Limits...
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

In vivo safety and functionality assessment of hESC-derived arterial and venous endothelial cells. a Representative hematoxylin and eosin (H&E) stained micrographs of Matrigel plug study showing absence of microvessels in Matrigel-only control, while the others show the formation of perfused microvessels by engrafted hESC-derived arterial and venous ECs. b Histochemical micrographs of microvessels formed by hESC-derived arterial and venous ECs stained for hCD31, hCollagen-IV, hEphrin-B2 and hEph-B4. c Representative photographs showing the presence or absence of teratoma formation in immunodeficient mice. d Representative H&E stained micrographs showing the formation of teratoma by H1-hESCs with evidence of differentiation to derivatives of three germ layers: ectoderm (hair follicles, <), mesoderm (cartilage, *), and endoderm (glandular structures, +). e Representative H&E stained micrographs showing the absence of teratoma formation by H1-hESCs derived arterial and venous ECs. Scale bars in (a) and (b) = 100 μm; in (d) and (e) = 500 μm. Art-EC Arterial endothelial cells, hESC Human embryonic stem cells, Ven-EC Venous endothelial cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: In vivo safety and functionality assessment of hESC-derived arterial and venous endothelial cells. a Representative hematoxylin and eosin (H&E) stained micrographs of Matrigel plug study showing absence of microvessels in Matrigel-only control, while the others show the formation of perfused microvessels by engrafted hESC-derived arterial and venous ECs. b Histochemical micrographs of microvessels formed by hESC-derived arterial and venous ECs stained for hCD31, hCollagen-IV, hEphrin-B2 and hEph-B4. c Representative photographs showing the presence or absence of teratoma formation in immunodeficient mice. d Representative H&E stained micrographs showing the formation of teratoma by H1-hESCs with evidence of differentiation to derivatives of three germ layers: ectoderm (hair follicles, <), mesoderm (cartilage, *), and endoderm (glandular structures, +). e Representative H&E stained micrographs showing the absence of teratoma formation by H1-hESCs derived arterial and venous ECs. Scale bars in (a) and (b) = 100 μm; in (d) and (e) = 500 μm. Art-EC Arterial endothelial cells, hESC Human embryonic stem cells, Ven-EC Venous endothelial cells

Mentions: After assessment of the in vitro functionality of the hESC-derived arterial and venous ECs, we investigated whether these ECs have the ability to form functional microvessels upon transplantation, by injecting ECs suspended in Matrigel subcutaneously into the dorsal region of immunodeficient mice. Matrigel plugs implanted without cells exhibited no evidence of microvessels within the matrix (Fig. 7a). On the other hand, the Matrigel plugs implanted along with hESC-Art-ECs or hESC-Ven-ECs showed the presence of microvessels within the matrix. Additionally, these microvessels were seen to be connected to the host circulation, as evidenced by the presence of red blood cells within the lumen (Fig. 7a). Furthermore, these microvessels were reactive for anti-human CD31 and anti-human collagen-IV, indicating that they were of human origin (Fig. 7b). Furthermore, the hESC-Art-ECs and hESC-Ven-ECs largely maintained their respective phenotypes in vivo, as evidenced by the expression of Ephrin-B2 and Eph-B4, respectively (Fig. 7b). However, few microvessels formed by hESC-Art-ECs showed the expression of venous markers and vice versa.Fig. 7


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)

In vivo safety and functionality assessment of hESC-derived arterial and venous endothelial cells. a Representative hematoxylin and eosin (H&E) stained micrographs of Matrigel plug study showing absence of microvessels in Matrigel-only control, while the others show the formation of perfused microvessels by engrafted hESC-derived arterial and venous ECs. b Histochemical micrographs of microvessels formed by hESC-derived arterial and venous ECs stained for hCD31, hCollagen-IV, hEphrin-B2 and hEph-B4. c Representative photographs showing the presence or absence of teratoma formation in immunodeficient mice. d Representative H&E stained micrographs showing the formation of teratoma by H1-hESCs with evidence of differentiation to derivatives of three germ layers: ectoderm (hair follicles, <), mesoderm (cartilage, *), and endoderm (glandular structures, +). e Representative H&E stained micrographs showing the absence of teratoma formation by H1-hESCs derived arterial and venous ECs. Scale bars in (a) and (b) = 100 μm; in (d) and (e) = 500 μm. Art-EC Arterial endothelial cells, hESC Human embryonic stem cells, Ven-EC Venous endothelial cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: In vivo safety and functionality assessment of hESC-derived arterial and venous endothelial cells. a Representative hematoxylin and eosin (H&E) stained micrographs of Matrigel plug study showing absence of microvessels in Matrigel-only control, while the others show the formation of perfused microvessels by engrafted hESC-derived arterial and venous ECs. b Histochemical micrographs of microvessels formed by hESC-derived arterial and venous ECs stained for hCD31, hCollagen-IV, hEphrin-B2 and hEph-B4. c Representative photographs showing the presence or absence of teratoma formation in immunodeficient mice. d Representative H&E stained micrographs showing the formation of teratoma by H1-hESCs with evidence of differentiation to derivatives of three germ layers: ectoderm (hair follicles, <), mesoderm (cartilage, *), and endoderm (glandular structures, +). e Representative H&E stained micrographs showing the absence of teratoma formation by H1-hESCs derived arterial and venous ECs. Scale bars in (a) and (b) = 100 μm; in (d) and (e) = 500 μm. Art-EC Arterial endothelial cells, hESC Human embryonic stem cells, Ven-EC Venous endothelial cells
Mentions: After assessment of the in vitro functionality of the hESC-derived arterial and venous ECs, we investigated whether these ECs have the ability to form functional microvessels upon transplantation, by injecting ECs suspended in Matrigel subcutaneously into the dorsal region of immunodeficient mice. Matrigel plugs implanted without cells exhibited no evidence of microvessels within the matrix (Fig. 7a). On the other hand, the Matrigel plugs implanted along with hESC-Art-ECs or hESC-Ven-ECs showed the presence of microvessels within the matrix. Additionally, these microvessels were seen to be connected to the host circulation, as evidenced by the presence of red blood cells within the lumen (Fig. 7a). Furthermore, these microvessels were reactive for anti-human CD31 and anti-human collagen-IV, indicating that they were of human origin (Fig. 7b). Furthermore, the hESC-Art-ECs and hESC-Ven-ECs largely maintained their respective phenotypes in vivo, as evidenced by the expression of Ephrin-B2 and Eph-B4, respectively (Fig. 7b). However, few microvessels formed by hESC-Art-ECs showed the expression of venous markers and vice versa.Fig. 7

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