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Dynamic changes in haematopiotic stem cells after myocardial infarction Elmestiri, Mostafa Mohamed 2008-10-03

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DYNAMIC CHANGES IN HAEMATOPIOTIC STEM CELLSAFTER MYOCARDIAL INFARCTIONbyMostafa Mohamed ElmestiriM.D. Alfateh University 1997M.B.B.C.H Alfateh University 1998A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFREQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinThe Faculty of Graduate Studies( Surgery )THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)APRIL 2008© Mostafa Mohamed Elmestiri 2008ABSTRACTObjectiveIncreases in the number of CD34+ stem cells and progenitor cells in blood and infarctedareas after acute myocardial infarction (AMI) are a documented phenomenon. However,no study has yet reported on the dynamic changes in specific populations of adult stemcells, such as c-kit +Lin- cells or ckit + Lin - Sca1 + (KLS cells), following AMI. This studyinvestigated the dynamic changes in these cells in multiple systems/organs following MIin mice.MethodsThe C57BU6J mice received either no surgery (normal control, n=6) or surgical ligationof the left anterior descending coronary artery to create AMI (n=24). On day-1 (n=7), -3(n=5), -6 (n=6), and -12 (n=6) after AMI, mononuclear cells were isolated from theblood, spleen, and bone marrow, and stained with Lineage-PEcy7, c-kit-PE, and Sca1-APC antibodies. The c-kit +Lin - cell and KLS cell populations in the mononuclear cellswere analyzed by FACS flowcytometry.ResultsThe pattern of changes in the c-kit + Lin - cells was very similar to that in the KLS cellsin the bone marrow, circulating blood, and spleen following AMI. There was a significantincrease in these cells on day-3 in the bone marrow (c-kit +Lin- cells: 1.470 ± 0.094% vscontrol 1.127 ± 0.019 `)/0, and KLS cells: 0.365 ± 0.012 % vs control 0.1848 ± 0.019 `)/0,p<0.05), which then slowly declined from day-6 to -12. In the blood, these cells,particularly the KLS cells, decreased slightly from day-1 to -12. On day-3, -6, and -12the cells increased continuously and significantly in the spleen, (on day 3, c-kit +Lin-iicells: 0.253 ± 0.0107 % vs control 0.1305 ± 0.014 %; it was 0.3212 ± 0.028 % on day-6). (on day-6 KLS cells: 0.1078 ± 0.076 % vs control 0.0425 ± 0.0064 % while on day12 it was 0.1174 ± 0.035 % p<0.05).ConclusionThis study provides for the first time the longest observation of the dynamic changes ofspecific sub-groups of adult stem cells (c-kit +Lin- cells and KLS cells) in multiplesystems following AMI. The study demonstrates that AMI results in significant changes,or mobilization, of these cells in the bone marrow, spleen, and blood. Significant andcontinuous accumulation of the cells in the spleen occurs following AMI, despite thedecreased level of the cells in the blood. The role of the spleen in stem cell mobilizationafter AMI is unclear and requires further investigation.iiiTABLE OF CONTENTSABSTRACT ^ iiTABLE OF CONTENTS ^ ivLIST OF FIGURES viiABBREVIATIONS ^ ixACKNOWLEDGEMENTS ^ xiDEDICATION ^ xiii1 INTRODUCTION ^ 12 KNOWLEDGE TO DATE ^ 3^2.1^Myocardial infarction 32.1.1^Definition ^  32.1.2^Epidemiology and prevalence ^ 32.1.3^Sequence of changes after myocardial infarction ^ 32.1 .4^Therapeutic strategies ^ 42.1.5^Rational of developing stem cell therapy^ 52.2^Stem Cells ^ 52.2.1^Definition and types of stem cells ^  52.2.2^Adult stem cells^ 62.2.3^Advantages and disadvantages of using adult stem cells ^ 72.2.4^Haematopoietic stem cells ^ 72.2.5^Roles of HSCs in repairing heart and blood vessels ^ 132.3^Dynamic changes in haematopoietic stem cells ^  132.3.1^Dynamic changes of HSCs under physiological conditions ^ 13iv^2.3.2^Dynamic changes of HSCs under pathophysiological conditions ^ 15^2.3.3^Clinical importance of stem cell mobilization ^  193 RATIONALE AND HYPOTHESIS ^ 20^3.1^Hypothesis ^ 20^3.2^Significance of the study ^ 204 MATERIALS & METHODS 22^4.1^Animals ^ 224.2 Animal modal and surgical procedures ^ 22^4.2.1^Animal model ^ 22^4.2.2^Anesthesia and mechanical ventilation ^ 22^4.2.3^Surgical Procedure ^ 23^4.2.4^Postoperative care 24^4.3^Animal euthanasia and sample collection ^ 24^4.4^Experimental groups ^ 24^4.5^Sample preparation for flowcytometry analysis ^ 25^4.5.1^Blood sample ^ 25^4.5.2^Spleen 25^4.5.3^Bone marrow ^ 25^4.5.4^Lysis of red blood cells (RBCs) ^ 26^4.5.5^Immunofluorescence staining 26^4.6^Quantification of hematopoietic stem cells by flowcytometry ^ 27^4.7^Statistical analyses^ 285 RESULTS ^ 39v^5.1^Effects of myocardial infarction on c-kit+/Lin- cells ^  39^5.1.1^Bone marrow c-kit+/Lin- cells ^ 39^5.1.2^Splenic c-kit+/Lin- cells 40^5.1.3^Blood c-kit+/Lin- cells^ 40^5.2^Effects of myocardial infarction on KLS cells ^ 415.2.1^Bone marrow KLS cells ^ 415.2.2^Splenic KLS cells 425.2.3^Blood KLS cells^ 436 DISCUSSION AND CONCLUSIONS ^ 45REFERENCES ^ 52APPENDIX 59viLIST OF FIGURESFigure 1. Anesthesia