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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells

Definitions

• the invention relates to autologous bone marrow stroma cells (MSCs) and the use of these cells in improving cardiac function in patients with heart failure.
• the present invention relates to the transplantation of MSCs into the myocardium to grow new muscle fibers.
• the specific aims of the invention include: 1) using cell labeling techniques to confirm the survival and differentiation of implanted MSCs, and to identify their phenotype by both morphology and molecular markers; 2) examining the effects of the micro-environment of the implanted MSCs, on their differentiation and phenotype expression; and 3) the functional contribution of MSCs when implanted into an ischemic segment of a myocardium.
• Heart failure is both common and deadly. In Canada, approximately 50,000 new cases of heart failure are diagnosed, and more than 300,000 patients currently suffer this condition. Heart failure is the only major cardiovascular disorder that is increasing in incidence and mortality at present, and in Class IV patients, one year mortality approaches 50% in spite of advances in drug therapy in recent years (L' ieri, C: Fixing the failing heart. Circulation 1997; 95:771-772).
• tissue engineering m which various cells are cultured m vi tro over biodegradable polymer scaffolds to create a 3- dimensional construct m vi tro, which can then be implanted to replace damaged tissues or organs.
• these replacement tissues would not require immunosuppression if autologous donor cells were used for tissue engineering.
• Advances are being made m constructing cardiovascular structures, such as arteries and cardiac valves. Attempts are also being made to engineer 3-d ⁇ mens ⁇ onal myocardial tissue blocks by seeding cardiac myocytes on a 3-d ⁇ mens ⁇ onal scaffold, and cultured m rotating bioreactors . Without concomitant creation of a coronary vascular system within these constructs, however, such tissue engineered myocardium cannot be used therapeutically m vi vo, as they will suffer rapid ischemic necrosis.
• Cellular cardio yoplasty is a potential future therapy for heart failure m which donor cells with the potential to differentiate into cardiac myocytes are implanted into the damaged myocardium m order to regenerate new muscle fibers (Chiu RC-J, Zibaitis A, Kao RL: Cellular cardiomyoplasty: Myocardial regeneration with satellite cell implantation. Ann Thorac Surg 1995; 60:12- 18) .
• Ann Thorac Surg 1995; 60:12- 18 a number of donor cells have been studied by various investigators, and are summarized below.
• fetal cardiomyocytes retain a capacity for proliferation.
• fetal cardiomyocytes implanted into the myocardial wall of adult animals have been shown to be successfully engrafted, and develop into cells which are morphologically and functionally indistinguishable with the native cardiac myocytes within the recipient heart. They form gap junctions which should allow them to be depolarized and contract synchronously as a syncytium (Soonpaa MH, Koh GY, Klug MG, et al.: Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 1994; 264: 98-101).
• fetal cardiomyocyte transplantation is likely to require millions of new cells to be efficacious, and continued proliferation of engrafted myocytes cannot be expected to expand the population, once they are removed from the donor embryos .
• Klug et al. transfected a transgene that confers resistance to a toxic drug into embryonic stem cells under the control of a cardiac specific promoter (Klug M.G., Soonpaa M.H., Koh G.Y., Field L.J. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J. Clin. Invest. 1996; 98:216-24). When embryoid bodies derived from these stably transfected embryonic stem cells were exposed to the toxic antibodies, only cardiac myocytes survived. These cells were harvested and injected into the myocardial wall of the adult mice, where they engrafted and formed appropriate cell-to-cell junctions, i.e.
• the adult cardiomyocytes are generally believed to be terminally differentiated and thus unable to proliferate. It has been previously reported that adult cardiomyocytes obtained from biopsy can be induced to proliferate in vi tro, while retaining some phenotypic characteristics of the cardiac myocytes, and they can be successfully engrafted into the myocardium (Li RK, Jia ZQ, Weisel RD et al. Cardiomyocyte transplantation improves heart function. Ann. Thor. Surg. 1996; 62:654-660). It was shown that such cells implanted into an ische ic myocardium could improve ventricular function.
• One aim of the present invention is to provide means to perform myocardial implantation without eliciting an immune response and without sacrificing the patient's skeletal muscle.
• Another aim of the present invention is to use autologous MSCs m myocardial implantation to improve cardiac function.
