Journal of Veterinary Science

The Korean Society of Veterinary Science (대한수의학회)
권호 표지
DOI QR코드

DOI QR Code

Derivation of endothelial cells from porcine induced pluripotent stem cells by optimized single layer culture system

  • Wei, Renyue (College of Life Science, Northeast Agricultural University) ;
  • Lv, Jiawei (College of Life Science, Northeast Agricultural University) ;
  • Li, Xuechun (College of Life Science, Northeast Agricultural University) ;
  • Li, Yan (College of Life Science, Northeast Agricultural University) ;
  • Xu, Qianqian (College of Life Science, Northeast Agricultural University) ;
  • Jin, Junxue (College of Life Science, Northeast Agricultural University) ;
  • Zhang, Yu (College of Life Science, Northeast Agricultural University) ;
  • Liu, Zhonghua (College of Life Science, Northeast Agricultural University)
  • Received : 2019.08.27
  • Accepted : 2019.11.21
  • Published : 2020.01.31
Download PDF
⟨ Previous Next ⟩

Abstract

Regenerative therapy holds great promise in the development of cures of some untreatable diseases such as cardiovascular diseases, and pluripotent stem cells (PSCs) including induced PSCs (iPSCs) are the most important regenerative seed cells. Recently, differentiation of human PSCs into functional tissues and cells in vitro has been widely reported. However, although porcine reports are rare they are quite essential, as the pig is an important animal model for the in vitro generation of human organs. In this study, we reprogramed porcine embryonic fibroblasts into porcine iPSCs (piPSCs), and differentiated them into cluster of differentiation 31 (CD31)-positive endothelial cells (ECs) (piPSC-derived ECs, piPS-ECs) using an optimized single-layer culture method. During differentiation, we observed that a combination of GSK3β inhibitor (CHIR99021) and bone morphogenetic protein 4 (BMP4) promoted mesodermal differentiation, resulting in higher proportions of CD31-positive cells than those from separate CHIR99021 or BMP4 treatment. Importantly, the piPS-ECs showed comparable morphological and functional properties to immortalized porcine aortic ECs, which are capable of taking up low-density lipoprotein and forming network structures on Matrigel. Our study, which is the first trial on a species other than human and mouse, has provided an optimized single-layer culture method for obtaining ECs from porcine PSCs. Our approach can be beneficial when evaluating autologous EC transplantation in pig models.

Keywords

Acknowledgement

We thank Dr. Jiangqiang Wang from Northeast Agricultural University for his valuable discussion during the manuscript preparation. Thanks also to Dr. Yu Zhang for financial support (University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province, UNPYSCT-2016010).

