In vivo vascularization of cell sheets provided better long‐term tissue survival than injection of cell suspension

Cell sheets have shown a remarkable ability for repairing damaged myocardium in clinical and preclinical studies. Although they demonstrate a high degree of viability as engrafted cells in vivo, the reason behind their survivability is unclear. In this study, the survival and vascularization of rat cardiac cell sheets transplanted in the subcutaneous tissue of athymic rats were investigated temporally. The cell sheets showed significantly higher survival than cell suspensions for up to 12 months, using an in vivo bioluminescence imaging system to detect luciferase‐positive transplanted cells. Terminal deoxynucleotidyl transferase dUTP nick‐end labelling (TUNEL) assay also showed a smaller number of apoptotic cells in the cell sheets than in the cell suspensions at 1 day. Rapid vascular formation and maturation were observed inside the cell sheets using an in vivo imaging system. Leaky vessels appeared at 6 h, red blood cells flowing through functional vessels appeared at 12 h, and morphologically matured vessels appeared at 7 days. In addition, immunostaining of cell sheets with nerve/glial antigen‐2 (NG2) showed that vessel maturity increased over time. Interestingly, these results correlated with the dynamics of cell sheet mRNA expression. Genes related to endothelial cells (ECs) proliferation, migration and vessel sprouting were highly expressed within 1 day, and genes related to pericyte recruitment and vessel maturation were highly expressed at 3 days or later. This suggested that the cell sheets could secrete appropriate angiogenic factors in a timely way after transplantation, and this ability might be a key reason for their high survival. Copyright © 2014 John Wiley & Sons, Ltd.

[1]  A. Mantovani,et al.  Granulocyte- and granulocyte– macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate , 1989, Nature.

[2]  Rinat Abramovitch,et al.  VEGF-Induced Adult Neovascularization: Recruitment, Retention, and Role of Accessory Cells , 2006, Cell.

[3]  D. Bodine,et al.  Bone marrow stem cells regenerate infarcted myocardium , 2003, Pediatric transplantation.

[4]  David M. Bodine,et al.  Bone marrow cells regenerate infarcted myocardium , 2001, Nature.

[5]  H. Mertsching,et al.  Influence of scaffold thickness and scaffold composition on bioartificial graft survival. , 2003, Biomaterials.

[6]  Daniel Berman,et al.  Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial , 2012, The Lancet.

[7]  J L West,et al.  Tissue engineering in the cardiovascular system: Progress toward a tissue engineered heart , 2001, The Anatomical record.

[8]  M. Skobe,et al.  Activation of the tie2 receptor by angiopoietin-1 enhances tumor vessel maturation and impairs squamous cell carcinoma growth. , 2002, The American journal of pathology.

[9]  Masayuki Yamato,et al.  Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[10]  L. Claesson‐Welsh,et al.  FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. , 2001, Trends in pharmacological sciences.

[11]  Muhammad Ashraf,et al.  Skeletal myoblasts for cardiac repair. , 2010, Regenerative medicine.

[12]  K. Tokoyoda,et al.  The role of CXCL12 in the organ-specific process of artery formation. , 2005, Blood.

[13]  Richard T. Lee,et al.  Stem-cell therapy for cardiac disease , 2008, Nature.

[14]  C. Betsholtz,et al.  Pericytes and vascular stability. , 2006, Experimental cell research.

[15]  A. Mantovani,et al.  In vitro and in vivo activation of endothelial cells by colony-stimulating factors. , 1991, The Journal of clinical investigation.

[16]  Mitsuo Umezu,et al.  Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Cell Culture Surfaces , 2002, Circulation research.

[17]  Thomas N. Sato,et al.  Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. , 1999, Science.

[18]  Shigeyoshi Itohara,et al.  Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis , 2000, Nature Cell Biology.

[19]  L. Reinlib,et al.  Cell transplantation as future therapy for cardiovascular disease?: A workshop of the National Heart, Lung, and Blood Institute. , 2000, Circulation.

[20]  A. Laplanche,et al.  Granulocyte and granulocyte-macrophage colony-stimulating factors , 1993, The Lancet.

[21]  T. Kawabe,et al.  Erythromycin-induced CXCR4 expression on microvascular endothelial cells. , 2009, American journal of physiology. Lung cellular and molecular physiology.

[22]  K. Alitalo,et al.  Vascular Endothelial Growth Factor-A and Platelet-Derived Growth Factor-B Combination Gene Therapy Prolongs Angiogenic Effects via Recruitment of Interstitial Mononuclear Cells and Paracrine Effects Rather Than Improved Pericyte Coverage of Angiogenic Vessels , 2008, Circulation research.

[23]  Christine L. Mummery,et al.  Embryonic Stem (es) Cells from Mice and Primates Can Differentiate into Any Cell Type in the Adult Body Stem Cells in Fetal and Adult Hearts Stem-cell-based Therapy and Lessons from the Heart Insight Review , 2022 .

[24]  Philippe Leboulch,et al.  Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2 , 2003, Nature Medicine.

[25]  Masayuki Yamato,et al.  Long-term survival and growth of pulsatile myocardial tissue grafts engineered by the layering of cardiomyocyte sheets. , 2006, Tissue engineering.

[26]  Lila R Collins,et al.  Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts , 2007, Nature Biotechnology.

[27]  C. Heldin,et al.  PDGF and vessel maturation. , 2010, Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les recherches sur le cancer.

[28]  L. Zentilin,et al.  Bone marrow cells recruited through the neuropilin-1 receptor promote arterial formation at the sites of adult neoangiogenesis in mice. , 2008, The Journal of clinical investigation.

[29]  Masayuki Yamato,et al.  Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. , 2011, Tissue engineering. Part A.

[30]  Peter Carmeliet,et al.  Regulation of angiogenesis by oxygen and metabolism. , 2009, Developmental cell.

[31]  T. Okano,et al.  Tissue Cardiomyoplasty Using Bioengineered Contractile Cardiomyocyte Sheets to Repair Damaged Myocardium: Their Integration with Recipient Myocardium , 2005, Transplantation.

[32]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[33]  Timothy J. Nelson,et al.  Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells. , 2009, Circulation.

[34]  E. Kobayashi,et al.  “Firefly Rats” as an Organ/Cellular Source for Long-Term In Vivo Bioluminescent Imaging , 2006, Transplantation.

[35]  T. Kawabe,et al.  Erythromycin-induced CXCR 4 expression on microvascular endothelial cells , 2009 .

[36]  W. Risau,et al.  Mechanisms of angiogenesis , 1997, Nature.

[37]  Masayuki Yamato,et al.  Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential. , 2006, Biochemical and biophysical research communications.

[38]  T. Okano,et al.  Cardiomyocyte bridging between hearts and bioengineered myocardial tissues with mesenchymal transition of mesothelial cells. , 2006, The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation.

[39]  T. Okano,et al.  Cell sheet engineering for myocardial tissue reconstruction. , 2003, Biomaterials.

[40]  K. Alitalo,et al.  Molecular regulation of angiogenesis and lymphangiogenesis , 2007, Nature Reviews Molecular Cell Biology.