Cellular Cardiomyoplasty Using Autologous Satellite Cells: from Experimental to Clinical Study Race L. Kao, Fumin Zhang(1), Zhi-Jian Yiang(1), Xiang Gao(2) and Chuanfu Li Department of Surgery, East Tennessee State University, Johnson City, TN, USA, (1) The First Affiliated Hospital of Nanjing Medical University, Nanjing, P.R. China and (2) Model Animal Research Center, Nanjing University, Nanjing, P.R. China Abstract Adult mammalian ventricular myocytes lack regenerative capability, consequently an in- jured heart is normally repaired by scar formation, hypertrophy of surviving myocytes, and hyperplasia of non-muscle cells. The possible existence of stem cells or progenitor cells for myocardium has been suggested recently, however it is clear that functionally significant myocardial regeneration has not been documented in diseased or injured heart. Contribution of other cells to the formation of ventricular myocytes appears to be negligible as evidenced by the consistent formation of scar after myocardial infarction. Satellite cells are adult stem cells responsible for growth, repair, and maintain homeostasis of skeletal muscle. We have been using autologous satellite cells for myocardial regeneration in dogs since 1989 and have applied this procedure to patients in 2001. Satellite cells have been successfully iso- lated, labeled, and implanted into injured heart with neomyocardial formation and func- tional improvement. Viable muscle cells with clear labeling are found in the infarct area af- ter cell implantation. The labeled muscle cells have intercalated disks at cellular junctions. Significant improvements in contractile function are only observed in the animals that have successful engraftment after cell transplantation. Marked improvement in ejection fraction, myocardial perfusion, and local contractility are also found for patients after cellular car- diomyoplasty using autologous satellite cells. Most importantly, a heart failure patient after cellular cardiomyoplasty without conjunctional surgical procedure has recovered from con- gestive heart failure with significant improvement in myocardial perfusion, contractility, and metabolic activity at sits of cell implantation. Transplantation of satellite cells into in- jured heart can be a new treatment for myocardial infarction and heart failure. Key words: cellular cardiomyoplasty, dog, heart attack, heart failure, man, satellite cells. Basic Appl Myol 13 (1): 23-28, 2003 Cardiovascular diseases remain the single largest also includes our clinical studies using patients’ own cause of morbidity and mortality in the Western world satellite cells implanted into the injured heart with or and the United States [1, 2]. It is estimated that 12.6 without simultaneous coronary artery bypass grafts. million Americans alive today have coronary artery disease and more than one million people will suffer a Materials and Methods heart attack every year. With longer life expectancy Experimental animal for the Western world, a further increase is anticipated Mongrel dogs weighing 20 to 30 kg were purchased for cardiovascular disease. Restoring blood flow, im- from a licensed vendor. The animals were housed in air- proving perfusion, reducing clinical symptoms, and conditioned rooms with free access to food and water at augmenting ventricular function are the common all times. Humane care and proper analgesic, anesthetic, treatments after myocardial infarction. Other than re- and tranquilizing drugs were provided when needed to placing the whole heart (cardiac transplantation) no all experimental animals. The “Principles of Laboratory standard clinical procedure is available to restore or Animal Care” formulated by the National Society for regenerate the damaged myocardium following a heart Medical Research and the “Guide for the Care and Use attack. This report summarizes the experimental stud- of Laboratory Animals” prepared by the National Re- ies of cellular cardiomyoplasty using autologous satel- search Council in 1996 were followed. The proposed lite cells in dogs with our new observations. This paper - 23- Satellite cells for myocardial regeneration study was approved by the University Committee on medium in a 25 cm2 culture flask. The isolated cells have Animal Care of East Tennessee State University. a doubling time of 20 to 22 hours and can easily go After fasting and preoperative antibiotic treatment, through 20 cell cycles and still retain their proliferation each dog was anesthetized with sodium pentobarbital and differentiation capabilities. The cells were sub- (15 mg/kg) and intubated with a cuffed endotracheal cultured every 3 to 4 days to maintain at low density for tube. After shaving the surgical sites and cleaning them continual proliferation without