Combined transplantation of skeletal myoblasts and bone marrow stem cells for myocardial repair in rats

European Journal of Cardio-thoracic Surgery 25 (2004) 627–634 www.elsevier.com/locate/ejcts Combined transplantation of skeletal myoblasts and bone marrow stem cells for myocardial repair in ratsq a Department of Cardiac Surgery, University of Innsbruck, Anichstrasse, A-6020 Innsbruck, Austria b Biochemical Pharmacology, University of Innsbruck, Innsbruck, Austria c Department of Hematology, University of Innsbruck, Innsbruck, Austria d Pharmacology and Toxicology, University of Vienna, Vienna, Austria Received 13 September 2003; received in revised form 3 December 2003; accepted 15 December 2003 Abstract Objectives: To prove whether intramyocardial transplantation of combined skeletal myoblasts (SM) and mononuclear bone marrow stem cells is superior to the isolated transplantation of these cell types after myocardial infarction in rats. Methods: In 67 male Fischer rats myocardial infarction was induced by direct ligature of the LAD. Seven days postinfarction baseline echocardiography and intramyocardial cell transplantation were performed. Via lateral thoracotomy 200 ml containing either 107 SMs or 107 bone marrow-derived mononuclear cells (BM-MNC) or a combination of 5 £ 106 of both cell types (MB) were injected in 10 –15 sites in and around the infarct zone. In controls (C) 200 ml of cell-free medium were injected in the same manner. Before injection both cell types were stained using a fluorescent cell linker kit (PKH, Sigma). In addition, SMs were transfected with green fluorescent protein. Nine weeks postinfarction follow-up echocardiography was performed and animals were sacrificed for further analysis. Results: At baseline echocardiography there was no difference in left ventricular ejection fraction (LVEF; C, SM, BM-MNC, MB: 60.1 ^ 3.2, 53.3 ^ 10.2, 53.1 ^ 8.7, 49 ^ 9.0%) and left ventricular end diastolic diameter (LVEDD; C, SM, BM-MNC, MB: 6.5 ^ 0.8, 5.17 ^ 0.8, 5.77 ^ 1.4, 6.25 ^ 0.8 mm) between the different therapeutic groups. Eight weeks after cell transplantation LVEDD was significantly increased in all animals except those that received a combination of myoblasts and bone marrow stem cells (MB; C, SM, BM-MNC, MB: 7.7 ^ 0.6 mm, P ¼ 0:001; 7.7 ^ 1.5 mm, P , 0:001; 7.7 ^ 1.1 mm, P ¼ 0:005; 6.6 ^ 1.7 mm, P ¼ 0:397). At the same time LVEF decreased significantly in the control group (C), stayed unchanged in animals that received bone marrow stem cells (BM-MNC) and increased in animals that received myoblasts (SM) and a combination of both cell types (MB; C, SM, BM-MNC, MB: 45.3 ^ 7.0%, P ¼ 0:05; 63.9 ^ 15.4%, P ¼ 0:044; 54.3 ^ 6.3%, P ¼ 0:607; 63.0 ^ 11.5%, P ¼ 0:039). Conclusions: The present data show that the concept of combining SMs with bone marrow-derived stem cells may be of clinical relevance by merging the beneficial effects of each cell line and potentially reducing the required cell quantity. Further studies are required to identify the exact mechanisms underlying this synergy and to allow full exploitation of its therapeutic potential. q 2004 Elsevier B.V. All rights reserved. Keywords: Cellular cardiomyoplasty; Skeletal myoblast; Bone marrow; Cell transplantation; Cardiomyopathy 1. Introduction q Presented at the Joint 17th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 11th Annual Meeting of the European Society of Thoracic Surgeons, Vienna, Austria, October 12– 15, 2003. Abbreviations: BM-MNC, bone marrow-derived mononuclear cell; IVS, interventricular septum; LVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejection fraction; SM, skeletal myoblast; vWF, von Willebrand Factor. * Corresponding author. Tel.: þ43-512-504-2501; fax: þ 43-512-5042502. E-mail address: (H.C. Ott). 1010-7940/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ejcts.2003.12.031 The adult heart lacks the potential of effective regeneration. Instead of tissue recovery the infarcted myocardium is transformed into a non-contractile fibrous scar. The following remodelling process leads to expansion of the initial infarct area and dilatation of the left ventricular lumen [1]. As one novel therapeutic option attempting to stop or even reverse the process of postinfarction remodelling and restoring left ventricular function, cellular H.C. Otta,*, N. Bonarosa, R. Marksteinerb, D. Wolfc, E. Margreiterb, T. Schachnera, G. Laufera, S. Heringd 628 H.C. Ott et al. / European Journal of Cardio-thoracic Surgery 25 (2004) 627–634 2. Materials and methods 2.1. Isolation and expansion of skeletal myoblasts and bone marrow stem cells Primary SMs were isolated from a male F344 Fischer rat (6 weeks of age). After intraperitoneal administration of ketamine (50 mg/100 g) and xylazine (1 mg/100 g) hind limb muscles (0.5 – 1.0 g) were dissected free from connective tissues, and minced into pieces of approximately 1 mm3. Muscle samples were enzymatically dissociated according to the cell dispersion technique described by Blau and Webster. Single satellite cells in suspension were manually collected with a micropipette under microscope control and transferred into the cell culture with 96-well plates. The cells were cultured in growth media and maintained in a proliferating state. Desmin was used as a marker to identify clones of myoblasts (Fig. 1A). To confirm the ability to differentiate, desmin-positive mononucleated myoblasts were cultured in differentiation media. Under these conditions myoblasts fused into multinucleated myotubes (Fig. 1B). After 1 week in differentiation medium spontaneous contractions of the myotubes were observed. Electrophysiologic analysis revealed myoblast characteristic Ba2þ currents through T-type calcium channels in myoblasts (Fig. 1C, left panel) with corresponding current – voltage relationship (Fig. 1C, right panel). Before intramyocardial injection, cells were labelled using a fluorescent cell linker kit (PKH26-GL, SigmaAldrich Co., Vienna, Austria) following the instructions of the manufacturer. Bone marrow stem cells were isolated from male F344 Fischer rats. After intraperitoneal administration of ketamine (50 mg/100 g) and xylazine (1 mg/100 g) both femurs Fig. 1. Characterisation of skeletal myoblasts before transplantation. Desmin was used as a marker to identify clones of myoblasts (A). Myoblasts fused into multinucleated myotubes if cultured in differentiation medium (B). Electrophysiological analysis revealed myoblast characteristic Ba2þ currents through T-type calcium channels in myoblasts (C, left panel) with the corresponding current–voltage relationship (C, right panel). cardiomyoplasty has been proposed and opened new perspectives for the treatment of ischemic heart disease [2]. In a number of myocardial injury models, different cell lines proved the potential to regenerate viable tissue after being transplanted into the infarcted heart [2 – 6]. Among those, skeletal myoblasts (SM) improved myocardial performance in vitro and in vivo, either delive (...truncated)