References

  1. Hadi HA, Carr CS, Al Suwaidi J. Endothelial dysfunction: cardiovascular risk factors, therapy, and outcome. Vasc Health Risk Manag 2005;1:183-198.
  2. Tsifaki M, Kelaini S, Caines R, Yang C, Margariti A. Regenerating the cardiovascular system through cell reprogramming; current approaches and a look into the future. Front Cardiovasc Med 2018;5:109. https://doi.org/10.3389/fcvm.2018.00109
  3. Lee SJ, Kim KH, Yoon YS. Generation of human pluripotent stem cell-derived endothelial cells and their therapeutic utility. Curr Cardiol Rep 2018;20:45. https://doi.org/10.1007/s11886-018-0985-8
  4. Pearson S, Sroczynska P, Lacaud G, Kouskoff V. The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF. Development 2008;135:1525-1535. https://doi.org/10.1242/dev.011767
  5. Sirard C, de la Pompa JL, Elia A, Itie A, Mirtsos C, Cheung A, Hahn S, Wakeham A, Schwartz L, Kern SE, Rossant J, Mak TW. The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo. Genes Dev 1998;12:107-119. https://doi.org/10.1101/gad.12.1.107
  6. Lin Y, Gil CH, Yoder MC. Differentiation, evaluation, and application of human induced pluripotent stem cell-derived endothelial cells. Arterioscler Thromb Vasc Biol 2017;37:2014-2025. https://doi.org/10.1161/atvbaha.117.309962
  7. Shi Y, Inoue H, Wu JC, Yamanaka S. Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov 2017;16:115-130. https://doi.org/10.1038/nrd.2016.245
  8. Harding A, Cortez-Toledo E, Magner NL, Beegle JR, Coleal-Bergum DP, Hao D, Wang A, Nolta JA, Zhou P. Highly efficient differentiation of endothelial cells from pluripotent stem cells requires the MAPK and the PI3K pathways. Stem Cells 2017;35:909-919. https://doi.org/10.1002/stem.2577
  9. 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;9:1514-1531. https://doi.org/10.1038/nprot.2014.102
  10. Song G, Li X, Shen Y, Qian L, Kong X, Chen M, Cao K, Zhang F. Transplantation of iPSc restores cardiac function by promoting angiogenesis and ameliorating cardiac remodeling in a post-infarcted swine model. Cell Biochem Biophys 2015;71:1463-1473. https://doi.org/10.1007/s12013-014-0369-7
  11. Kim E, Hwang SU, Yoo H, Yoon JD, Jeon Y, Kim H, Jeung EB, Lee CK, Hyun SH. Putative embryonic stem cells derived from porcine cloned blastocysts using induced pluripotent stem cells as donors. Theriogenology 2016;85:601-616. https://doi.org/10.1016/j.theriogenology.2015.09.051
  12. Ezashi T, Telugu BP, Alexenko AP, Sachdev S, Sinha S, Roberts RM. Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S A 2009;106:10993-10998. https://doi.org/10.1073/pnas.0905284106
  13. Wu Z, Chen J, Ren J, Bao L, Liao J, Cui C, Rao L, Li H, Gu Y, Dai H, Zhu H, Teng X, Cheng L, Xiao L. Generation of pig induced pluripotent stem cells with a drug-inducible system. J Mol Cell Biol 2009;1:46-54. https://doi.org/10.1093/jmcb/mjp003
  14. Wang J, Gu Q, Hao J, Jia Y, Xue B, Jin H, Ma J, Wei R, Hai T, Kong Q, Bou G, Xia P, Zhou Q, Wang L, Liu Z. Tbx3 and Nr5α2 play important roles in pig pluripotent stem cells. Stem Cell Rev Rep 2013;9:700-708. https://doi.org/10.1007/s12015-013-9439-2
  15. Li X, Shan ZY, Wu YS, Shen XH, Liu CJ, Shen JL, Liu ZH, Lei L. Generation of neural progenitors from induced Bama miniature pig pluripotent cells. Reproduction 2013;147:65-72. https://doi.org/10.1530/REP-13-0196
  16. Gu M, Nguyen PK, Lee AS, Xu D, Hu S, Plews JR, Han L, Huber BC, Lee WH, Gong Y, de Almeida PE, Lyons J, Ikeno F, Pacharinsak C, Connolly AJ, Gambhir SS, Robbins RC, Longaker MT, Wu JC. Microfluidic single-cell analysis shows that porcine induced pluripotent stem cell-derived endothelial cells improve myocardial function by paracrine activation. Circ Res 2012;111:882-893. https://doi.org/10.1161/CIRCRESAHA.112.269001
  17. Almalki SG, Agrawal DK. ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells. Stem Cell Res Ther 2017;8:113. https://doi.org/10.1186/s13287-017-0568-4
  18. Almalki SG, Llamas Valle Y, Agrawal DK. MMP-2 and MMP-14 silencing inhibits VEGFR2 cleavage and induces the differentiation of porcine adipose-derived mesenchymal stem cells to endothelial cells. Stem Cells Transl Med 2017;6:1385-1398. https://doi.org/10.1002/sctm.16-0329