differentiation. When with alcohol, the electrocardiogram was recorded using forming multinucleated myotubes were desired, the satel- the PageWriter cardiograph and blood pressures were lite cells were cultured with medium 199 containing 2% measured with Millar micro-tip pressure transducers horse serum and 1% antibiotic antimycotic solution until (Millar Instruments, Inc., Houston, TX, USA). Anesthe- myotube formation to document myogenic capability. sia was maintained by 1% halothane and the surgical Labeling of cultured satellite cells area was prepared with Betadine. The number of ani- mals used for each study was detailed in the Results. To identify and follow the transplanted satellite cells at the site of injured heart, it is necessary to have long- Patient selection term stable labeling of the cells with high intensity and After obtaining approval from Department of Health specificity. Satellite cells enriched and proliferated by (equal to FDA of USA) and Hospital Review Board culture can be labeled with fluorescent microspheres (same as IRB of USA), patient selection and signed con- (Polyscience Inc., Warrington, PA), 3H-thymidine sent forms were established. Male patients (55 to 74 years (Amersham Life Science Inc., Arlington Heights, IL), of age) with recurrent exertion angina, abnormal ECG 5-bromo-2’-deoxyuridine (BrdU), 4’6-diamidino-2- indicating history of myocardial infarction, and coronary phenylindole (DAPI), dialkycarbocyanine (DiI) or angiography showing significant stenosis or blockage other membrane markers (Molecular Probes, Inc., were recruited for the study. 2D-echocardiography, Eugene, OR) as reported by us and other investigators 99mTc-MIBI, and /or 18F-deoxyglucose were used to de- [3-13]. The disadvantage of all these labeling proce- termine the changes in ventricular function, myocardial dures is the loss of labeling intensity as the cell divides perfusion, and metabolic activities before and after cellu- (assuming equal division into daughter cells, 50% lar cardiomyoplasty. lower intensity for each cell cycle). Therefore, better Isolation and culture of satellite cells labeling methods need be developed. The lacZ gene which encodes β-galactosidase in E. coli Under full anesthesia and sterile surgical conditions, a and green fluorescent protein (GFP) have been used to biopsy sample (15 ~ 20 g) from canine tibialis anterior label the cultured satellite cells. The mammalian reporter muscle or a small sample (2 ~ 4 g) from right vastus lat- vectors lacZ and GFP (pCMVβ) from Clontch Labora- eralis muscle of the patients was obtained for cell isola- tory Inc. (Palo Alto, CA) and Lipofectamine from Gibco tion. The muscle sample was rinsed in 70% ethanol fol- BRL (Gaithersburg, MD) have been purchased for the ++ lowed by Hank’s basal salt solution without Ca and procedure. Very good labeling efficiency can be achieved ++ Mg but contained 1% penicillin-streptomycin and 1% using Lipofectanime, however AdenoLacZ and Ade- 3 amphotericin B. The tissue was minced (~ 1 mm ) be- noGFP from Quantum Biotechnologies (Montreal, Que- fore incubation in 10 volumes of enzyme solution in bec, Canada) have outstanding labeling efficiency and the sterile 50 ml plastic tubes. The enzyme solution was label can be maintained in cultured cells for more than made of buffered medium 199 containing 1% colla- eight weeks. Unfortunately, the AdenoGFP cannot be genase and 0.2% hyaluronidase filtered through a 0.2 µ used for long-term in vivo study due to the strong in- filter and equilibrated with 95% O2 : 5% CO2. After 15 flammatory reactions and expression of viral proteins. o minutes of incubation at 37 C in a shaking water bath, The humanized Renilla green fluorescent protein the released satellite cells were harvested by pouring the (hrGFP) using helper-free adeno-associate virus (AAV- solution through layers of sterile gauze. The remaining hrGFP) purchased from Stratagene (La Jolla, CA) is o tissue was incubated in the enzyme solution at 37 C for used to label the cultured satellite cells. Since AAV can another 15 minutes to complete the enzymatic liberation insert the DNA of interest into the host genome, long- of satellite cells from muscle. term stable expression has been achieved using this The isolated satellite cells were pelleted by centrifuga- method [14]. Use AAV to label the cells has the advan- tion (650 x G for 10 min.) and washed with culture me- tages of long-term expression, non-pathogeneicity, in- dium consisting of medium 199 with 10% fetal bovine ducing no immune response, physical stability, and the serum and 1% antibiotic antimycotic solution (Sigma ability to transduce dividing and non-dividing cells [14- Chemical Co., St. Louis, MO). The viability of isolated 16]. The humanized GFP is selected