• Another aim of the present invention is to use autologous MSCs m myocardial implantation to effect the growth of new muscle fibers. - 7 -
• Another aim of the present invention is to provide means for using a cell labeling technique to confirm the survival and differentiation of the implanted MSCs, and to identify MSCs phenotype by both morphology and molecular markers .
• Another aim of the present invention is to provide a means for using autologous marrow stem cells for improving cardiac function, wherein said autologous marrow stem cells are introduced in si tu into a myocardium.
• the bone marrow stroma micro-environment is a complex network of cells and extracellular matrix, which maintains the hematopoietic system throughout the life of the individual. Hematopoietic cells are found in the spaces between the marrow stroma.
• the marrow stroma cells are functionally defined as capable of supporting hematopoiesis (Lichtman MD: The relationship of stromal cells to hemopoietic cells in marrow. In Long Term Bone Marrow Cul ture, pp. 57-96, DG Wright, JS Greenberger (eds) , Alan R.
• mesengenic process for self-repair (Caplan Al : The mesengenic process. Clinics Plast Surg 1994;21:429-435).
• the body has developed two major strategies for tissue replacement and renewal. The first way the body attempts cell repair is predicated on the remaining proliferative capacity of differentiated, functioning cells, such as hepatocytes and endothelial cells. The second way is by their regeneration from residual cycling stem cells.
• This category is the blood cells. All cells of the hematopoietic lineage are derived from a limited number of self-renewing multi-potent cells which respond to the appropriate cytokmes and growth factors for differentiation.
• bone marrow also contains cells that meet the criteria for stem cells of non-hematopoietic tissue. These cells are referred to as mesenchymal stem cells, because of their ability ro differentiate into cells that can roughly be defined as mesenchyma. These cells are also known as marrow stroma cells (MSCs) , because they appear to arise from the supporting structures found m bone marrows .
• MSCs marrow stroma cells
• the donor cells accounted for 1.5 to 12% of the differentiated cells m bone, cartilage, and lung m addition to marrow and spleen. It appeared that the donor MSCs first replaced a portion of the MSCs m the bone marrow of the recipient mice. The MSCs then participated m a normal biological cycle m which MSCs m the bone marrow served as a continuing source of progenitor cells for a variety of mesenchymal tissues in the body. These findings suggested that the progeny of MSCs acquired the phenotype of different target tissues, either before they left the marrow, or after they have entered the micro-environment of the tissue itself through "milieu-dependent differentiation".
• MSCs participate in the "mesengenic process" which continues throughout life (Caplan A.I.: The mesengenic process. Clinics Plast. Surg. 1994; 21:429-435). This process functions to continually rejuvenate various mesenchymal tissues and ensure rapid repair of tissue injuries. Such repair involves the recapitulation of the same cellular transitional events observed during embryonic development on the scaffolding of the pre-existing macro-morphology and its signaling systems. Clinical applications of these findings have been attempted (Horwitz EM, et al . Transplantability and therapeutic effects of bone marrow- derived mesenchymal cells in children with osteogenesis imperfecta. Nature Medicine 5, 309-313, 1999) .
• MSCs have been shown to differentiate to cardiac myocytes in vi tro under proper conditions (Makino S. Fukuda K, Miyoshi S, et al . : Cardiomyocytes can be generated from marrow stromal cells in vi tro . J. Clin. Invest. 1999; 103:697-705). After immortalizing MSCs by prolonged culture in vi tro, they were able to identify a single clone of adherent fibroblast like cells which, when treated with 5- azacytidine, would reproducibly differentiate into adjoining myocytes with synchronous beating.
• cardiomyogenic cells acquired many morphologic features of cardiac muscle, including sarcomeres, one to three centrally located nuclei, and atrial granules. They also expressed several cardiac specific genes, including the GATA4 and Nkx2.5 transcription factors and the brain natriuretic peptide (BNP) as well as atrial natriuretic factor (ANF) genes. They stained positive with anti-myosm, anti-desmm, and anti-actinm antibodies. In addition, they displayed cardiac like action potentials with a shallow resting membrane potential, long action potential duration, as well as a late diastolic slow depolarization current.
• BNP brain natriuretic peptide
• AMF atrial natriuretic factor
• the myocardium belongs to mesenchymal tissues.
• the myocardium was investigated as a target recipient of MSCs for effecting m vi vo MSCs differentiation for improving cardiac function.