  19. Schlegel F, Appler M, Halling M, Smit FE, Mohr FW, Dhein S, Dohmen PM. Reprogramming bone marrow stem cells to functional endothelial cells in a mini pig animal model. Med Sci Monit Basic Res 2017;23:285-294. https://doi.org/10.12659/MSMBR.905081
  20. Zhao H, Nie J, Zhu X, Lu Y, Liang X, Xu H, Yang X, Zhang Y, Lu K, Lu S. In vitro differentiation of spermatogonial stem cells using testicular cells from Guangxi Bama mini-pig. J Vet Sci 2018;19:592-599. https://doi.org/10.4142/jvs.2018.19.5.592
  21. 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;22:1893-1906. https://doi.org/10.1089/scd.2012.0590
  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. https://doi.org/10.1186/1756-0500-7-273
  23. Park C, Afrikanova I, Chung YS, Zhang WJ, Arentson E, Fong Gh G, Rosendahl A, Choi K. A hierarchical order of factors in the generation of FLK1- and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells. Development 2004;131:2749-2762. https://doi.org/10.1242/dev.01130
  24. Winnier G, Blessing M, Labosky PA, Hogan BL. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 1995;9:2105-2116. https://doi.org/10.1101/gad.9.17.2105
  25. Chang H, Huylebroeck D, Verschueren K, Guo Q, Matzuk MM, Zwijsen A. Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects. Development 1999;126:1631-1642. https://doi.org/10.1242/dev.126.8.1631
  26. Huber TL, Zhou Y, Mead PE, Zon LI. Cooperative effects of growth factors involved in the induction of hematopoietic mesoderm. Blood 1998;92:4128-4137. https://doi.org/10.1182/blood.v92.11.4128
  27. Zhang P, Li J, Tan Z, Wang C, Liu T, Chen L, Yong J, Jiang W, Sun X, Du L, Ding M, Deng H. Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. Blood 2008;111:1933-1941. https://doi.org/10.1182/blood-2007-02-074120
  28. Sriram G, Tan JY, Islam I, Rufaihah AJ, Cao T. Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions. Stem Cell Res Ther 2015;6:261. https://doi.org/10.1186/s13287-015-0260-5
  29. Adams WJ, Zhang Y, Cloutier J, Kuchimanchi P, Newton G, Sehrawat S, Aird WC, Mayadas TN, Luscinskas FW, Garcia-Cardena G. Functional vascular endothelium derived from human induced pluripotent stem cells. Stem Cell Reports 2013;1:105-113. https://doi.org/10.1016/j.stemcr.2013.06.007
  30. Patsch C, Challet-Meylan L, Thoma EC, Urich E, Heckel T, O'Sullivan JF, Grainger SJ, Kapp FG, Sun L, Christensen K, Xia Y, Florido MH, He W, Pan W, Prummer M, Warren CR, Jakob-Roetne R, Certa U, Jagasia R, Freskgard PO, Adatto I, Kling D, Huang P, Zon LI, Chaikof EL, Gerszten RE, Graf M, Iacone R, Cowan CA. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat Cell Biol 2015;17:994-1003. https://doi.org/10.1038/ncb3205
  31. Cha Y, Heo SH, Ahn HJ, Yang SK, Song JH, Suh W, Park KS. Tcea3 regulates the vascular differentiation potential of mouse embryonic stem cells. Gene Expr 2013;16:25-30. https://doi.org/10.3727/105221613X13776146743343
  32. 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;3:768-776. https://doi.org/10.1038/nprot.2008.42
  33. Ikuno T, Masumoto H, Yamamizu K, Yoshioka M, Minakata K, Ikeda T, Sakata R, Yamashita JK. Efficient and robust differentiation of endothelial cells from human induced pluripotent stem cells via lineage control with VEGF and cyclic AMP. PLoS One 2017;12:e0173271. https://doi.org/10.1371/journal.pone.0173271
  34. Chen T, Margariti A, Kelaini S, Cochrane A, Guha ST, Hu Y, Stitt AW, Zhang L, Xu Q. MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling. Stem Cells 2015;33:1405-1418. https://doi.org/10.1002/stem.1930
  35. Ginsberg M, James D, Ding BS, Nolan D, Geng F, Butler JM, Schachterle W, Pulijaal VR, Mathew S, Chasen ST, Xiang J, Rosenwaks Z, Shido K, Elemento O, Rabbany SY, Rafii S. Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGFβ suppression. Cell 2012;151:559-575. https://doi.org/10.1016/j.cell.2012.09.032
  36. Morita R, Suzuki M, Kasahara H, Shimizu N, Shichita T, Sekiya T, Kimura A, Sasaki K, Yasukawa H, Yoshimura A. ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells. Proc Natl Acad Sci U S A 2015;112:160-165. https://doi.org/10.1073/pnas.1413234112