system and induction chamber ^ 29Figure 2. Induction of anesthesia in the induction chamber prior to intubation of themouse ^ 30Figure 3. Setting up for intubation. ^  31Figure 4. Harvard ventilator for mice 32Figure 5. Animal preparation for surgery after intubation. ^ 33Figure 6. Exposure of the heart and identification of the LAD anatomy^ 34Figure 7. Closure of the thoracotomy after ligation of the LAD ^ 34Figure 8. Recovered mouse placed in a new cage. ^ 35Figure 9. Sections of the heart with myocardial infarction showing pale color ^ 36Figure 10. Mouse heart showing the infarcted area (pale color) in the left ventricular ^ 37Figure 11. Quantitative evaluation of c-kit+ Lineage-/Sca-1+ (KLS) mononuclear cells byFACS analysis, and representative FACS data obtained from bone marrow,spleen, and blood. Middle penal represents Lineage negative mononuclearcells selected as the lower 5% Lineage staining gated from mononuclear cellregion (upper penal). Lower penal represents Lineage negative mononuclearcells with both c-Kit and Sca-1 positive staining that were judged as KLS cells.38Figure 12. Changes in percentage of c-kit+/Lin- cells in bone marrow at different timepoints following AMI. * p < 0.5 vs control (Ctrl) ^ 39Figure 13. Changes in percentage of c-kit+/Lin- cells in spleen at different time pointsfollowing MI. * p < 0.5 vs control (Ctrl). ^ 40viiFigure 14. Change in percentage of c-kit+lin- cells in blood at different time pointsfollowing AMI. ^ 41Figure 15. Changes in percentage of KLS cells in BM at different time points followingAMI. * p < 0.5 vs control (Ctrl). ^  42Figure 16. Changes in percentage of KLS cells in spleen at different time pointsfollowing AMI. * p < 0.5 vs control (Ctrl). ^ 43Figure 17. Changes in percentage of KLS cells in blood at different time points followingAMI. ^ 44Supplementary Figure 1 KLS cells in three systems ^ 59Supplementary Figure 2 C-kit+ Lin- cells in three systems 59viiiABBREVIATIONSAMI^ACUTE MYOCARDIAL INFARCTIONANOVA^ANALYSIS OF VARIANCEARF ACUTE RENAL FAILUREBM^BONE MARROWBMSC BONE MARROW STEM CELLSBrUdr^THYMIDINE ANALOGUE 5-BROMO-2'-DEOXYURIDINECCAC CANADIAN COUNCIL ON ANIMAL CARECHF^CONGESTIVE HEART FALIURECXCR4^G-PROTEIN-LINKED CHEMOKINE RECEPTOReC-SOD^EXTRACELLULAR SUPEROXIDE DISMUTASEEDTA ETHYLENEDIAMINETETRAACETIC ACIDESC^EMBRYONIC STEM CELLFACS FLUORESCENCE-ACTIVATED CELL SORTINGG-CSF^GRANULOCYTE-COLONY STIMULATING FACTORG-F GROWTH FACTORGM-CSF^GRANULOCYTE/MACROPHAGE COLONY STIMULATINGHGF HEPATOCYTE GROWTH FACTORHIF-1^HYPDXIA-INDUCIBLE FACTOR 1HSCs HEMATOPOIETIC STEM CELLSI.P^INTRAPERITONEALLYI.V INTRA-VENOUSLYIL -8^INTERLEUKIN- 8IL-1 INTERLEUKIN- 1IL-11^INTERLEUKIN -11IL-12 INTERLEUKIN -12IL-3^INTERLEUKIN -3IL-7 INTERLEUKIN-7KLS^C-KIT +,LIN -,SCA-1+LAD LEFT ANTERIOR DESCENDING CORONARY ARTERYLIN -^LINEAGE NEGATIVEMI MYOCARDIAL INFARCTIONMIP-1A^MACROPHAGE INFLAMMATORY PROTEIN-1 AMMPS MATRIX METALLOPROTEINASESixMNC^MONONUCLEAR CELLPB PERIPHERAL BLOODPBS^PHOSPHATE-BUFFERED SALINERBC RED BLOOD CELL.Sca-1^STEM CELL ANTIGEN -1SCF STEM CELL FACTORSDF-1 a^STROMAL CELL-DERIVED FACTOR-1ALPHASEM STANDARD ERROR OF THE MEANSIF^STRESS INTENSITY FACTORSM STAINING MEDIUMTTC^2,3,5 TRIPHENYLTETRAZOLIUM CHLORIDEVEG-F VASCULAR ENDOTHELIAL GROWTH FACTORxACKNOWLEDGEMENTSIn the name of Allah, the Most Gracious, the Most Merciful. All praises and thanks are toAllah who gave me the strength, patience, and ability to complete this work. All prayersand peace be upon Mohammed, the Prophet and Messenger of Allah.I acknowledge the tremendous influence of my parents, who tirelessly providedsupport, encouragement, prayers and blessings until my work was completed. I will beindebted to them forever for the many sacrifices they made for me.I also wish to express my gratitude to my supervisor Dr. Jian Ye, for guiding methrough this research and for being a tireless mentor. He was always available,supportive, and helpful. Additionally, I would like to express appreciation to my co-supervisor, Dr. Bruce McManus, for his intelligence, insight and generosity throughoutthe preparation of this research. I would also thank Dr. Sam Lichtenstein, Dr. IsmailLaher, and Dr. John Luo, all of who generously gave of their valuable time andcontributed many helpful suggestions.I greatly acknowledge the support of my family here in Vancouver: my wife for herunderstanding, patience and encouragement, and my sons, Mohamed and Mohanned,all of whom gave me the strength to finish this research; and Monem, my brother, who Iwish to thank for his generosity and continuous support; and also my brothers andsisters at home for their endless efforts on my behalf.I would like to thank the staff of the James Hogg I CAPTURE Centre forCardiovascular and Pulmonary Research, Vancouver, especially Dr. Luogia Yang andDr. Sall, the staff at the Bio Medical Research Center UBC, the Brain Research CenterUBC, the Department of Cardiac Surgery at St Paul's Hospital especially Dr. Huq