• bone marrow stoma cells were employed m m vivo myocardium implantation and found to effect growth of new muscle fibers and improve overall cardiac function.
• a use of autologous MSCs for improving cardiac function wherein said MSCs are transplanted in si t u into a myocardium, and differentiate into cardiomyocytes, fibroblasts and endothelial cells.
• a method of improving cardiac function m a patient with heart failure without eliciting an immune response and without sacrificing the patient's skeletal muscle which comprises the step of transplanting autologous bone marrow stroma cells (MSCs) into said patient's myocardium to grow new muscle fibers.
• MSCs autologous bone marrow stroma cells
• the method may further comprise the step of using a cell labeling technique to confirm survival and differentiation of implanted MSCs, and to identify said MSCs phenotype by both morphology and molecular markers.
• the method may further comprise examining the effects of the micro-environment of implanted MSCs on their differentiation and phenotype expression.
• the method may further comprise examining functional contribution of MSCs implanted into an lschemic segment of the myocardium.
• the transplanting may be effected m the myocardium m si tu, m the myocardium artery or using a catheter from within the myocardium.
• the transplanting may also be effected m association with angiogenesis factors.
• a method of treating cardiac failure comprising (a) retrieving bone marrow from a patient suffering from cardiac failure; (b) isolating marrow stroma cells from said bone marrow;
• autologous marrow stroma cells for examining the effects of a myocardial micro-environment on marrow stroma cell differentiation, wherein said autologous marrow stem cells are introduced m si tu into an ischemic segment of a myocardium of an animal model.
• MSCs autologous bone marrow stroma cells
• Fig. 1 illustrates four days after implantation of
• Top panel Hematoxylin and eosin stain
• Lower panel Fluorescent microscopy picture, showing
• Fig. 2 illustrates four weeks post-implantation
• Fig. 3 illustrates specimen four weeks after MSC implantation into the myocardium; Top panel: Hematoxylin and eosin stain; Lower panel: Fluorescent cells originated from MSCs labeled with DAPI in vi tro; Fig. 4 illustrates rat MSCs morphology in culture, and in particular, a phase contrast photomicrograph of twice-passaged culture of MSCs just before implantation.
• Fig. 5 illustrates histochemical staining for ⁇ -galactosidase activity of rat MSCs in culture. The transfected MSCs showed clear staining for ⁇ -galactosidase activity. Transfection efficiency of the MSCs was approximately 100%.
• Scale bar represents 60 ⁇ m;
• Fig. 6 illustrates ⁇ -gal positive cells trapped within a coronary capillary immediately after MSCs injection. Staining for ⁇ -galactosidase activity followed by H & E stain. Arrow, a capillary endothelial cell. Scale bar represents 15 ⁇ m; Fig.
• FIG. 7 illustrates ⁇ -gal positive cells with i morphology outside infarct scar 4 weeks after MSCs injection. Staining for ⁇ -galactosidase activity followed by H & E stain. Arrows, intercalated disk-like structure. Arrowhead, the nucleus of a ⁇ -gal positive cell. Scale bar represents 15 ⁇ m;
• Fig. 8 illustrates ⁇ -gal positive cells with fibroblast-like morphology m the myocardial scar 4 weeks after MSCs injection. Staining for ⁇ -galactosidase activity followed by H & E stain. Arrows, ⁇ -gal positive cells. Scale bar represents 1.5mm. (Inset) Higher magnification of the area m square. Scale bar represents 30 ⁇ m; Fig. 9 illustrates ⁇ -gal positive cells incorporated into endocardium 4 weeks after MSCs injection. Staining for ⁇ -galactosidase activity followed by Eosin stain. Arrow, endocardium. Scale bar represents 15 ⁇ m;
• Figs. 10A & 10B illustrate ⁇ -gal positive cells incorporated into coronary capillaries 4 weeks after MSCs injection. Staining for ⁇ -galactosidase activity followed by H & E stain.
• A Outside the mfarct scar. Arrow, a capillary with ⁇ -gal positive cells m the subendocardial fibrosis area. Arrowhead, normal myocardium. Asterisk, endoventricular space. Scale bar represents 600 ⁇ m. (Inset) Higher magnification. Arrow, the cross section of the same capillary with ⁇ -gal positive cells. Scale bar represents 15 ⁇ m.
