Cellular cardiomyoplasty is a cell therapy approach

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1 In Vitro Preprogramming of Marrow Stromal Cells for Myocardial Regeneration Bindu Bittira, MD, Jin-Qiang Kuang, BS, Abdulaziz Al-Khaldi, MD, Dominique Shum-Tim, MD, and Ray C.-J. Chiu, MD, PhD Division of Cardiac Surgery, McGill University Health Center, Montreal, Quebec, Canada Background. We have previously reported that marrow stromal stem cells (MSCs), when implanted into myocardium, can undergo milieu-dependent differentiation to express phenotypes similar to the cells in the immediate microenvironment. We tested the hypothesis that by in vitro preprogramming of MSCs, we may be able to guide their differentiation to express a therapeutically desirable phenotype that is different from those in their microenvironment. Methods. MSCs were isolated from isogenic Lewis rats, culture expanded, and labeled with beta-gal using retrovirus carrying the lac-z gene. A subset of the transfected MSCs was then treated with 5-aza-2 deoxycytidine (5- aza). Three weeks after the left ventricles were cryoinjured, either 5-aza-pretreated (n 10) or untreated (n 8) MSCs were injected into the myocardial scar. The hearts were harvested 4 to 8 weeks later and stained immunohistochemically for phenotypic markers. Results. The labeled MSCs within the scars that were 5-aza pretreated appeared to be morphologically distinct from the untreated ones. The treated cells (8/10 rats) appeared more myotube-like, with elongated nuclei, linearly aligned with one another, and stained positive for the cardiomyocyte-specific marker troponin I-C. Untreated MSCs (5/8 rats), in contrast, were poorly differentiated, and some appeared to express other phenotypes seen in the scar tissue. Conclusions. Our findings indicate that in cellular cardiomyoplasty using MSCs, one may select different strategies to achieve specific therapeutic goals. By milieudependent differentiation, unmodified MSCs may augment myocardial angiogenesis and myogenesis, whereas converting scar into myogenic tissue may be facilitated by preprogramming of MSCs before implantation. (Ann Thorac Surg 2002;74: ) 2002 by The Society of Thoracic Surgeons Cellular cardiomyoplasty is a cell therapy approach used to augment the myocardium of a failing heart by implanting myogenic cells to replace lost cardiomyocytes [1]. A number of progenitor cells and stem cells are being studied as potential donor cell sources for cellular cardiomyoplasty. Embryonic stem cells have been guided to form pulsatile syncytial cardiomyocytes in culture [2] and to form stable intracardiac grafts [3]. Autologous myoblasts obtained from a patient s own skeletal muscle (ie, the satellite cells) are being used in ongoing phase I clinical trials [4] after nearly a decade of experimental studies [1 5]. More recently, the bone marrow stomal cells, now confirmed to be multipotent adult stem cells [6], have been shown to be capable of differentiating in culture to express a cardiomyocyte phenotype [7]. In vivo implantation studies carried out in our laboratory [8] had suggested the importance of the microenvironment in determining the differentiation of marrow stromal stem cells (MSCs). In that experiment, MSCs were implanted into the left ventricular wall using epicardial needle punctures. Cells that remained within the myocardial needle scar differentiated poorly, whereas those that had Presented at the Thirty-eighth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 28 30, Address reprint requests to Dr Chiu, The Montreal General Hospital/ MUHC, 1650 Cedar Ave, Room C9-169, Montreal, Quebec, Canada H3G 1A4; rchiu@po-box.mcgill.ca. infiltrated out into the adjacent normal myocardium expressed a cardiomyogenic phenotype. This implies that signals from the in situ environment may play a major role in determining the differentiation and maturation [9] of implanted MSCs. Thus, if our therapeutic goal is to implant cells directly into myocardial scar tissue in order to replace it with contractile myofibers, the adult stem cells derived from bone marrow may require preprogramming in vitro before the implantation, such that these cells become committed to differentiate