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Ann Thorac Surg 2002;74:1154-1160
© 2002 The Society of Thoracic Surgeons
a Division of Cardiac Surgery, McGill University Health Center, Montreal, Quebec, Canada
* Address reprint requests to Dr Chiu, The Montreal General Hospital/MUHC, 1650 Cedar Ave, Room C9-169, Montreal, Quebec, Canada H3G 1A4
e-mail: rchiu{at}po-box.mcgill.ca
Presented at the Thirty-eighth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 28–30, 2002.
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Abstract |
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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 milieu-dependent differentiation, unmodified MSCs may augment myocardial angiogenesis and myogenesis, whereas converting scar into myogenic tissue may be facilitated by preprogramming of MSCs before implantation.
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Introduction |
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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 cardiac-specific 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, 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.
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Material and methods |
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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 µg/mg streptomycin, 0.25 µg amphotericin B; all obtained from Gibro Laboratories [Boston, MA]) in a humidified atmosphere of 5% CO2. 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 ß-galactosidase enzyme. These cells were cultured in DMEM with 10% fetal calf serum and antibiotics (50 U/mL penicillin G and 50 µg/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 µL 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 0.45-µm 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 ß-galactosidase activity, in order to determine the percentage of cells expressing ß-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 ß-gal activity was performed with a solution containing 1 mg/mL 5-bromo-4-chloro-3-indoyl-ß-D-galactoside (X-gal), 2% dimethylsulfoxide, 20 mmol/L K3Fe(CN)6, 20 mmol/L K4Fe(CN)6.3H2O, and 2 mmol/L magnesium chloride. The cells were then incubated at 37°C and protected from light for 16 hours. The presence of blue-labeled 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 µmol/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% CO2. 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 3 x 106 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 cm3 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 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 ß-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 ß-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 µm 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, ß-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.
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Results |
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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 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).
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Comment |
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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 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 5aza-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.
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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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.
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. E-mail: 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.
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