• B In the mfarct scar. Arrow, the oblique section of a capillary with ⁇ -gal positive cells. Scale bar represents 15 ⁇ m; and
• Fig. 11 illustrates histochemical stain for connexm-43 m the intercalated discs (arrows) , demonstrating the presence of gap junction unique to myocardium m the labeled (blue) myocytes.
• MSCs are infused into coronary artery and appear to repopulate the heart. Further, signals originating in the cardiac milieu appear to modify the developmental program of the infused MSCs. Studies conducted in accordance with the present invention further confirm the residence of MSCs outside of the capillary bed, and illustrate the structural interactions between the host myocardial tissues and the implanted MSCs.
• MCSs were retrovirally transfected with ⁇ -gal reporter gene for cell labeling.
• retrovirus has less immunological response and longer gene expression (Onifer SM, White LA, Whittemore SR, Holets VR.
• the transfection efficiency in culture is approximately 100% without obvious adverse effect on the cell growth.
• MSCs were trapped within the coronary capillaries in the non-infarct area.
• the reason MSCs could not be found in the infarct scar at this time may be related to the complete occlusion of involved coronary artery (Left coronary artery) .
• the MSCs could be found both in the infarcted scar and in non-infarct area outside the vascular structure.
• the mechanism of the translocation of MSCs from the vascular lumen into the myocardial interstitium is unknown. MSCs may migrate out of the vasculature and move from the non-infarct area to the infarction scar.
• MSCs in different myocardial microenvironments clearly have different fates.
• they express the phenotypes of normal cardiomyocytes and connected with surrounding host cardiomyocytes by intercalated disk-like structure.
• they appear primarily fibroblast-like.
• This homing ability and the capability to acquire the phenotypes of different target tissues suggest that the microenvironment plays a significant role for the differentiation of these cells.
• the normal myocardial microenvironment appears to enable newly arrived cells to be exposed, in an appropriate sequential manner, to various cardiomyogenic specific growth factors and differentiation molecules, such that the infused MSCs could develop into fully mature cardiomyocytes.
• the fibroblast-like MSCs seen in the infarction scar could have differentiated into primary fibroblast, which are mature mesenchymal cells or they could still maintain the multipotent differentiation ability for future maturation.
• ⁇ -gal positive donor cells differentiate into endothelium, which was incorporated into capillaries in the infarct and non-infarct areas. Accoringly, these marrow- derived endothelial progenitor cells are likely to be involved in the angiogenesis and vasculogenesis in the remodeling process of myocardial infarction. Thus furhter suggesting the potential of use of MSCs for implantation into the myocardium to improve heart function.
• Both localized site-specific and global delivery of autologous MSCs may be of potential therapeutic benefit in view of different cardiac pathology.
• intra- coronary delivery of MSCs may be more suitable for the treatment of heart failure due to diffuse cardiomyopathy .
• the present invention demonstrates that when expanded marrow-derived stromal cells are delivered to the coronary circulation of an infarcted heart, they are capable of populating the heart and differentiating along several lineages including cardiomyocytes, fibroblast and endothelial cells.
• infracted heart muscle can signal mobilization of MSCs to enter circulation, and reach the coronary artery, where they may participate in myocyte replenishment, reactive fibrosis and scar formation as well as angiogenesis in the post-infarct pathophysiological remodeling process, involving both the infarcted segment and the remote non- infarcted areas. Additional studies in accordance with the present invention will further elucidate the role of MSCs in myocardial infarction and the clinical applications of MSCs implantation. It is fully contemplated that the findings presented in accordance with the present invention will enable therapeutic modulation of the remodeling process after myocardial infarction, in both animals and humans .
• Fig. 1 illustrates a microphotograph obtained 4 days after implantation.
• the implant site shows a needle track created during the process of injecting the MSCs, with some inflammatory response and fibrosis within the needle track. Fluorescent microscopic examination demonstrated the presence of the labeled MSCs implanted.
• Fig. 1 illustrates a microphotograph obtained 4 days after implantation.
• the implant site shows a needle track created during the process of injecting the MSCs, with some inflammatory response and fibrosis within the needle track. Fluorescent microscopic examination demonstrated the presence of the labeled MSCs implanted.