into a myogenic lineage in spite of being surrounded by fibrous scar. In 1999, Makino and associates [7] treated a single clone of MSCs in culture with 5-azacytidine, a hypomethylating agent, and found they differentiated into cells with morphological and electromechanical features of cardiomyocytes. Wakitani and associates [10] also confirmed that culture-propogated rat MSCs treated with 5-azacytidine could be induced to differentiate in vitro into myogenic and adipocytic phenotypes. Tomita and associates [11], in an in vivo study using MSCs treated with 5-azacytidine, reported cardiomyogenic differentiation of the implanted cells with expression of the cardiacspecific protein troponin I. Although the exact mechanism of such action by 5-azacytidine has not been elucidated, it has been proposed that 5-azacytidine converts cells by hypomethylating regulatory loci on genes, 2002 by The Society of Thoracic Surgeons /02/$22.00 Published by Elsevier Science Inc PII S (02)

2 Ann Thorac Surg BITTIRA ET AL 2002;74: MSCs FOR MYOCARDIAL REGENERATION 1155 thereby establishing lineages of stem cells with restricted potential into muscle, cartilage, or fat cells [12]. Thus in this study, we tested the hypothesis that in vitro treatment of MSCs with 5-aza-2 deoxycytidine (5-aza), a bioactive in vivo metabolite of 5-azacytidine, may preprogram these cells and facilitate their differentiation into cardiomyocytes within scar tissue, as compared with that with nonpretreated MSCs. Material and Methods Animals Male isogenic Lewis rats were obtained from Charles River Laboratories (Wilmington, MA). These rats were used as donors and recipients to avoid immunological rejection. All animals received humane care in compliance with the Guide for the Care and the Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996, and the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Isolation and Culture of Marrow-Derived Stromal Cells Isolation and primary culture of MSCs from the donor rats were performed according to Caplan s method [13]. After an overdose of pentobarbital (100 mg/kg) given intraperitoneally, the femoral and tibial bones were collected and both ends of the long bones were cut away from the diaphyses. The bone marrow plugs were hydrostatically expelled from the bones using complete medium, consisting of Dulbecco s modified Eagle s medium (DMEM) containing selected lots of 10% calf serum and antibiotics (100 U/mL penicillin G, 100 g/mg streptomycin, 0.25 g amphotericin B; all obtained from Gibro Laboratories [Boston, MA]) in a humidified atmosphere of 5% CO 2. The marrow plugs were disaggregated and the dispersed cells were centrifuged and resuspended twice in complete medium. These cells in 10 ml of complete medium were then introduced into tissue culture dishes. Medium was replaced every 3 days and the nonadherent cells were discarded. Each primary culture was passaged twice to three new plates, and the cell density of the colonies was grown to approximately 90% confluence. The passaged MSCs were used for transfection and for 5-aza pretreatment. Once these cells were nearly confluent, they were labeled. MSC Labeling With LacZ LacZ-GP AM12 amphotropic retrovirus producer cells were obtained from Dr Jacques Galipeau s Laboratory (McGill University, Lady Davis Institute for Medical Research, Montreal, QC, Canada). These cells produce a replication-defective retrovirus containing the reporter (LacZ) gene that encodes for the bacterial -galactosidase enzyme. These cells were cultured in DMEM with 10% fetal calf serum and antibiotics (50 U/mL penicillin G and 50 g/ml streptomycin; Wisent Inc [St. Bruno, Quebec, Canada]). The cells were allowed to proliferate until at least a 70% confluence was achieved before using them for transduction in order to achieve a high retrovirus titer. Twenty-four hours before transduction, the marrow stromal cells were trypsinized with 0.05% Trypsin 0.53 mmol/l EDTA (Gibco Labs) and replated. The next day, these cells were transduced with LacZ retroviral particles twice per day for 3 consecutive days with lipofectamine (3 L of lipofectamine 2 mg/ml solution for each 1 ml of virus medium). At each transduction, the MSC medium was replaced