andthe Center of Surgical Research in the Department of Surgery especially Dr. Alice Mui,Ms. Joanne Clifton, and Ms. Rachel Cadelifia. As well as gaining valuable experienceworking with colleagues at these centers, the time I spent among them was enjoyableand valuable in many ways.xiI would like to thank all my friends and colleagues at MSA-UBC, especially Dr.Hesham Soliman, Dr. Mohamed Rehan, Dr. Hafed Bascal, and Mr. Sabri Frage for theirhelp in completing this work.Finally, I would like to extend my thanks to the Ministry of Education in Libya and theLibyan Cultural Section in Canada, especially Mr. Abdenor H. Timesguida, ProgramDirector, and Mrs. Azza El Barery, for their great support throughout my program ofstudy.xiiDEDICATIONTo you, my younger brother, Dr. Al Sadek Elmestiri, who died a year ago, Idedicate this modest effort.To you, who waited eagerly to congratulate me on my degree.To you, destined to die from a heart attack as you walked out of your cliniclate at night, caring for others as usual, and inattentive to yourself.To you, who loved me more than yourself, I confess that I love you morethan my self and every other being.To you, who spared no money or effort to support me since I left home.I swear by God that the tears have not left my eyes as I write these wordsin your memory.1 INTRODUCTIONCardiovascular diseases are the leading cause of morbidity and mortality in the Westernworld. Myocardial infarction (MI) leads to extensive damage to myocytes and toremodeling of the extra cellular matrix, which, in turn, leads to ventricular dysfunction.Myocardial injury is associated with an intense inflammatory response and healingprocess, characterized by the release of many growth factors (GF), cytokinase, andchemokinase from damaged tissue, which activate the remodeling process in the heart(Nian, Lee et al. 2004). These growth factors and inflammatory agents also circulate tothe bone marrow (BM) to induce dynamic changes in bone marrow stem cells (BMSC)(Kaplan, Psaila et al. 2007).Recently, a number of studies have addressed the role of adult stem cells,especially hematopoietic stem cells (HSCs), in cardiac tissue repair after acutemyocardial infarction (AMI) or other heart diseases. It has been reported that anyinflammation, trauma or surgical condition applied to mice, might result in increasedlevels of stem cells (Fu and Liesveld 2000). Orlic and Kajstura found that HSCs frombone marrow could repair damaged myocytes after AMI when they were transplanteddirectly into the injured myocardium (Orlic, Kajstura et al. 2001) or mobilized from bonemarrow to the heart (Orlic, Kajstura et al. 2001). Adult stem cell mobilization andinvolvement in microvascular repair has been further demonstrated in a number ofexperimental and clinical studies (Rajantie, Ilmonen et al. 2004).For improving myocardial repair and function, adult stem cells from bone marrowcan either be directly transplanted into, or mobilized to, the damaged myocardium.However, the major disadvantage of direct transplantation is that the majority of theinjected BM stem cells die quickly after local transplantation, although recent studies1have shown that after local injection, preconditioned BMSCs have a better survival ratethan non-preconditioned BMSCs. It has been shown recently that mobilization of stemcells from bone marrow to the damaged myocardium can enhance tissue repairfollowing a heart attack. (Tomoda and Aoki 2003). Adult stem cells may improve cardiacfunction via revascularization, repair of damaged heart cells, or regeneration ofmyocytes (Canepa, Coviello et al. 2006; Velazquez 2007).Although there are few studies reporting the changes in stem cells/progenitor cellsin a single system following tissue ischemia (Patschan, Krupincza et al. 2006) (Ii,Nishimura et al. 2005), there are no reports on simultaneous dynamic changes inspecific sub-groups of adult stem cells in different systems or organs in the sameanimals following AMI.The aim of the study was to investigate simultaneous dynamic changes in HSCs inmultiple systems/organs in the same animal affected by AMI. The results will lead to abetter understanding of stem cell mobilization or activation following AMI and thedevelopment of better strategies for cell therapy for myocardial repair following AMI.22 KNOWLEDGE TO DATE2.1 Myocardial infarction2.1.1 DefinitionMyocardial infarction is a condition characterized by irreversible necrosis ofcardiomyocytes secondary to prolonged ischemia. This usually results from animbalance of oxygen supply and demand (Gowda, Khan et al. 2003). A prolongedperiod of myocardial ischemia leads to extensive injury to myocytes and remodeling ofthe extra-cellular matrix resulting in ventricular dysfunction and heart failure (Sutton andSharpe 2000).2.1.2 Epidemiology and prevalenceAcute myocardial infarction and heart failure remain the leading cause of morbidity andmortality worldwide (Thom, Haase et al. 2006). In