• FIG. 2 shows labeled cells in the injection site, immunolabeled with myosin-slow antibodies which stains red; DAPI labeled fluorescent cells within needle track made during implantation; and immunohistochemical stain with antibody against slow myosin heavy chain, which shows red color.
• the triangle marker points to the native cardiac myocyte, and arrows point to the appearance of myosin molecules in the cytoplasm of implanted cells.
• Fig. 3 is a photograph taken a short distance away from the implant needle track, showing migrated or infiltrated MSCs appearing to have differentiated fully and were incorporated into cardiac muscle fibers, morphologically indistinguishable with the native myocardium. Clear labeling of these cells can be demonstrated under fluorescent microscopy. Morphologically, they appear identical to the native myocardial fibers. Photographs were taken from the myocardium adjacent to the needle track, where the implanted cells had migrated or infiltrated.
• rat receiving cardiac cell implant there will be an isogenic rat to serve as the donor of bone marrow stroma cells.
• Another group of sham operated rats (controls) will undergo identical surgical procedures as the experimental animals, but will receive injection of cell culture media without MSCs.
• Donor rats will be sacrificed, and their femoral and tibial bones will be used to isolate, select and culture MSCs m vi tro for 2 weeks using the technique described below. Then the cells will be collected, labeled and injected into the myocardium of the experimental recipient rats .
• sample size of 10 was based on our preliminary study as this is a highly reliable model that resulted in minimal operative mortality. In the future, wall motion studies will be performed to calculate the sample size required.
• Isolation and primary culture of MSCs will be performed according to Caplan' s method.
• pentobarbital 100 mg/kg given intraperitoneally
• MTC Pharmaceuticals Cambridge, Ontario
• Meticulous dissection of the long bones will be carried out in order to remove soft tissue to ensure that myogenic precursors are not carried into the bone marrow preparation.
• Both ends of the bones will be cut away from the diaphysis with bone scissors.
• the bone marrow plugs will be hydrostatically expelled from the bones by insertion of 18-gauge needles fastened to 10 ml syringes filled with complete medium; the needles are inserted into - 26 -
• the marrow plugs are disaggregated by sequential passage through 18-gauge, 20-gauge and 22-gauge needles and these dispersed cells are centrifuged and resuspended twice in complete medium. Cell viability is assessed by the trypan blue exclusion test.
• 5xl0 7 cells in 7-10 ml of complete medium are to be introduced into 60 mm polystyrene tissue culture dishes (Corning, Inc., Corning, NY), which are coated in advance with a layer of laminin (Sigma) to promote marrow stroma cell adherence.
• laminin Sigma
• the dishes will become nearly confluent and the adherent cells can be released from the dishes with 0.25% trypsin in 1 mmol/L sodium ethylenediaminetetraacetic acid (Gibco Laboratories, Grand Island, NY), split 1:2, and seeded onto fresh plates. After these twice passaged cells become nearly confluent, they can be harvested and used for implantation experiments described below after being labeled with DAPI.
• the "complete medium” mentioned above for our culture consists of Dulbecco's modified Eagle's medium (DMEM, Gibco Laboratories) containing selected lots of 10% fetal calf serum (FCS; JR Scientific Inc., Woodland, CA) , and antibiotics (Gibco Laboratories; penicillin G, 100 U/ml; streptomycin 100 ⁇ g/ml, amphotericin B 0.25 ⁇ g/ml) at 37°C in a humidified atmosphere of 5% C0 ; .
• DMEM Dulbecco's modified Eagle's medium
• FCS fetal calf serum
• antibiotics Gibco Laboratories
• penicillin G 100 U/ml
• Retroviral vectors permit insertion of foreign synthetic genetic information m target cells. Reporter genes such as ⁇ -galactosidase, can therefore be stably integrated m chromosomal DNA. Expression of these transgenes permits ambiguous identification, by classic histochemical or fluorescence microscopy, of gene modified cells. As an example, retroviral labeling of hematopoietic stem cells is routinely carried out m the study of bone marrow transplantation m rodents.
• Undifferentiated progeny cells of a widely different phenotype are readily detectable up to one year after bone marrow transplantation.
• retroviral labeled stroma cells that survive, proliferate or differentiate following cardiac implantation, will preserve and express the transgene as is classically observed m animals transplanted with retrovirus labeled hematopoietic stem cells.