with the supernatant from the LacZ-GP AM12 cells (after being filtered through a m filter). Seventy-two hours after the last transduction, MSCs were trypsinized and part of the cells were plated in a 35-mm dish for histochemical staining for -galactosidase activity, in order to determine the percentage of cells expressing -galactosidase. The medium was aspirated from the plates and the cells rinsed with phosphate-buffered saline (PBS). The cells were fixed at 4 C in fix solution (2% formaldehyde and 0.2% glutaraldehyde in PBS) for 15 minutes and rerinsed with PBS. Staining for -gal activity was performed with a solution containing 1 mg/ml 5-bromo-4-chloro-3-indoyl- -D-galactoside (X-gal), 2% dimethylsulfoxide, 20 mmol/l K 3 Fe(CN) 6,20 mmol/l K 4 Fe(CN) 6.3H 2 O, and 2 mmol/l magnesium chloride. The cells were then incubated at 37 C and protected from light for 16 hours. The presence of bluelabeled cells was then confirmed under phase microscopy. MSC Pretreatment With 5-aza-2 deoxycytidine (5-aza) MSCs that were harvested and labeled were treated for 24 hours with 5-aza (0.3 mol/l solution in 10% DMEM) within the culture dish. The cells were incubated overnight in a 37 C incubator with a humidified atmosphere of 5% CO 2. The following day, the supernatant was removed and replaced with 10% DMEM solution for 24 hours. The cells were treated once more with 5-aza for 24 hours and the growth medium was replaced. Preparation of Cells for Injection Cells isolated from the bone marrow were cultured in complete medium in tissue culture dishes. After labeling, the medium was aspirated and both the 5-aza-treated and untreated cells in each dish were washed with 6 ml of Hank s balanced salt solution (HBSS). The HBSS was aspirated and 2 ml of trypsin-edta was added to detach the cells from the bottom of the dish. The detached cell suspension was then placed in a flask with 2 ml of complete medium and placed in a hemocytometer for counting. A volume consisting of cells was then collected and centrifuged at 2,500 rpm for 5 minutes. The supernatant was discarded and the cell pellet resuspended in 0.5 cm 3 of complete medium. Implantation of MSCs The rat ventricles were cryoinjured with a 0.5-cm diameter metal probe, which was immersed into liquid nitrogen at 80 C. The probe was placed onto the rat ventricle for 20-second periods, with a 5-second rest. This was

3 1156 BITTIRA ET AL Ann Thorac Surg MSCs FOR MYOCARDIAL REGENERATION 2002;74: repeated twice for a total cryoinjury time of 1 minute. Three weeks after cryoinjury was induced, 10 rats underwent injection with pretreated MSCs, whereas 8 rats underwent injection with untreated MSCs in the following fashion. Anesthesia was induced and maintained. Animals were intubated with an 18-gauge intravenous catheter and connected to a Harvard rodent ventilator (Harvard Apparatus Co. Inc, Boston, MA) at 85 breaths/ minute. The heart was reexposed via a 1.5-cm left thoracotomy incision. Under direct vision, the MSC suspension was injected into the left ventricular wall with a 28-gauge needle. The implantation site was marked by 8-0 polypropylene sutures. The wound was closed in layers and the animals were sacrificed at various intervals. Histology and Histochemical Staining for - Galactosidase Activity The rats in both groups were sacrificed at various time points ranging from 4 to 8 weeks after cell implantation. Three rats were sacrificed at 4 weeks, 3 rats at 6 weeks, and 4 rats at 8 weeks. The hearts were harvested and rinsed with PBS and perfusion fixed in 2% paraformaldehyde in PBS. The staining for -galactosidase activity was performed as described above, but with the addition of 0.02% Nonidet P-40 and 0.01% deoxycholate to the staining solution. The gross cardiac specimens were stained for 6 hours at 37 C. After X-gal staining, the lateral wall of the left ventricle containing the scar was separated from the surrounding normal myocardium, and both segments were embedded in paraffin. Coronal sections 5 m in thickness were mounted on a set of gelatin-coated glass slides such that serial sections could be used for different stains. A series of sections from each heart specimen were stained with hematoxylin and eosin to show