Canada, for example, congestiveheart failure (CHF) affects 400,000 annually resulting in up to 1.38 million hospital daysa year. 25 - 40% of these patients will die within one year of diagnosis . In the USA, upto 10 million people have a history of myocardial infarction, angina pectoris or both.2.1.3 Sequence of changes after myocardial infarctionFollowing acute myocardial infarction both the infarcted and unaffected myocardialregions undergo progressive changes over the hours, days and weeks followinginitiation of myocardial Infarction (Rodriguez-Calvo, Tourret et al. 2001). During the firsthour heart contractility ceases, followed by the cessation of blood supply to the coronaryarteries, subsequently causing necrosis of the sub-endocardium.In the next 3-6 hours the necrotic region grows outward towards the epicardium,followed by necrosis across the entire ventricular wall (Qin, Liang et al. 2005).3Alterations in the appearance of the infarcted tissue begins about six hours afterthe onset of cell death. Many abnormalities in cell biochemistry and underlyingstructures begin occurring within 20 minutes following AMI (Javadov and Karmazyn2007).The coagulation necrotic process starts 4-12 hours after the infarction. This ischaracterized by cell swelling, organelle breakdown and protein de-naturation (Ciulla,Paliotti et al. 2004). Inflammatory cells, including neutrophils and lymphocytes, will enterthe infarcted area after 18 hours.Myocardial infarction triggers all the characteristics of an inflammatory responseto initiate the healing process (Vermeiren, Claeys et al. 2000). After 3-4 days,granulation tissue appears at the edges of the infarcted zone; this tissue is responsiblefor the formation of scar tissue and new capillaries . After 2-3 months, the infarction' willhave healed, leaving a thin, firm, non-contracting region of the ventricular wall (Burkeand Virmani 2007).2.1.4 Therapeutic strategiesThe optimal medical treatments for heart disease, including angiotensin convertingenzyme inhibitors, beta-blockers, and aldactone, have shown to decrease symptomsand improve outcomes (Koelling and Eagle 2008). However, clinical trials from 1992 to2000 have shown that there was no significant decrease in mortality in patients withCHF following these treatments (Lee, Mamdani et al. 2004). Heart transplantation is aneffective therapy, but due to the fixed number of donors it is not available to all patientsin need (Steinman, Becker et al. 2001). In order to address the increasing incidence ofheart failure, stem cell therapy has been proposed as an alternative treatment for heartdisease and is currently under extensive investigation (Condorelli and Catalucci 2007).42.1.5 Rational of developing stem cell therapyThe idea of using stem cell therapy to treat heart disease results from the observationthat myocardial infarction leads to structural changes which depress cardiac function(Cleutjens and Creemers 2002). The cardiomyocytes that survive myocardial ischemiaprimarily respond to cellular hypertrophy rather than proliferation due to the limitedmitotic capacity of adult cardiomyocytes (Vandervelde, van Luyn et al. 2005).Under physiological circumstances this limited mitotic capacity restricts the repairof the ischemic myocardium; ultimately the infarcted tissue will be replaced by fibrotictissue (Bian, Popovic et al. 2007). The fibrotic tissue can destroy normal contractilefunction and result in decreased cardiac performance and heart failure. One wayresearchers are thinking about how to repair the myocardium is to replace lost cardiactissue with healthy myocardial tissue. A potential source for regeneration of myocardialcells are stem cells.A therapeutic approach aimed at promoting blood vessel formation (angiogenesis)and the formation of new heart muscle fibers (myocardial regeneration) (Orlic, Kajsturaet al. 2001) is a potentially attractive alternative to heart transplantation.2.2 Stem Cells2.2.1 Definition and types of stem cellsStem cells are non-differentiated, immature cells and are also referred to asunspecialized cells. They have the unique ability to divide throughout their lifetime andcan evolve into cells with a highly specialized function and take the place of dead or lostcells. This description of stem cells refers to their contribution to the renewal and repairof body tissue (Tuch 2006).5There are two broad categories of stem cell: embryonic stem cells (ESC), whichderive from blastocysts, and adult stem cells, which are found in adult tissue. In adeveloping embryo, stem cells can differentiate into all of the specialized embryonictissues and in adult organisms they act as a repair system for the body by replenishingspecialized cells, but can also maintain the normal turnover of regenerative systems,such as blood and skin (Mimeault, Hauke et al. 2007).Progenitor stem cells, which are a group of stem cells deriving from primary(immature) stem cells, differ from primary