Journal of Artificial Organs

, Volume 11, Issue 3, pp 141–147 | Cite as

Fibroblast sheets co-cultured with endothelial progenitor cells improve cardiac function of infarcted hearts

  • Hiroshi Kobayashi
  • Tatsuya Shimizu
  • Masayuki Yamato
  • Kayoko Tono
  • Haruchika Masuda
  • Takayuki Asahara
  • Hiroshi Kasanuki
  • Teruo Okano
Original Article


We have already confirmed that cell sheet transplantation can improve damaged heart function via continuous cytokine secretion. In this study, we hypothesized that cytokine-secreting cell sheets co-cultured with an endothelial cell source may be more effective for repairing ischemic myocardium. Confluent rat fibroblasts cultured on temperature-responsive culture dishes were harvested as contiguous cell sheets by temperature reduction. Green fluorescent protein (GFP)-positive endothelial progenitor cells (EPCs) were seeded on fibroblast sheets to create co-cultured cell sheets, and sandwich-like constructs were engineered by stacking of the co-cultured cell sheets. These constructs were transplanted into rat myocardial infarction models. Cardiac function and histology were assessed in four groups: the sham operation (C) group, the isolated EPC injection (E) group, the transplantation of triple-layer fibroblast sheets (F) group, and the transplantation of triple-layer sandwich-like constructs (E + F) group. Echocardiography showed significant improvement of the fractional shortening in the E + F group in comparison with the C group (0.25 ± 0.05 vs. 0.16 ± 0.02). On histological examination, significantly less connective tissue formation was observed in the E, F, and E + F groups when compared to the C group (C, E, F, and E + F groups: 53 ± 2%, 41 ± 4%, 40 ± 4%, and 32 ± 7%, respectively). Additionally, increased blood vessel formation was detected in the E, F, and E + F groups compared with the C group (C, E, F, and E + F groups: 1.9% ± 0.6%, 6.7% ± 0.6%, 7.8% ± 0.9%, and 10.2% ± 2.4%, respectively). Furthermore, GFP-staining demonstrated that the newly formed blood vessels were composed of the co-cultured EPCs. Transplantation of cell sheets co-cultured with an endothelial cell source may be a new therapeutic strategy for myocardial tissue regeneration.