nuclei, cytoplasm, and connective tissue, and another series of sections stained with picrosirius red to show the presence of connective and fibrous tissue and to confirm cell implantation within the region of scar. Other serial sections from each heart were selected for immunohistochemical staining for: sarcomeric myosin heavy chain molecules with MF20 (Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Dept. of Biological Sciences); connexin 43 (Zymed Laboratories Inc, San Francisco, CA); troponin I-C (Santa-Cruz Biotechnology Inc, Santa Cruz, CA); and for anti-alpha monoclonal smooth muscle actin (Sigma Laboratories). Briefly, after deparaffinization, sections were placed in boiled citrate buffer (ph 6.0). After blocking in normal serum, sections were treated with the respective monoclonal antibodies overnight and with secondary antibodies the following day. Diaminobenzidine (DAB) was then used as a chromogen for light microscopy. Counterstaining of sections by hematoxylin and eosin was also performed. Cells derived from MSCs were identified by their blue nuclei (ie, -gal labeled) with an Olympus microscope (BX-FLA; Olympus). Digital images, transferred to a computer equipped with Image Pro Software (Media Cybernetics, Silver Spring, MD), were subsequently printed (Stylus Photo 700; Epson, Long Beach, CA) for publication. Results The MSCs in culture were observed under phase microscopy and assessed for proliferation and morphological change at each culture medium change. The hematopoietic cells were not adherent and mostly removed by the second change of medium. Approximately three to four passages after first culturing the cells, they were expanded to over 25 million cells from the initial 250 to 500 that had adhered to the culture dish. The transfection efficiency of the reporter gene Lac Z was nearly 100%. After treatment with 5-aza, the cell cultures were observed for morphological changes. Because 5-aza-treated MSCs were implanted within 3 days of treatment, there was no evidence of myotube formation or multinucleated cells in isolated colonies or groupings as described by others [10]. Although the concentration of 0.3 mol/l of 5-aza gave the highest incidence of myogenic conversion when compared with a dose of 10 mol/l of 5-aza, at least 2 weeks was required before a morphological change in vitro was observed. Because the focus of this study was to determine in vivo morphological changes after cell injection, MSCs in culture were not observed on a long-term basis. There were no deaths from the cryoinjury procedure and all rats survived to their cell implantation date. There were no deaths from the cell implantation technique and all rats survived until their dates of sacrifice. After harvesting, gross examination of the cryoinjured hearts revealed a concentric scar in the left anterior wall of the myocardium that was clearly delineated from the normal myocardium. The hearts were stained with X-gal solution, and gross examination revealed sparse areas of blue discoloration in the regions of cell implantation. When serial cross-sections of the hearts were evaluated, labeled MSCs could be identified in all 18 hearts in the scar region, with a few MSCs also seen migrated into the adjacent normal myocardium. Sections were stained with hematoxylin and eosin to characterize the injured myocardium and surrounding viable muscle tissue. The undifferentiated phenotypes of the untreated cells (Fig 1) could be seen in comparison with the treated cells (Fig 2). By 4 weeks, most of the cryoinjured area was devoid of inflammatory cells and was replaced by fibrous and connective tissue, as seen with picrosirius red staining. The presence of labeled MSCs was seen in the distribution of the scar, with very few seen to migrate into the surrounding viable tissue. The aza-treated cells appeared morphologically distinct from the untreated cells in the scar tissue. By 4 to 6 weeks, they had a tendency to elongate and align with one another in close approximation (Fig 3A, 3B). By 8 weeks, there was still progressive elongation with a rod-shaped morphology and the grafted MSCs tended to align themselves parallel to the direction of stretch. The untreated MSCs remained clumped and circular in morphology with a large nucleus-to-cytoplasm ratio. To investigate the cardiomyogenic