stem cells, which have a reduceddifferentiation capacity. More importantly, progenitor cells lack the ability for self-renewal, one of the main characteristics of adult stem cells (Morrison, Uchida et al.1995). Due to ethical issues surrounding the use of embryonic stem cells, the studyfocuses on adult rather than embryonic stem cells.2.2.2 Adult stem cellsThere are two main groups of adult stem cells: haematopoietic stem cells (HSCs) andmesenchymal stem cells (MSCs).Haematopoietic stem cells have the ability to give rise to all blood cells and arecapable of self-renewal (Guo, Lubbert et al. 2003). HSCs may also differentiate to avariety of specialized cells under special conditions by the process oftransdifferentiation. (Uher and Vas 2002). Additionally, HSCs are able to mobilize out ofbone marrow into circulating blood (Nakano 2003).Mesenchymal stem cells are multipotent stem cells which can differentiate into avariety of cell types in in-vitro or in-vivo conditions. These cell types include osteoblasts,chondrocytes, myocytes, adipocytes, neuron cells, and pancreatic beta cells (Jiang,Jahagirdar et al. 2002).62.2.3 Advantages and disadvantages of using adult stem cellsGenerally speaking, adult stem cells are limited to differentiating into different cell typesof their tissue of origin. However, some evidence suggests that adult stem cell plasticitymay still exist (Uher and Vas 2002).Compared to adult stem cells, large numbers of embryonic stem cells can berelatively easily grown in culture (Wiles and Keller 1991), whereas adult stem cells areusually rare in mature tissues and methods for expanding their numbers in cell culturehave not yet been well worked out; this is an important distinction between adult andembryonic stem cells. The difficulty of growing adult stem cells is a disadvantage aslarge numbers of them are needed for stem cell replacement therapies (Ulloa-Montoya,Verfaillie et al. 2005).The use of adult stem cells instead of embryonic stem cells avoids some ethicalissues. A further advantage in the use of adult stem cells especially in the clinical field isthat use of the patient's own adult stem cells ensures that the cells would not berejected by the immune system. This represents a significant advantage as immunerejection is a difficult problem that can only be resolved with immunosuppressive drugs(Li and Xie 2005).2.2.4 Haematopoietic stem cellsHaematopoietic stem cells are pluripotent cells that typically reside in the bone marrowand have the capability to differentiate and develop into various blood lineages (Balint,Malesevic et al. 1998). A small population of HSCs remains undifferentiated and self-renews to serve as the source for future blood cell development, as well as being ableto transdifferentiate into various tissues. It is estimated that about 1 in 100,000 of wholebone marrow cells are HSCs (Yoshimoto, Chang et al. 2005). Sources of HSCsA. Bone marrow and peripheral bloodIn adults, under steady state conditions, the majority of HSCs reside in the bonemarrow. Additionally, HSCs receive their regulatory messages from themicroenvironment in the bone marrow. For this reason, the localization ofhaematopiosis is usually restricted to bone marrow (Heissig, Hattori et al. 2002).However, cytokine and other growth factors (G-F) agents can result in the release oflarge numbers of HSCs into the blood. The mobilized HSCs in peripheral blood currentlyreplace bone marrow HSCs sourced under clinical conditions because it is easier toharvest peripheral blood (PB) than to harvest bone marrow (Childs, Chernoff et al.2000).B. Umbilical cord bloodSince the late 1980s, umbilical cord blood has been recognized as an important clinicalsource of HSCs. Blood from the placenta and umbilical cord is a rich source ofhaematopoietic stem cells, cells which are typically discarded after birth (Ebrahim2002). Identification of HSCsA. HSC markersStem cell markers are given their names based on the molecules that bind to thesurface receptors (Civin, Strauss et al. 1984). Previous researchers identified stem cellbased on cell size and density (Sutherland, Eaves et al. 1989). However, more recentefforts focus on the absences of cell surface protein markers as defined by monoclonalantibodies ( Lineages negative) and presence of other cell surface antigens.8For HSCs in mice, these protein markers include panels of 8 to 14 differentmonoclonal antibodies that recognize cell surface proteins present on differentiatedhematopoietic lineages. Where as the antigen markers are Sca-1 (van de Rijn, Heimfeldet al. 1989), CD27, CD34, CD38, CD43, CD90.1 (Thy-1.1), CD117(c-Kit) (D'Arena,Musto et al. 1998). For example, the cell that has the receptor stem cell antigen -1 on itssurface is identified as Sca-1 (van de Rijn, Heimfeld et al. 1989).Most of the stem cells are usually identified by shorthand, with a combination ofmarker names reflecting the presence (+) or absence (-) of those markers. None