• important questions regarding bio-distribution of transplanted stroma cells can be addressed. Unambiguous identification of "labeled stroma cells”, as well as their differentiated progeny, local and distant to the site of implantation m the injured heart, will become possible.
• cortisol cortisol cortisol cortisol
• the recipient rats both experimental and control, are isogenic Lewis rats weighing 175 to 200 grams. Anesthesia is induced and maintained with isoflurane (MTC Pharmaceuticals) .
• the animals are intubated with an 18- gauge intravenous catheter and connected to a Harvard rodent ventilator (Harvard Apparatus Co., Inc., South Natick, Mass.) at 85 breaths per minute.
• the heart is exposed via a 1.5 cm left thoracotomy incision. Under direct vision, using a 5-0 prolene suture, the anterior descending coronary artery which is visible in the epicardium is ligated proximally.
• the thoracotomy is closed with 4-0 monofilament sutures.
• the muscle and skin layers are closed with 4-0 absorbable sutures and the animals are returned to their cages with filter tops. After the learning curve, the operative mortality is virtually nil.
• the recipient rats which underwent coronary artery ligation 2 weeks previously will undergo a second operation. Anesthesia and thoracotomy will be performed in the manner described above. Under direct vision, the MSC suspension is injected into the lateral wall of the left ventricle with a 20-gauge needle, both at the center of the ischemic segment of the myocardium, and at the border zone at the junction between the infarcted and normal myocardium.
• the thoracotomy closure and post-operative care are similar to that described above, and the animals sacrificed at intervals after this procedure, as stated earlier.
• the sham operated control rats will undergo an identical procedure as described for cell implantation in the experimental animals. The only difference is that instead of injecting cultured MSCs, an identical volume of culture media (component described above) will be injected.
• Transthoracic Doppler echocardiographic studies will be performed in the rats every week following implantation.
• the rats are anesthetized as described above, the chest wall shaved, and echocardiography performed using our echo system equipped with a 7.5 MHZ transducer (Hewlett Packard Sonos 2500) .
• a 2-dimensional short axis view of the left ventricle is obtained at the level of the papillary muscle to record M-mode tracing.
• Anterior and posterior end- diastolic and end-systolic wall thickness and LV diameters are measured using the American Society of Echocardiography Lineage Method, from at least three consecutive cardiac cycles. The changes in wall thickness and ventricular segmental wall motion and diameter will be recorded on videotape, and assessed by blinded independent echocardiographers .
• the heart specimens obtained from the recipient rats at various intervals will be perfused with 100 ml of saline through the posterior wall of the left ventricle, avoiding the transplant area, then processed for frozen sections.
• the lateral wall of the left ventricle is isolated from the remainder of the heart. Sections 6 ⁇ m thick are cut from the hearts and successive sections collected by gelatine coated glass slides. This ensures that different stains could be applied on successive sections of the tissue cut through the transplanted area (Figs. 1 and 3) .
• One of the sections is mounted and stained with X-Gal, to identify and view the ⁇ -gal labeled donor cells.
• An adjacent section is stained with hematoxylin and eosin as described in the manufacturer's specification (Sigma Diagnostics) to depict nuclei, cytoplasm and connective tissue. Other adjacent sections will be immunolabeled using various antibodies for immunohistochemical evaluation in order to identify phenotypic expression at the molecular level.
• MSCs Bone marrow stromal cells
• the cells were routinely cultured in complete medium consisting of Dulbecco's modified Eagle's medium (DMEM) containing selected lots of 10% fetal calf serum and antibiotics (100 U/mL penicillin G, lOO ⁇ g/mL streptomycin and 0.25 ⁇ g/mL amphotericin B; all obtained from Gibro laboratories) at 37°C in a humidified atmosphere of 5% C0 2 .
• DMEM Dulbecco's modified Eagle's medium
• P+E86 murine ectropic retrovirus-packaging cells which are derived from NIH 3T3 mouse fibroblasts, were obtained from Dr. Denis Cournoyer (McGill University, Montreal, QC, Canada) (Momparler RL, Laliberte J, Eliopoulos N, Beause journey C, Cournoyer D. Transfection of murine fibroblast cells with human cytidine deaminase cDNA confers resistance to cytosine arabinoside. Anti-Cancer Drugs 1996;7:266-274).