4 Ann Thorac Surg BITTIRA ET AL 2002;74: MSCs FOR MYOCARDIAL REGENERATION 1157 Fig 1. Cross-section through cryoinjured myocardium with untreated MSCs at 4 weeks, stained with X-gal and hematoxylin and eosin. Note the large nucleus-to-cytoplasm ratio of the cells with unpatterned distribution (100 ). Fig 2. Cross-section through cryoinjured myocardium with 5-aza-treated MSCs at 4 weeks, stained with X-gal and hematoxylin and eosin. Cells are linearly arranged with one another and with the anatomic distribution of adjacent viable myocardial fibers (100 ). Fig 3. (A) Cross-section through cryoinjured myocardium with untreated MSCs at 5 weeks, stained with picrosirius red. Note clumped and unorganized distribution of MSCs (200 ). (B) Cross-section through cryoinjured myocardium with 5-aza-treated MSCs at 5 weeks, stained with picrosirius red. MSCs are elongated in shape with a linear distribution to one another (200 ). Fig 4. (A and B) Cross-sections through cryoinjured myocardium with 5-aza-treated MSCs at 6 weeks, stained with antibody for troponin I. Arrows denote presence of troponin within cytoplasm, seen as the brown pigment (400 ).

5 1158 BITTIRA ET AL Ann Thorac Surg MSCs FOR MYOCARDIAL REGENERATION 2002;74: Fig 5. Cross-sections through cryoinjured myocardium with untreated MSCs at 6 weeks, stained with antibody for troponin I. Note relatively little presence of brown pigment within cytoplasms of cells (400 ). Fig 6. Cross-section through cryoinjured myocardium with 5-azatreated MSCs at 6 weeks, stained with antibody for connexin 43. Note presence of connexin 43 in adjacent viable myocardium (arrows) but not found within implantation area (200 ). differentiation of these MSCs, both subsets of cells were stained for troponin I, a cardiomyocyte-specific marker [14]. By as early as 4 weeks, there was considerably more troponin production within the cytoplasm of the treated cells (Fig 4A, 4B) in comparison with the untreated cells (Fig 5), but no evidence of any myosin heavy chain production in both cell populations at any of the time points studied. Electromechanical integration of grafted MSCs into the myocardium was investigated by immunostaining for one of the major components of the intercalated disk, connexin 43 [27]. Despite 5-aza pretreatment, there was no presence of any connexin 43 seen at any of the time points (Fig 6). In keeping with previous studies in our laboratory, however, sections stained for anti-alpha smooth muscle actin identified the transformation of some labeled MSCs, both untreated and treated, into smooth muscle phenotypes (Fig 7). [17], the MSCs were injected into periinfarct myocardium, and they likewise found MSC-derived cardiomyocytes within the scar, presumably also as the result of cellular migration from the implant sites into the fibrous scar tissue. Nevertheless, one implantation strategy favored by many [5 11] is to implant donor cells directly within the scar, with the goal of replacing scar with contractile Comment As described above, our earlier studies in which MSCs were implanted directly into the myocardium suggested that the differentiation of these marrow-derived adult stem cells was guided by the in vivo signals from the microenvironment in situ [8]. Our more recent studies indicate that if MSCs are delivered to the heart through the vascular system, either by intravenous [15] or intracoronary artery [16] injection, these cells are capable of leaving the vascular lumen and migrating toward the site of myocardial injury. In the process of such migration, they can differentiate into various phenotypes. Thus, with this mode of cell delivery, MSC-derived cells expressing cardiomyocytic markers may be found within myocardial scar tissue. In the experiments by Orlic and associates Fig 7. Cross-section through cryoinjured myocardium with 5-azatreated MSCs at 6 weeks, stained with antibody for smooth muscle actin. Note presence of small arterioles staining positive with brown pigment, indicating presence of alpha smooth muscle content. Arrow denotes a MSC that has incorporated within a small arteriole (200 ).