ofthese markers recognize functional stem cell activity. However, combinations (typicallywith 3 to 5 different markers) allow for the purification of near-homogenous populationsof HSCs. C-kit +, Sca-1+ and lineage negative property, for example, have beenidentified as the most primitive HSCs in bone marrow ((Chen, Li et al. 2002).B. C-kit+ and KLS sub-groupsSca-1 (stem cell antigen-1) is a member of the Ly-6 antigen family expressed on the cellsurface of multipotent HSCs. Sca-1 is the most recognized HSC marker in mice with Ly-6 haplotypes (van de Rijn, Heimfeld et al. 1989), whereas c-kit is a membrane-boundtyrosine kinase present on the cell surface of HSCs. The over expression of c-kit hasbeen observed in several stem cells, especially bone marrow HSCs (Dagher, Hiatt et al.1998). The KLS are those cells that are c-kit positive, Sca-I positive, and absolutelynegative for lineage expression . KLS cells are a population of cells identified as givingrise to life-long myeloid and lymphoid mature cells in mice (Brummendorf, Orlic et al.2001).Researchers have found that there are several indications that KLS cellsrepresent a pure population of HSCs. Firstly, over 90% of KLS cells from individual9blast-cell colonies in methyl-cellulose culture were found correlating with very primitivebone marrow activity (Tadokoro, Ema et al. 2007). Secondly, the frequency of KLS cellsin the bone marrow (0.01%) is the same as estimates of HSC frequency. Finally, HSCactivity has not been found in any population of cells outside the KLS gates (Surdez,Kunz et al. 2005).For these reasons, KLS cells are considered to be the most common activepopulation of bone marrow HSCs and are invariably mentioned alongside HSCs in thisproject. Characteristic of HSCsA. Self-renewalOne essential feature of HSCs is their ability to self-renew, i.e. to make copies with thesame or very similar potential (Eaves, Miller et al. 1999). This is an essential property ofmost stem cells as their presence is essential to the production of the many matureblood cells. However, it is still unclear which key signals allow self-renewal. One linkthat has been noted is telomerase, the enzyme necessary for maintaining telomeres,which are the DNA regions at the end of chromosomes which protect them fromaccumulating damage from DNA replication (Stein, Zhu et al. 2004). Expression oftelomerase is associated with self-renewal activity; however, the absence of telomerasereduces the self renewing capacity of mice HSCs (Zhu, Zhang et al. 2005).B. DifferentiationHSCs can differentiate into progenitor and mature adult blood cells. This differentiation,together with the option to self-renew, defines the core function of this type of stem cell10(Spivakov and Fisher 2007). Differentiation is driven and guided by an intricate networkof growth factors and cytokines. Differentiation, rather than self-renewal, is the usualoutcome for HSCs when stimulated by many factors to which they have been shown torespond (Attema, Papathanasiou et al. 2007). It appears that once they commit todifferentiation, HSCs cannot revert to a self-renewing state. Thus, specific signals,provided by growth factors and cytokines appear to be necessary to maintain HSCsduring the organism's lifetime (Veiby, Mikhail et al. 1997).C. MigrationMigration of HSCs occurs at specific times during development (i.e. during seeding ofthe fetal liver, spleen and, eventually, bone marrow) and under certain conditions (e.g.cytokine-induced mobilization) later in life (Wright, Wagers et al. 2001). This issue willbe discussed in detail in section 2.3.D. ApoptosisApoptosis is a mechanism by which cells can actively self-destruct without causinginflammation. Apoptosis is an essential feature in all multicellular organisms and is auseful mechanism required to regulate HSC metabolism. The effect of apoptosis hasbeen demonstrated in transgenic mouse experiments in which HSC numbers doubledwhen the apoptotic threshold was increased (Domen 2001).E. PlasticityStudies of stem cell transdifferentiation have been highly controversial (Ho and Punzel2003). In adults, homeostatic cell replacement and tissue regeneration have been11considered to maintain tissue specificity, such as in those tissues and organs that retainstem cell dependence. It is understood that tissue-resident stem cells generate onlythose mature cell types corresponding to their tissue of origin and do not cross tissueboundaries to generate cell types of different lineages (Bordignon and Roncarolo 2002)but recent experiments have challenged this notion.The lineage commitment of various adult stem cell populations may, undercertain circumstances, transdifferentiate to contribute to a much wider spectrum ofdifferentiated cells than previously expected (Joshi and Enver 2002).Transdifferentiation is the conversion of a cell of one tissue lineage into a cell of anentirely different lineage, followed by a corresponding loss of