Tissue engineering Cell sheet Endothelial progenitor cells Heart 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Reinlib L, Field L. Cell transplantation as future therapy for cardiovascular disease? A workshop of the National Heart, Lung, and Blood Institute. Circulation 2000;101:E182–187PubMedGoogle Scholar
  2. 2.
    Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol 2005;23:845–856PubMedCrossRefGoogle Scholar
  3. 3.
    Zandonella C. Tissue engineering: the beat goes on. Nature 2003; 421:884–886PubMedCrossRefGoogle Scholar
  4. 4.
    Hagege AA, Marolleau JP, Vilquin JT, Alheritiere A, Peyrard S, Duboc D, Abergel E, Messas E, Mousseaux E, Schwartz K, Desnos M, Menasche P. Skeletal myoblast transplantation in ischemic heart failure: long-term follow-up of the first phase I cohort of patients. Circulation 2006;114:I108–113PubMedCrossRefGoogle Scholar
  5. 5.
    Meyer GP, Wollert KC, Lotz J, Steffens J, Lippolt P, Fichtner S, Hecker H, Schaefer A, Arseniev L, Hertenstein B, Ganser A, Drexler H. Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months’ follow-up data from the randomized, controlled BOOST (BOne marrOw transfer to enhance ST-elevation infarct regeneration) trial. Circulation 2006;113: 1287–1294PubMedCrossRefGoogle Scholar
  6. 6.
    Muller-Ehmsen J, Peterson KL, Kedes L, Whittaker P, Dow JS, Long TI, Laird PW, Kloner RA. Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function. Circulation 2002;105:1720–1726PubMedCrossRefGoogle Scholar
  7. 7.
    Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 2001;33:907–921PubMedCrossRefGoogle Scholar
  8. 8.
    Suzuki K, Murtuza B, Beauchamp JR, Smolenski RT, Varela-Carver A, Fukushima S, Coppen SR, Partridge TA, Yacoub MH. Dynamics and mediators of acute graft attrition after myoblast transplantation to the heart. FASEB J 2004;18: 1153–1155PubMedGoogle Scholar
  9. 9.
    Cohen S, Leor J. Rebuilding broken hearts. Biologists and engineers working together in the fledgling field of tissue engineering are within reach of one of their greatest goals: constructing a living human heart patch. Sci Am 2004;291:44–51PubMedCrossRefGoogle Scholar
  10. 10.
    Eschenhagen T, Zimmermann WH. Engineering myocardial tissue. Circ Res 2005;97:1220–1231PubMedCrossRefGoogle Scholar
  11. 11.
    Shimizu T, Yamato M, Kikuchi A, Okano T. Cell sheet engineering for myocardial tissue reconstruction. Biomaterials 2003;24: 2309–2316PubMedCrossRefGoogle Scholar
  12. 12.
    Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y. Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromol Chem Rapid Comm 1990;11:571–576CrossRefGoogle Scholar
  13. 13.
    Okano T, Yamada N, Sakai H, Sakurai Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J Biomed Mater Res 1993;27: 1243–1251PubMedCrossRefGoogle Scholar
  14. 14.
    Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, Kikuchi A, Umezu M, Okano T. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 2002;90:e40PubMedCrossRefGoogle Scholar
  15. 15.
    Miyagawa S, Sawa Y, Sakakida S, Taketani S, Kondoh H, Memon IA, Imanishi Y, Shimizu T, Okano T, Matsuda H. Tissue cardiomyoplasty using bioengineered contractile cardiomyocyte sheets to repair damaged myocardium: their integration with recipient myocardium. Transplantation 2005;80:1586–1595PubMedCrossRefGoogle Scholar
  16. 16.
    Memon IA, Sawa Y, Fukushima N, Matsumiya G, Miyagawa S, Taketani S, Sakakida SK, Kondoh H, Aleshin AN, Shimizu T, Okano T, Matsuda H. Repair of impaired myocardium by means of implantation of engineered autologous myoblast sheets. J Thorac Cardiovasc Surg 2005;130:1333–1341PubMedCrossRefGoogle Scholar
  17. 17.
    Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, Ishino K, Ishida H, Shimizu T, Kangawa K, Sano S, Okano T, Kitamura S, Mori H. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 2006;12: 459–465PubMedCrossRefGoogle Scholar
  18. 18.
    Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, Masaki H, Mori Y, Iba O, Tateishi E, Kosaki A, Shintani S, Murohara T, Imaizumi T, Iwasaka T. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 2001;104: 1046–1052PubMedCrossRefGoogle Scholar
  19. 19.
    Tang YL, Zhao Q, Qin X, Shen L, Cheng L, Ge J, 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 2005;80:229–236; discussion 236–237PubMedCrossRefGoogle Scholar
  20. 20.
    Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705PubMedCrossRefGoogle Scholar
