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

Stem Cell Research & Therapy

Tong Cao¹  Abdul Jalil Rufaihah⁴  Intekhab Islam²  Jia Yong Tan⁵  Gopu Sriram³ 

[1] Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore;Oral and Maxillofacial Surgery Disciplines, Faculty of Dentistry, National University of Singapore, Singapore 119083, Singapore;Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Groove, #06-06 Immunos, Singapore 138648, Singapore;Singapore-Technion Alliance For Research and Technology (START) Regenerative Medicine Laboratory, Campus for Research Excellence And Technological Enterprise (CREATE), Singapore 138602, Singapore;Oral Sciences Disciplines, Faculty of Dentistry, National University of Singapore, Singapore 119083, Singapore

关键词: Feeder-free;    Serum-free;    Venous;    Arterial;    Endothelial differentiation;    Human embryonic stem cells;   

Others  :  1235185
DOI  :  10.1186/s13287-015-0260-5

 received in 2015-03-27, accepted in 2015-08-26,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Sriram et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

【 参考文献 】

• [1]Torres-Vazquez J, Kamei M, Weinstein BM: Molecular distinction between arteries and veins. Cell Tissue Res 2003, 314(1):43-59.
• [2]Aird WC: Phenotypic heterogeneity of the endothelium: II. Representative vascular beds. Circ Res 2007, 100(2):174-90.
• [3]Coultas L, Chawengsaksophak K, Rossant J: Endothelial cells and VEGF in vascular development. Nature 2005, 438(7070):937-45.
• [4]Rosa V, Toh WS, Cao T, Shim W: Inducing pluripotency for disease modeling, drug development and craniofacial applications. Expert Opin Biol Ther 2014, 14(9):1233-40.
• [5]Kane NM, Xiao Q, Baker AH, Luo Z, Xu Q, Emanueli C: Pluripotent stem cell differentiation into vascular cells: a novel technology with promises for vascular re(generation). Pharmacol Ther 2011, 129(1):29-49.
• [6]Mercola M, Colas A, Willems E: Induced pluripotent stem cells in cardiovascular drug discovery. Circ Res 2013, 112(3):534-48.
• [7]Vinoth KJ, Manikandan J, Sethu S, Balakrishnan L, Heng A, Lu K, et al.: Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening. J Biotechnol. 2014, 184:154-68.
• [8]Ferreira LS, Gerecht S, Shieh HF, Watson N, Rupnick MA, Dallabrida SM, et al.: Vascular progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle like cells and form vascular networks in vivo. Circ Res 2007, 101(3):286-94.
• [9]Li Z, Wilson KD, Smith B, Kraft DL, Jia F, Huang M, et al.: Functional and transcriptional characterization of human embryonic stem cell-derived endothelial cells for treatment of myocardial infarction. PLoS One 2009, 4(12):e8443.
• [10]James D, Nam HS, Seandel M, Nolan D, Janovitz T, Tomishima M, et al.: Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFbeta inhibition is Id1 dependent. Nat Biotechnol 2010, 28(2):161-6.
• [11]Levenberg S, Ferreira LS, Chen-Konak L, Kraehenbuehl TP, Langer R: Isolation, differentiation and characterization of vascular cells derived from human embryonic stem cells. Nat Protoc 2010, 5(6):1115-26.
• [12]Hill KL, Obrtlikova P, Alvarez DF, King JA, Keirstead SA, Allred JR, et al.: Human embryonic stem cell-derived vascular progenitor cells capable of endothelial and smooth muscle cell function. Exp Hematol 2010, 38(3):246-57.
• [13]Rufaihah AJ, Huang NF, Jame S, Lee JC, Nguyen HN, Byers B, et al.: Endothelial cells derived from human iPSCS increase capillary density and improve perfusion in a mouse model of peripheral arterial disease. Arterioscler Thromb Vasc Biol 2011, 31(11):e72-9.
• [14]Margariti A, Winkler B, Karamariti E, Zampetaki A, Tsai TN, Baban D, et al.: Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and re-endothelialization in tissue-engineered vessels. Proc Natl Acad Sci U S A 2012, 109(34):13793-8.
• [15]White MP, Rufaihah AJ, Liu L, Ghebremariam YT, Ivey KN, Cooke JP, et al.: Limited gene expression variation in human embryonic stem cell and induced pluripotent stem cell-derived endothelial cells. Stem Cells 2013, 31(1):92-103.