tissue-specific markersand functions of the original cell type and acquisition of markers and function of thetransdifferentiated cell type (Costa and Shaw 2007).The suggestion that adult stem cells may transdifferentiate has in turn given riseto the concept of stem cell plasticity, which holds that the lineage determination of adifferentiating stem cell may not be rigidly defined but is, instead, flexible, therebyallowing these cells to respond to a variety of microenvironmental regenerative signals(Blau, Peterson et al. 1997) (Wagers and Weissman 2004). It has been claimed thathaematopiotic bone marrow cells can differentiate not only into blood cells but also intomuscle cells (both skeletal myocytes and cardiomyocytes) (Orlic, Hill et al. 2002), braincells (Bonilla, Alarcon et al. 2002), liver cells (Austin and Lagasse 2003), skin cells(Fathke, Wilson et al. 2004) lung cells (van Haaften and Thebaud 2006) (Kotton,Summer et al. 2003), kidney cells (Lin, Cordes et al. 2003), intestinal cells (Piscaglia, DiCampli et al. 2003) and pancreatic cells (Hess, Li et al. 2003); but until now themechanism of transdifferention has been unclear.122.2.5 Roles of HSCs in repairing heart and blood vesselsMany studies have shown that HSCs can contribute to myocardial repair (Weissbergand Qasim 2005), but whether HSCs repair myocardium through trans-differentiation,cell fusion, paracrine effect, or other mechanisms is still under question.Cardiac stem cells, human embryonic stem cells and adult bone marrow stemcells (BMSCs) have been shown to participate in myocardial repair processes and torepopulate the infarcted myocardium (Orlic 2003). Under normal conditions, BMSCs arerarely seen in tissue and organs. However, acute myocardial infarction (AMI) enhancestheir mobilization into blood circulation and localization in the damaged tissue to repairischemic myocardium (Wojakowski and Tendera 2005). Moreover, BMSCs can bedirectly transplanted into the infarcted area by intra-coronary infusion, catheter basedintra-myocardial injection, or direct intra-myocardial injection (Leone and Crea 2006).The mobilized or transplanted BMSCs significantly improve left ventricular function,probably due to better regional perfusion and transdifferentiation of stem cells intocardiomyocytes (Rosenstrauch, Poglajen et al. 2005).2.3 Dynamic changes in haematopoietic stem cells2.3.1 Dynamic changes of HSCs under physiological conditions2.3.1.1 Migration of HSCs during early lifeIt has been confirmed that HSC migration appears to occur at least twice duringprenatal development, with both migrations likely to be associated with haematopoieticstem cell expansion (Christensen, Wright et al. 2004).The first migration involves the colonization of the fetal liver haematopoietic cells(Bonifer, Faust et al. 1998). Prior to their localization in the fetal liver, haematopoietic13stem cells can be isolated from the yolk sac even before completion of the circulatorysystem (Samokhvalov, Samokhvalova et al. 2007).The second presumed migration is from the fetal liver to the spleen and bonemarrow during embryonic development (Dzierzak, Medvinsky et al. 1998).Haematopoietic stem cells migrate from the fetal liver via blood circulation, enter thespleen and bone marrow (BM), and repopulate this tissue with high levels of immatureand maturing cells of all lineages. The mature cells are released into the circulationwhen needed and at the same time maintain a small pool of undifferentiated (immature)stem cells within the bone marrow during embryonic development. Reportedly, fetal liverHSCs are the precursors of HSCs in adult bone marrow (Kim, He et al. 2006).Studies have demonstrated that umbilical cord blood cells contain relatively highlevels of immature HSC progenitor cells (0.5%), including a minority of more primitive,undifferentiated cells (Lewis 2002). This suggests that high levels of haematopoieticstem cells migrate via blood circulation during the late-stage of embryonic development. Migration of HSCs during adult lifeIt has been noted that bone marrow serves as the microenvironment that supports adultHSC proliferation and migration. Adult HSCs can migrate spontaneously under specialconditions, as in the case of malignant disease, to initiate extramedullary hematopoiesis(Dingli, Mesa et al. 2004). Adult HSCs have also been shown to migrate spontaneouslyin some genetically altered animals (Wright, Wagers et al. 2001).Mobilization is a kinetic process by which adult HSCs are made to migrate fromthe bone marrow into the bloodstream. This process mimics the enhancement of thephysiological release of stem cells and progenitors from the bone marrow reservoir in14response to stress signals (Heissig, Hattori et al. 2002). HSCs are probably onlyoccasionally migratory under certain physiological conditions, such as stress and heavyexercise. In adult mammals it is believed that low frequencies of HSCs can be found inthe blood due to