization of hESC-derived venous and arterial endothelial cells. a Profiles of transcripts related to endothelial, arterial and venous phenotypes among Ven-ECs and Art-ECs derived from H1-hESCs, H9-hESCs and primary cells (PC: HUVECs and HCAECs). Gene expression levels were normalized to corresponding β-ACTIN values and are represented as relative to undifferentiated hESCs. b Representative photomicrographs of H1-Ven-ECs, H1-Art-ECs, H9-Ven-ECs, H9-Art-ECs, HUVECs, and HCAECs show the cobblestone morphology of ECs under phase contrast microscopy, and immunofluorescence images demonstrate the expression of pan-endothelial markers CD31, VE-Cadherin, and von Willebrand factor (vWF), uptake of Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) and formation of cord-like structures over Matrigel (Green-CalceinAM). Scale bars = 150 μm. Error bars show standard deviation; n ≥ 3. *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, Ven-EC Venous endothelial cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: Characterization of hESC-derived venous and arterial endothelial cells. a Profiles of transcripts related to endothelial, arterial and venous phenotypes among Ven-ECs and Art-ECs derived from H1-hESCs, H9-hESCs and primary cells (PC: HUVECs and HCAECs). Gene expression levels were normalized to corresponding β-ACTIN values and are represented as relative to undifferentiated hESCs. b Representative photomicrographs of H1-Ven-ECs, H1-Art-ECs, H9-Ven-ECs, H9-Art-ECs, HUVECs, and HCAECs show the cobblestone morphology of ECs under phase contrast microscopy, and immunofluorescence images demonstrate the expression of pan-endothelial markers CD31, VE-Cadherin, and von Willebrand factor (vWF), uptake of Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) and formation of cord-like structures over Matrigel (Green-CalceinAM). Scale bars = 150 μm. Error bars show standard deviation; n ≥ 3. *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, Ven-EC Venous endothelial cells
Mentions: Under serum-containing conditions, high concentrations (50 ng/ml) of VEGF have been reported to aid arterial differentiation, while lower concentrations (10 ng/ml) aid venous commitment of mouse ESCs and human iPSCs [27–29]. However, differentiation of hESCs to arterial and venous ECs and, specifically, differentiation under serum-free conditions has not been reported so far. The CD34+CD31+ endothelial progenitors were sorted and further differentiated towards endothelial subtypes in serum-free conditions using commercially available ESFM. Serum-containing endothelial medium typically requires supplementation with FBS (2–5 %), insulin, heparin, ascorbic acid, hydrocortisone, insulin-like growth factor, bFGF, EGF and VEGF, but the serum-free endothelial medium as per manufacturer’s instructions requires supplementation with bFGF (20 ng/ml) and EGF (10 ng/ml) only. Hence we initially carried out the differentiation of the CD34+CD31+ cells in ESFM supplemented with bFGF and EGF for 3–6 passages (Fig. 4a). Differentiation under these conditions yielded 98–99 % CD34+/CD31+/VE-CAD+ ECs (Fig. 4b). Real time RT-PCR analysis demonstrated upregulation of all transcripts associated with endothelial lineage (Fig. 5a). Additionally, immunocytochemistry revealed the expression of CD31, VE-CAD and vWF (Fig. 5b). Further analysis into the arterial and venous phenotype markers showed almost 80–90 % of the cells to be positive for venous markers (NRP2, EPH-B4) while only ~2–10 % of the cells differentiated from H1/H9-hESCs expressed NRP1 and DLL4, and ~13–17 % expressed CXCR4 (Fig. 4b). The endothelial, arterial and venous marker expression profiles were similar to those expressed by human umbilical vein endothelial cells (HUVECs) (Figs. 4 and 5).These observations suggest the commitment of CD34+CD31+ cells towards venous endothelial phenotype and these would be referred to as hESC-Ven-ECs (H1/H9).Fig. 4

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