• [16]Kaupisch A, Kennedy L, Stelmanis V, Tye B, Kane NM, Mountford JC, et al.: Derivation of vascular endothelial cells from human embryonic stem cells under GMP-compliant conditions: towards clinical studies in ischaemic disease. J Cardiovasc Transl Res 2012, 5(5):605-17.
• [17]Tan JY, Sriram G, Rufaihah AJ, Neoh KG, Cao T: Efficient derivation of lateral plate and paraxial mesoderm subtypes from human embryonic stem cells through GSKi-mediated differentiation. Stem Cells Dev 2013, 22(13):1893-906.
• [18]Heng BC, Haider H, Sim EK, Cao T, Ng SC: Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro. Cardiovasc Res 2004, 62(1):34-42.
• [19]Kidwai FK, Liu H, Toh WS, Fu X, Jokhun DS, Movahednia MM, et al.: Differentiation of human embryonic stem cells into clinically amenable keratinocytes in an autogenic environment. J Invest Dermatol 2013, 133(3):618-28.
• [20]Fu X, Toh WS, Liu H, Lu K, Li M, Cao T: Establishment of clinically compliant human embryonic stem cells in an autologous feeder-free system. Tissue Eng Part C Methods 2011, 17(9):927-37.
• [21]Ng ES, Davis R, Stanley EG, Elefanty AG: A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nat Protoc 2008, 3(5):768-76.
• [22]Naujok O, Lentes J, Diekmann U, Davenport C, Lenzen S: Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. BMC Res Notes. 2014, 7:273. BioMed Central Full Text
• [23]Naujok O, Diekmann U, Lenzen S: The generation of definitive endoderm from human embryonic stem cells is initially independent from Activin A but requires canonical Wnt-signaling. Stem Cell Rev 2014, 10(4):480-93.
• [24]Shelton M, Metz J, Liu J, Carpenedo Richard L, Demers S-P, Stanford William L, et al.: Derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells. Stem Cell Reports 2014, 3(3):516-29.
• [25]Turner DA, Rue P, Mackenzie JP, Davies E, Arias A: Brachyury cooperates with Wnt/ss-Catenin signalling to elicit Primitive Streak like behaviour in differentiating mouse ES cells. BMC Biol 2014, 12(1):63. BioMed Central Full Text
• [26]Pick M, Azzola L, Mossman A, Stanley EG, Elefanty AG: Differentiation of human embryonic stem cells in serum-free medium reveals distinct roles for bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and fibroblast growth factor 2 in hematopoiesis. Stem Cells 2007, 25(9):2206-14.
• [27]Yurugi-Kobayashi T, Itoh H, Schroeder T, Nakano A, Narazaki G, Kita F, et al.: Adrenomedullin/cyclic AMP pathway induces Notch activation and differentiation of arterial endothelial cells from vascular progenitors. Arterioscler Thromb Vasc Biol 2006, 26(9):1977-84.
• [28]Rufaihah AJ, Huang NF, Kim J, Herold J, Volz KS, Park TS, et al.: Human induced pluripotent stem cell-derived endothelial cells exhibit functional heterogeneity. Am J Transl Res 2013, 5(1):21-35.
• [29]Lanner F, Sohl M, Farnebo F: Functional arterial and venous fate is determined by graded VEGF signaling and notch status during embryonic stem cell differentiation. Arterioscler Thromb Vasc Biol 2007, 27(3):487-93.
• [30]Yamamizu K, Matsunaga T, Uosaki H, Fukushima H, Katayama S, Hiraoka-Kanie M, et al.: Convergence of Notch and beta-catenin signaling induces arterial fate in vascular progenitors. J Cell Biol 2010, 189(2):325-38.
• [31]Lawson ND, Vogel AM, Weinstein BM: sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell 2002, 3(1):127-36.
• [32]Lawson ND, Scheer N, Pham VN, Kim CH, Chitnis AB, Campos-Ortega JA, et al.: Notch signaling is required for arterial-venous differentiation during embryonic vascular development. Development 2001, 128(19):3675-83.
• [33]Aranguren XL, Luttun A, Clavel C, Moreno C, Abizanda G, Barajas MA, et al.: In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells. Blood 2007, 109(6):2634-42.
• [34]Cooley LS, Edwards DR: New insights into the plasticity of the endothelial phenotype. Biochem Soc Trans 2011, 39(6):1639-43.
• [35]Moyon D, Pardanaud L, Yuan L, Breant C, Eichmann A: Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo. Development 2001, 128(17):3359-70.
