Objective: This study aim to enhance endothelial differentiation of human embryonic stem cells (hESCs) by transduction of an adenovirus (Ad) vector expressing hVEGF₁₆₅ gene (Ad-hVEGF₁₆₅). Purified hESC-derived CD133⁺ endothelial progenitors were transplanted into a rat myocardial infarct model to assess their ability to contribute to heart regeneration. Methods: Optimal transduction efficiency with high cell viability was achieved by exposing differentiating hESCs to viral particles at a ratio of 1:500 for 4 h on three consecutive days. Results: Reverse transcription-PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF₁₆₅-transduced cells. Additionally, flow cytometric analysis of CD133, a cell surface marker, revealed an approximately fivefold increase of CD133 marker expression in Ad-hVEGF₁₆₅-transduced cells compared with the nontransduced control. Within a rat myocardial infarct model, transplanted CD133⁺ endothelial progenitor cells survived and participated, both actively and passively, in the regeneration of the infarcted myocardium, as seen by an approximately threefold increase in mature blood vessel density (13.62 ± 1.56 vs 5.11 ± 1.23; p < 0.01), as well as significantly reduced infarct size (28% ± 8.2% vs 76% ± 5.6%; p < 0.01) in the transplanted group compared with the culture medium-injected control. There was significant improvement in heart function 6 weeks post-transplantation, as confirmed by regional blood-flow analysis (1.72 ± 0.612 ml/min/g vs 0.8 ± 0.256 ml/min/g; p < 0.05), as well as echocardiography assessment of left ventricular ejection fraction (60.855% ± 7.7% vs 38.22 ± 8.6%; p < 0.05) and fractional shortening (38.63% ± 9.3% vs 25.2% ± 7.11%; p < 0.05). Conclusion: hESC-derived CD133⁺ endothelial progenitor cells can be utilized to regenerate the infarcted heart.

Keywords:

Bibliography

1 Reubinoff BE, Pera MF, Fong CY et al.: Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol.18,399–404 (2000).
Medline CASGoogle Scholar
2 Thomson JA, Itskovitz-Eldor J, Shapino SS et al.: Embryonic stem cell lines derived from human blastocysts. Science282,1145–1147 (1998).
Medline CASGoogle Scholar
3 Carpenter MK, Inokuma MS, Denham J et al.: Enrichment of neurons and neural precursors from human embryonic stem cells. Exp. Neurol.172,383–397 (2001).
Medline CASGoogle Scholar
4 Toh WS, Yang Z, Liu H et al.: Effects of BMP2 and culture conditions on the extent of chondrogenesis from human embryonic stem cells. Stem Cells25,950–960 (2007).
Medline CASGoogle Scholar
5 Kaufman DS, Hanson ET, Lewis RL et al.: Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl Acad. Sci. USA98,10716–10721 (2001).
Medline CASGoogle Scholar
6 Mummery C, Ward D, van den Brink CE et al.: Cardiomyocytes differentiation of mouse and human embryonic stem cells. J. Anat.200,233–242 (2002).
Medline CASGoogle Scholar
7 Kehat I, Kenyagin-Karsetin D, Snir M et al.: Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest.108,407–414 (2001).
Medline CASGoogle Scholar
8 Gerecht-Nir S, Ziskind A, Cohen S et al.: Human embryonic stem cells as an in vitro model for human vascular development and the induction of vascular differentiation. Lab. Invest.83,1811–1820 (2003).
MedlineGoogle Scholar
9 Levenberg S, Golub JS, Amit M et al.: Endothelial cells derived from human embryonic stem cells. Proc. Natl Acad. Sci. USA99,4391–4396 (2002).
Medline CASGoogle Scholar
10 Guillaume DJ, Johnson MA, Li XJ et al.: Human embryonic stem cell-derived neural precursors develop into neurons and integrate into the host brain. J. Neurosci. Res.84(6),1165–1176 (2006).
Medline CASGoogle Scholar
11 Caspi O, Huber I, Kehat I et al.: Transplantation of human embryonic stem cell-derived cardiomyocytes improves myocardial performance in infarcted rat hearts. J. Am. Coll. Cardiol.50(19),1884–1893 (2007).
MedlineGoogle Scholar
12 Oyamada N, Itoh H, Sone M et al.: Transplantation of vascular cells derived from human embryonic stem cells contributes to vascular regeneration after stroke in mice. J. Transl. Med.30(6),54–67 (2008).
Google Scholar
13 Heng BC, Cao T, Haider HK et al.: An overview and synopsis of techniques for directing stem cell differentiation in vitro. Cell Tissue Res.315(3),291–303 (2004).
MedlineGoogle Scholar
14 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 Cell25(9),2206–2214 (2007).
Medline CASGoogle Scholar
15 Heng BC, Cao T: Milieu-based versus gene-modulatory strategies for directing stem cell differentiation – major issue of contention in transplantation medicine. In vitro Cell. Dev. Biol. Anim.42(3–4),51–53 (2006).
Medline CASGoogle Scholar
16 Räty JK, Lesch HP, Wirth T, Ylä-Herttuala S: Improving safety of gene therapy. Curr. Drug Saf.3(1),46–53 (2008).
Medline CASGoogle Scholar
17 Heng BC, Hong YH, Cao T: Modulating gene expression in stem cells without recombinant DNA and permanent genetic modification. Cell Tissue Res.321(2),147–150 (2005).
Medline CASGoogle Scholar
18 Zwaka TP, Thomson JA: Homologous recombination in human embryonic stem cells. Nat. Biotechnol.21,319–321 (2003).
Medline CASGoogle Scholar
19 Siemen H, Nix M, Endl E et al.: Nucleofection of human embryonic stem cells. Stem Cells Dev.14,378–383 (2005).
Medline CASGoogle Scholar
20 Koumbi D, Clement JC, Sideratou Z et al.: Factors mediating lipofection potency of a series of cationic phosphonolipids in human cell lines. Biochim. Biophys. Acta1760(8),1151–9 (2006).
Medline CASGoogle Scholar
21 Kawabata K, Sakurai F, Koizumi N et al.: Adenovirus vector-mediated gene transfer into stem cells. Mol. Pharm.3(2),95–103 (2006).
Medline CASGoogle Scholar
22 Gropp M, Itsykson P, Singer O et al.: Stable genetic modification of human embryonic stem cells by lentiviral vectors. Mol. Ther.7(2),281–287 (2003).
Medline CASGoogle Scholar
23 Clements MO, Godfrey A, Crossley J et al.: Lentiviral manipulation of gene expression in human adult and embryonic stem cells. Tissue Eng.12(7),1741–1751 (2006).
Medline CASGoogle Scholar
24 Kawabata K, Sakurai F, Koizumi N, Hayakawa T, Mizuguchi H: Adenovirus vector-mediated gene transfer into stem cells. Mol. Pharm.3(2),95–103 (2006).
Medline CASGoogle Scholar
25 Wilber A, Linehan JL, Tian X, Woll PS et al.: Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer. Stem Cells25(11),2919–2927 (2007).
Medline CASGoogle Scholar
26 Ma Y, Ramezani A, Lewis R et al.: High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells21,111–117 (2003).
Medline CASGoogle Scholar
27 Lakshmipathy U, Pelacho B, Sudo K et al.: Efficient transfection of embryonic and adult stem cells. Stem Cells22,531–543 (2004).
MedlineGoogle Scholar
28 Brokhman I, Pomp O, Shaham L et al.: Genetic modification of human embryonic stem cells with adenoviral vectors: differences of infectability between lines and correlation of infectability with expression of the coxsackie and adenovirus receptor. Stem Cells Dev.18(3),447–456 (2008).
Google Scholar
29 Risau W, Flamme I: Vasculogenesis. Annu. Rev. Cell Dev. Biol.11,73–91 (1995).
Medline CASGoogle Scholar
30 Risau W: Mechanisms of angiogenesis. Nature386,671–674 (1997).
Medline CASGoogle Scholar