• the GP+E86 cells were transfected with the purified plasmid DNA pMFG-LacZ in a 10:1 molar ratio using the standard calcium phosphate transfection kit
• the LacZ gene encodes for the production of bacterial ⁇ -galactosidase. These cells were plated at 25% confluence for 48 hours. The second passaged MSCs growth medium was replaced with the supernatant from the GP+E86 cells (containing the replication-defective retrovirus carrying the ⁇ -gal reporter gene) to transfect the MSCs overnight and then replaced with normal complete medium for the following day. After three times of transfection, MSCs were then collected
• Staining for ⁇ -gal was accomplished at 37°C for 16 hr in a solution containing 1 mg/ml 5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside (X-gal) , 2% dimethylsulfoxide, 10 mM potassium ferricyanide, 10 mM potassium ferrocyanide, 1 mM magnesium chloride, and 0.02% Nonidet P-40 in PBS, pH7.3.
• the hearts were excised and sliced along short axis of left ventricle to 3 mm thick sections in series, and fixed in 2% paraformaldehyde in PBS for 2 hours. The sections were then cryoembedded after protection with 20% sucrose in PBS overnight. The other 10 rats were taken for their final experiments 4 weeks after the MSCs infusion. After overdose with pentobarbital, the hearts were exposed and injected with 100 ml saline (0.9%) through the apex of the left ventricle, then perfusion-fixed with 2% paraformaldehyde in phosphate buffered saline (PBS) . The hearts were excised, sliced and prepared as mentioned above.
• PBS phosphate buffered saline
• Cryosections 6 ⁇ m in thickness were collected in each 3 mm section sample across a set of gelatin-coated glass slides. One of every 10 cryosections was collected for histochemical staining for ⁇ -galactosidase activity as mentioned above. The sections were then counter-stained with hematoxylin and eosin. Tissue sections were examined with an Olympus microscope (BX-FLA, Olympus) . Digital images, transferred to a computer equipped with Image Pro software (Media Cybernetics, MA) , were subsequently printed.
• the rats were sacrificed at the following intervals after the MSCs infusion: immediately for 2 rats; and 4 - 36 -
• ⁇ -gal positive cells We failed to identify any ⁇ -gal positive cells in the infarction zone at this time.
• some ⁇ -gal positive cells could be found within the normal myocardial area outside the infarction scar (Fig. 7) . They have centrally located nuclei and are connected among themselves and with surrounding host cardiomyocytes ( ⁇ -gal negative cells) by intercalated disklike structure, which are characteristics of normal cardiomyocytes.
• the ⁇ -gal positive cells also could be detected individually or in clusters within the myocardial scar (Fig. 8) . They appear unorganized and scattered in the infarction scar with fibroblast-like morphology, similar to that of the surrounding ⁇ -gal negative (host) fibroblast cells.
• Some ⁇ -gal positive cells were found incorporated into endocardium (Fig. 9) and coronary capillary endotheliu within or outside the infarct scar area (Figs. 10A & 10B) .
• Tissue sections showing labeled cells with histological features of cardiomyocytes were further studied immunohistochemically using antibodies against connexin 43, a major constituent protein of gap junctions in the intercalated discs of cardiac myofibers.
• the demonstration of such junctional structure (Fig. 11) further confirms the phenotype of the differentiated labeled cells, and their integration into the native cardiac myofibers.
• MSCs are determined to traffic through the circulatory system to the injured heart, and are capable of forming cardiomyocytes and other types of cells, depending on the specific microenvironment.
• endothelial progenitor cells in the MSCs population may be involved in the post- infarction neovascularization process.
• MSCs display myocardial cell differentiation properties in vivo and provide a promising therapeutic use in improving myocardial healing following infarction. Labeled cardiac myocytes and fibers were present in the implant site, which exhibited positive immunohistochemical stains in normal or immature cardiac myocytes.
• MSCs autologous marrow stroma cells
• the present invention has application in the area of experimental research, where by MSCs may be employed in a variety of in vivo animal models to further study the influence of the micro environments on stem cell differentiation, and on cellular signaling mechanisms. Further echocardiographic studies are expected to demonstrate improved systolic thickening of the ischemic ventricular wall segment, and reduced ventricular size and remodeling, as had been reported following the implantation of other donor cells.

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