6 Ann Thorac Surg BITTIRA ET AL 2002;74: MSCs FOR MYOCARDIAL REGENERATION 1159 muscle tissue. Current clinical trials using skeletal myoblasts [4] utilize this technique of implantation. However, when MSCs are implanted directly within the scar, they are in a nonmyogenic milieu. Thus, our study reported here tests the hypothesis that for such an implant approach, in vitro preprogramming of the donor MSCs may enhance their myogenic differentiation. Our findings appear consistent with those reported by Tomita and associates (11), although they used 5- aza-cytidine whereas we used its active metabolite, 5-aza-2 deoxycytidine. Our donor MSCs were pretreated with 5-aza before implantation into the cryoinjured ventricular myocardium. The phenotypic outcome was compared with a control group in which the donor MSCs were not pretreated in vitro. Whereas there was evidence of cardiomyogenic differentiation in both groups, the proportion of cells that differentiated into a cardiomyocytic phenotype was greater in the 5-aza-treated group. More striking than the development of immunohistochemical evidence for cardiomyogenic markers was the morphological and structural relationships that had formed among the 5-aza-treated cells in comparison with the untreated ones. These troponin I-c positive cells are often lineally oriented, rather than randomly scattered. These findings appear consistent with the hypothesis that pretreating MSCs with 5-aza before local implantation into a scar tissue is desirable. However, even though a previous study using this approach was reported to improve ventricular function of an infarcted heart, the exact mechanism of such improvement remains unclear. There was no evidence of gap junction formation between neo-myofibers within the scar and the native myocardium outside of the infarct, making synchronous contraction unlikely [18]. At present, the beneficial functional effects of intracardiac implantation of cells seem to be related to the reduction of scar expansion and ventricular dilatation, and not augmentation of contractile force by the newly formed muscle tissue. Thus, further exploration of other strategies for cell implantation, such as periinfarct implantation [17, 19] and cellular administration via intravenous (15) or intracoronary [16] routes, should be worthwhile. The technical assistance of Minh Duong, BS, is deeply appreciated. References 1. Chiu RCJ, Zibaitis A, Kao RL. Cellular cardiomyoplasty: myocardial regeneration with satellite cell implantation. Ann Thorac Surg 1995;60: Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001;108: Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98: Menache P, Vilquin J-T, Desnos M, et al. Early results of autologous skeletal myoblast transplantation in patients with severe ischemic heart failure. Circulation 2001;104:II Taylor DA, Atkins BZ, Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nature Med 1998;4: Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284: Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103: Wang JS, Shum-Tim D, Galipeau J, Chedrawy E, Eliopoulos N, Chiu RCJ. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg 2000;120: Edelman GM. Topobiology: an introduction to molecular embryology. New York: Basic Books, Publ., Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 1995:18; Tomita S, Li R-K, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100(Suppl II): Konieczny SF, Emerson CP Jr. 5-Azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: evidence for regulatory genes controlling determination. Cell 1984;38: Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9: Adams JED, Bodor GS, Davila-Roman VG. Cardiac troponin I: a marker with high specificity for cardiac injury. Circulation 1993;88: Bittira B, Kuang J-Q, Piquer S, Shum-Tim D, A1-Khaldi A, Chiu RCJ. The pathophysiological roles of bone marrow stromal cells (MSCs) in myocardial infarction. Circulation 2001;104(Suppl II): Wang J-S, Shum-Tim D, Chedrawy E, Chiu RCJ. The coronary delivery of marrow stromal cells for myocardial regeneration: pathophysiologic and therapeutic implications. J Thorac Cardiovasc Surg 2001;122: Orlic D, Kajstura J, Chiment S, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410: Chiu RCJ. Therapeutic cardiac angiogenesis and myogenesis: the promises and challenges on a new frontier [Editorial]. J Thorac Cardiovasc Surg 2001;122: Matsushita T, Oyamada M, Kurata H, et al. Formation of cell junctions between grafted and host cardiomyocytes at the border zone of rat myocardial infarction. Circulation 1999; 100(Suppl II): DISCUSSION DR GINO GEROSA (Padova, Italy): Did you apply the same technology also to the peripheral stem cells? Did you apply the same technology using 5-aza to the peripheral stem cells? DR BITTIRA: No. We just looked at the bone marrow stromal cells and we looked at the marrow stromal component of that, not the hemopoietic stem cells.