• [36]Nunes SS, Rekapally H, Chang CC, Hoying JB: Vessel arterial-venous plasticity in adult neovascularization. PLoS One 2011, 6(11):e27332.
• [37]Harvey NL, Oliver G: Choose your fate: artery, vein or lymphatic vessel? Curr Opin Genet Dev 2004, 14(5):499-505.
• [38]Oliver G, Srinivasan RS: Endothelial cell plasticity: how to become and remain a lymphatic endothelial cell. Development 2010, 137(3):363-72.
• [39]Nolan DJ, Ginsberg M, Israely E, Palikuqi B, Poulos MG, James D, et al.: Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration. Dev Cell 2013, 26(2):204-19.
• [40]Sahara M, Hansson EM, Wernet O, Lui KO, Spater D, Chien KR: Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells. Cell Res 2014, 24(7):820-41.
• [41]Orlova VV, van den Hil FE, Petrus-Reurer S, Drabsch Y, Ten Dijke P, Mummery CL: Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. Nat Protoc 2014, 9(6):1514-31.
• [42]Sturgeon CM, Ditadi A, Awong G, Kennedy M, Keller G: Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 2014, 32(6):554-61.
• [43]Borchin B, Chen J, Barberi T: Derivation and FACS-mediated purification of PAX3+/PAX7+ skeletal muscle precursors from human pluripotent stem cells. Stem Cell Rep 2013, 1(6):620-31.
• [44]Cao N, Liang H, Huang J, Wang J, Chen Y, Chen Z, et al.: Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res 2013, 23(9):1119-32.
• [45]Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, et al.: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A 2012, 109(27):E1848-57.
• [46]Bai H, Gao Y, Arzigian M, Wojchowski DM, Wu WS, Wang ZZ: BMP4 regulates vascular progenitor development in human embryonic stem cells through a Smad-dependent pathway. J Cell Biochem 2010, 109(2):363-74.
• [47]Song SH, Jung W, Kim KL, Hong W, Kim HO, Lee KA, et al.: Distinct transcriptional profiles of angioblasts derived from human embryonic stem cells. Exp Cell Res 2013, 319(8):1136-45.
• [48]Narazaki G, Uosaki H, Teranishi M, Okita K, Kim B, Matsuoka S, et al.: Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells. Circulation 2008, 118(5):498-506.
• [49]Boyer-Di Ponio J, El-Ayoubi F, Glacial F, Ganeshamoorthy K, Driancourt C, Godet M, et al.: Instruction of circulating endothelial progenitors in vitro towards specialized blood–brain barrier and arterial phenotypes. PLoS One 2014, 9(1):e84179.
• [50]Lippmann ES, Al-Ahmad A, Azarin SM, Palecek SP, Shusta EV: A retinoic acid-enhanced, multicellular human blood–brain barrier model derived from stem cell sources. Sci Rep. 2014, 4:4160.
• [51]Lippmann ES, Azarin SM, Kay JE, Nessler RA, Wilson HK, Al-Ahmad A, et al.: Derivation of blood–brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 2012, 30(8):783-91.
• [52]Kurian L, Sancho-Martinez I, Nivet E, Aguirre A, Moon K, Pendaries C, et al.: Conversion of human fibroblasts to angioblast-like progenitor cells. Nat Methods 2013, 10(1):77-83.
• [53]Samuel R, Daheron L, Liao S, Vardam T, Kamoun WS, Batista A, et al.: Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells. Proc Natl Acad Sci U S A 2013, 110(31):12774-9.
• [54]Lei T, Jacob S, Ajil-Zaraa I, Dubuisson JB, Irion O, Jaconi M, et al.: Xeno-free derivation and culture of human embryonic stem cells: current status, problems and challenges. Cell Res 2007, 17(8):682-8.
• [55]Rodin S, Antonsson L, Niaudet C, Simonson OE, Salmela E, Hansson EM, et al.: Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nat Commun. 2014, 5:3195.
• [56]Wang Y, Chou BK, Dowey S, He C, Gerecht S, Cheng L: Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions. Stem Cell Res 2013, 11(3):1103-16.
• [57]Sampsell-Barron T: Xeno-free adaptation and culture of human pluripotent stem cells. Methods Mol Biol. 2013, 1001:81-97.
• [58]Bergstrom R, Strom S, Holm F, Feki A, Hovatta O: Xeno-free culture of human pluripotent stem cells. Methods Mol Biol. 2011, 767:125-36.
• [59]Rajala K, Lindroos B, Hussein SM, Lappalainen RS, Pekkanen-Mattila M, Inzunza J, et al.: A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells. PLoS One 2010, 5(4):e10246.