  21. 21.
    Kawamoto A, Tkebuchava T, Yamaguchi J, Nishimura H, Yoon YS, Milliken C, Uchida S, Masuo O, Iwaguro H, Ma H, Hanley A, Silver M, Kearney M, Losordo DW, Isner JM, Asahara T. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003;107:461–468PubMedCrossRefGoogle Scholar
  22. 22.
    Nagaya N, Nishikimi T, Yoshihara F, Horio T, Morimoto A, Kangawa K. Cardiac adrenomedullin gene expression and peptide accumulation after acute myocardial infarction in rats. Am J Physiol Regul Integr Comp Physiol 2000;278:R1019–1026PubMedGoogle Scholar
  23. 23.
    Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275: 964–967PubMedCrossRefGoogle Scholar
  24. 24.
    Walter DH, Rittig K, Bahlmann FH, Kirchmair R, Silver M, Murayama T, Nishimura H, Losordo DW, Asahara T, Isner JM. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation 2002;105:3017–3024PubMedCrossRefGoogle Scholar
  25. 25.
    Isik S, Er E, Soysal Y, Imirzalioglu N. Prolongation of skin xenograft survival with modified cultured fibroblasts. Plast Reconstr Surg 2003;111:275–282; discussion 283–285PubMedGoogle Scholar
  26. 26.
    Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R, Feigenbaum H, Gutgesell H, Reichek N, Sahn D, Schnittger I, Silverman NH, Tajik AJ. Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms. J Am Soc Echocardiogr 1989;2:358–367PubMedGoogle Scholar
  27. 27.
    Kushida A, Yamato M, Konno C, Kikuchi A, Sakurai Y, Okano T. Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces. J Biomed Mater Res 1999;45: 355–362PubMedCrossRefGoogle Scholar
  28. 28.
    Kondoh H, Sawa Y, Miyagawa S, Sakakida-Kitagawa S, Memon IA, Kawaguchi N, Matsuura N, Shimizu T, Okano T, Matsuda H. Longer preservation of cardiac performance by sheet-shaped myoblast implantation in dilated cardiomyopathic hamsters. Cardiovasc Res 2006;69:466–475PubMedCrossRefGoogle Scholar
  29. 29.
    Sekiya S, Shimizu T, Yamato M, Kikuchi A, Okano T. Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential. Biochem Biophys Res Commun 2006;341:573–582PubMedCrossRefGoogle Scholar
  30. 30.
    Mansbridge JN, Liu K, Pinney RE, Patch R, Ratcliffe A, Naughton GK. Growth factors secreted by fibroblasts: role in healing diabetic foot ulcers. Diabetes Obes Metab 1999;1:265–279PubMedCrossRefGoogle Scholar
  31. 31.
    Pinney E, Liu K, Sheeman B, Mansbridge J. Human threedimensional fibroblast cultures express angiogenic activity. J Cell Physiol 2000;183:74–82PubMedCrossRefGoogle Scholar
  32. 32.
    Li RK, Jia ZQ, Weisel RD, Mickle DA, Choi A, Yau TM. Survival and function of bioengineered cardiac grafts. Circulation 1999;100: II63–69PubMedGoogle Scholar
  33. 33.
    Bursac N, Papadaki M, Cohen RJ, Schoen FJ, Eisenberg SR, Carrier R, Vunjak-Novakovic G, Freed LE. Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies. Am J Physiol 1999;277:H433–444PubMedGoogle Scholar
  34. 34.
    Leor J, Aboulafia-Etzion S, Dar A, Shapiro L, Barbash IM, Battler A, Granot Y, Cohen S. Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium? Circulation 2000;102: III56–61PubMedGoogle Scholar
  35. 35.
    Eschenhagen T, Fink C, Remmers U, Scholz H, Wattchow J, Weil J, Zimmermann W, Dohmen HH, Schafer H, Bishopric N, Wakatsuki T, Elson EL. Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J 1997;11:683–694PubMedGoogle Scholar
  36. 36.
    Zimmermann WH, Melnychenko I, Wasmeier G, Didie M, Naito H, Nixdorff U, Hess A, Budinsky L, Brune K, Michaelis B, Dhein S, Schwoerer A, Ehmke H, Eschenhagen T. Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts. Nat Med 2006;12:452–458PubMedCrossRefGoogle Scholar

Copyright information

© The Japanese Society for Artificial Organs 2008

Authors and Affiliations

  • Hiroshi Kobayashi
    • 1
    • 3
  • Tatsuya Shimizu
    • 1
  • Masayuki Yamato
    • 1
  • Kayoko Tono
    • 2
  • Haruchika Masuda
    • 2
  • Takayuki Asahara
    • 2
    • 4
  • Hiroshi Kasanuki
    • 3
  • Teruo Okano
    • 1
  1. 1.Institute of Advanced Biomedical Engineering and ScienceTokyo Women’s Medical UniversityShinjuku-ku, TokyoJapan
  2. 2.Department of Regeneration MedicineTokai University School of MedicineKanagawaJapan
  3. 3.Department of CardiologyTokyo Women’s Medical UniversityTokyoJapan
  4. 4.Stem Cell Translational ResearchInstitute of Biomedical Research and Innovation/RIKEN Center for Developmental BiologyKobeJapan

Personalised recommendations