7 1160 BITTIRA ET AL Ann Thorac Surg MSCs FOR MYOCARDIAL REGENERATION 2002;74: DR GEROSA: Are you planning to do that? DR BITTIRA: No, we do not have a plan to do that. DR EUGENE SIM (Singapore): Thank you for your excellent paper. How did you prepare the bone marrow to get the stem cells, because the bone marrow stroma contains a lot of cells. What proportion of those cells do you think are actually stem cells, because that is very controversial? The second question I wanted to ask you is, what concentration of 5-azacytidine did you use and for how long, because since Makino s work, a lot of groups have actually tried doing the same thing, but they have not been uniformly successful in reproducing this directed differentiation of stem cells. DR BITTIRA: Those are two good questions. Thank you. There are actually two groups in the literature that have used 5-azacytidine pretreatment. Fukuda s paper has actually looked at different concentrations, as well as Dr Wakitani s paper, which looked at varying concentrations of 5-azacytidine, anywhere from 0.1 to 10 micromolar, and they found that the highest rate of conversion to a myogenic phenotype was found at 0.3 micromolar per liter, which is the concentration that we used in our study. After our bone marrow stromal cells are isolated, we treated them for 24 hours with a concentration of 0.3 micromolar and then rinsed our cells and did the same pretreatment 24 hours later. So we had actually two treatments with a 24-hour gap inbetween them. We did not actually look at how many of these cells converted to a myogenic phenotype, although we know from those previous studies that 30% of those treated with 5-azacytidine actually will convert to a myogenic phenotype. As to how exactly we identified our stromal cells from the other cells, we used a method described by Kaplan, known now as Kaplan s method, where we harvested the bone marrow very crudely; however, when we plated the cells we found that the cells that settled and stuck to the bottom of the Petri dish were the cells we used later on in our study and have been known to be mesenchymal stem cells. So after repeated passaging and washing, the hematopoietic component of these cells was washed away and we were left with these bone marrow stromal cells. You are right, it is a crude method, but it is not something we wanted to quantify as to how many would actually have a myogenic potential later on, but it is a method that has been written and shown to work. DR SIM: Did you try to identify whether these cells are CD-34 positive or CD-34 negative or whether they are lineage positive or negative, because there is a lot of controversy also in what kind of cells are actually the stem cells, and I am not sure anyone really knows. Did your group try to do that? DR BITTIRA: No, we did not actually look at that. This was strictly sort of an observational study to look at whether the mode of implantation, intravenous versus direct injection, would have a role to play in the ultimate phenotype; but you are right, there is controversy with that, and we did not look at any lineage specificity, the CK ligand or CD-34 positivity, no. Discussion by Todd K. Rosengart, MD., Evanston, Illinois. tkrosen@enh.org DR TODD ROSENGART (Evanston, IL): Assuming that the improvements you are observing are related to functional changes, is this on the basis of scaffolding or stabilizing of the scar, or in fact do you have any evidence to suggest that there is contractile function that is contributing? DR BITTIRA: The functional component of this study will actually be presented at the AATS, so I will not reveal those results, but it is promising that the function is improved with 5-azacytidine pretreatment. As you know, the marrow stromal cells can develop into a number of different cell types (smooth muscle cells, cardiomyocytes), so it is likely there is a scaffolding component to help with the remodeling of the scar as well as maybe some angiogenic component as well, because we have seen a lot of these cells within the vessel walls. So there could be a number of things going on.