31 Jujo K, Ii M, Losordo DW: Endothelial progenitor cells in neovascularization of infarcted myocardium. J. Mol. Cell. Cardiol.45(4),530–544 (2008).
Medline CASGoogle Scholar
32 Kawamoto A, Tkebuchava T, Yamaguchi J et al.: Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation107(3),461–468 (2003).
MedlineGoogle Scholar
33 Takahashi H, Shibuya M: The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin. Sci. (Lond.)109(3),227–241 (2005).
Medline CASGoogle Scholar
34 Roy H, Bhardwaj S, Yla-Herttuala S: Biology of vascular endothelial growth factors. FEBS Lett.580(12),2879–2887 (2006).
Medline CASGoogle Scholar
35 Miller-Kasprzak E, Jagodzinski PP: Endothelial progenitor cells as a new agent contributing to vascular repair. Arch. Immunol. Ther. Exp. (Warsz.)55(4),247–259 (2007).
Medline CASGoogle Scholar
36 Shmelkov SV, St Clair R, Lyden D et al.: AC133/CD133/Prominin-1. Int. J. Biochem. Cell Biol.37(4),715–719 (2005).
Medline CASGoogle Scholar
37 Urbich C, Dimmeler S: Endothelial progenitor cells: characterization and role in vascular biology. Circ. Res.95(4),343–353 (2004).
Medline CASGoogle Scholar
38 Hristov M, Weber C: Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. J. Cell. Mol. Med.8(4),498–508 (2004).
MedlineGoogle Scholar
39 Tatsis N, Ertl HC: Adenoviruses as vaccine vectors. Mol. Ther.10,616–629 (2004).
Medline CASGoogle Scholar
40 Ye L, Haider HKH, Jiang S, et al.: In vitro functional assessment of human skeletal myoblasts after transduction with adenoviral bicistronic vector carrying human VEGF165 and angiopoietin-1. J. Heart Lung Transplant.24,1393–1402 (2005).
MedlineGoogle Scholar
41 Jiang S, Haider HKH, Idris NM et al.: Supportive interaction between cell survival signaling and angiocompetent factors enhances donor cell survival and promotes angiomyogenesis for cardiac repair. Circ. Res.99(7),776–784 (2006).
Medline CASGoogle Scholar
42 Takahashi H, Shibuya M: The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin. Sci. (Lond.)109(3),227–241 (2005).
Medline CASGoogle Scholar
43 Rufaihah AJ, Haider HK, Heng BC et al.: Directing endothelial differentiation of human embryonic stem cells via transduction with an adenoviral vector expressing the VEGF(165) gene. J. Gene Med.9(6),452–461 (2007).
Medline CASGoogle Scholar
44 Wang I, Huang I: Adenovirus technology for gene manipulation and functional studies. Drug Discov. Today5,10–16 (2000).
Medline CASGoogle Scholar
45 Benihoud K, Yeh P, Perricaudet M: Adenovirus vectors for gene delivery. Curr. Opin. Biotechnol.10,440–447 (1999).
Medline CASGoogle Scholar
46 Olivetti G, Capasso JM, Meggs LG et al.: Cellular basis of chronic ventricular remodeling after MI in rats. Circ. Res.68(3),856–869 (1991).
Medline CASGoogle Scholar
47 Shi Q, Rafii S, Wu MH et al.: Evidence for circulating bone marrow-derived endothelial cells. Blood92,362–367 (1998).
Medline CASGoogle Scholar
48 Kawamoto A, Gwon HC, Iwaguro H et al.: Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation103,634–637 (2001).
Medline CASGoogle Scholar
49 Kalka C, Masuda H, Takahashi T et al.: Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl Acad. Sci. USA28,3422–3427 (2000).
Google Scholar
50 Kocher AA, Schuster MD, Szabolcs MJ et al.: Neovascularization of ischemic myocardium by human bone marrow-derived angioblasts prevents cardiomyocytes apoptosis, reduces remodeling and improves cardiac function. Nat. Med.7,430–436 (2001).
Medline CASGoogle Scholar
51 Takashi E, Ashraf M: Pathologic assessment of myocardial necrosis and apoptosis after ischemia and reperfusion with molecular and morphological markers. J. Mol. Cell. Cardiol.32,209–224 (2000).
Medline CASGoogle Scholar
52 Gehling UM, Ergün S, Schumacher U et al.: In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood95,3106–3112 (2000).
Medline CASGoogle Scholar
53 Kamihata H, Matsubara H, Nishiue T et al.: Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands and cytokines. Circulation104,1046–1052 (2001).
Medline CASGoogle Scholar
54 Tang YL, Zhao Q, Qin X, Shen L, Cheng L, Phillips MI: Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction. Ann. Thorac. Surg.80,229–237 (2005).
MedlineGoogle Scholar
55 Zhang S, Zhang P, Guo J et al.: Enhanced cytoprotection and angiogenesis by bone marrow cell transplantation may contribute to improved ischemic myocardial function. Eur. J. Cardiothorac. Surg.25,188–195 (2004).
MedlineGoogle Scholar
56 Heba G, Krzeminski T, Pore M, Grzyb J, Ratajska A, Dembinska-Kiec A: The time course of tumour necrosis factor-α, inducible nitric oxide synthase and vascular endothelial growth factor expression in an experimental model of chronic myocardial infarction in rats. J. Vasc. Res.38,288–300 (2001).
Medline CASGoogle Scholar

Vol. 5, No. 2

Supplemental Materials

Metrics

Downloaded 378 times

History

Published online 8 March 2010

Published in print March 2010

Information

Keywords

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

PDF download

Therapeutic angiogenesis by transplantation of human embryonic stem cell-derived CD133+ endothelial progenitor cells for cardiac repair

Abstract

Bibliography

Therapeutic angiogenesis by transplantation of human embryonic stem cell-derived CD133⁺ endothelial progenitor cells for cardiac repair