spontaneous, slow migration of small numbers of HSCs from the bonemarrow (Hirayama, Yamaguchi et al. 2003).As HSCs develop and mature within the bone marrow, blood cells leave the BMmicroenvironment and egress into the peripheral blood. The majority of undifferentiatedHSCs remain in the BM and a small population of the undifferentiated HSCs leave theBM and circulate into the peripheral blood (Pegg 1976); the reason for the release ofthese HSCs remains unknown. It is possible that the physiological role of thesecirculating HSCs may be to seed other organs.2.3.2 Dynamic changes of HSCs under pathophysiological conditions2.3.2.1 Migration secondary to intrinsic factor " ischemic condition"Several intrinsic factors play important roles in HSC mobilization from bone marrowniches. Among the important intrinsic factors is the condition of ischemia. It has beenreported that after any tissue ischemia there is a release of inflammatory cytokines,growth factors, surface receptors, proteolytic enzymes, and nuclear proteins (Frijns andKappelle 2002). Different organs such as the heart, brain, kidney, liver and limbs havebeen shown to recruit stem cells during ischemic conditions.HSCs have been shown to migrate to areas of injury and subsequentlyparticipate in the establishment of the repair process. Physiological triggers alone areoften insufficient to mobilize HSCs to induce complete healing in the presence of severeischemia (Goldstein, Gallagher et al. 2006). During various pathological conditions,particularly ischemia, the trafficking of the HSCs is directed by hypoxic tissue via15hypoxic inducible factor 1-a. (Ramirez-Bergeron and Simon 2001). Ischemia, hypoxiaand some cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF), were known stimuli in the mobilization of HSCs. Multiple mechanisms areinvolved in the ischemia-induced liberation of HSCs from the bone-marrow pool.A. Heart ischemiaAfter a myocardial infarction, the inflammatory response is very active. The degree ofintensity of the inflammatory process is important in the healing process (Fuster,Badimon et al. 1992). Usually the infiltrated inflammatory cells secondary to MI are richsources of cytokines and growth factors that play important roles in the cardiacremodeling process and mobilization of extra stem cells from bone marrow to the site ofinjury as part of the intrinsic mechanism (de Muinck and Simons 2005). The myocardialgrowth factors stromal cell-derived factor-1alpha "SDF-1 a" expression was seen tosignificantly increase in the early phase after MI (Wang, Haider et al. 2006).Granulocyte colony stimulating factor (G-CSF) increased by more than 50 times after MIin injured tissues. This factor is known to help mobilize HSCs from bone marrow(Kocher, Schuster et al. 2001). Ischemic cardiac tissue can also secrete vascularendothelial growth factor ( VEGF ), which also functions as a mobilizer for stem cellsfrom bone marrow (Kalka, Tehrani et al. 2000). These three inflammatory cytokines arenot only associated with the regulation of the inflammatory response after myocardialischemia, but also involved in the induction of mobilized stem cells toward the injuredmyocardium.B. Ischemia in other organsStudies have shown that expression of chemokinase (stroma derived —factors) SDF-laincreases following focal cerebral ischemia. SDF-1 a can increase mobilization of BM-16derived cells to damaged areas of the brain, resulting in stimulation of cell repair in thepenumbra of the ischemic brain (Shyu, Lin et al. 2007). It is known that endogenousextracellular superoxide dismutase (ecSOD) plays an essential role for post ischemicneovascularization after limb ischemia. In recent studies it has been found thatrecruitment of inflammatory cells into ischemic tissues and the number of stem cells,such as c-kit+ and CD31 4- cells, in both peripheral blood and bone marrow significantlydecreased in mice lacking ecSOD-/- after hind limb ischemia (Kim, Lin et al. 2007). Incontrast, hind limb ischemia of normal mice stimulates a significant increase in ecSODactivity in ischemic tissues where ecSOD protein is highly expressed at arterioles and ininflammatory cells. This indicates that ecSOD is involved in stem cell mobilization.Post-ischemic stem cell mobilization was also demonstrated following acute renalischemia in an animal model. A recent study from Patschan's group (Patschan,Krupincza et al. 2006) showed that a unilateral renal ischemia in mice for a period of 25minutes results in transient accumulation of stem cells in the spleen starting within 3-6hours, but not in peripheral blood during the 7-day follow-up after renal ischemia. Thisstudy presents for the first time a chronological analysis of the dynamics of stem cellmobilization in the case of acute renal ischemia.Other studies have also demonstrated that peripheral blood HSC levels wereelevated after extensive liver resection (Dalakas, Newsome et al. 2005) and in patientswith alcoholic hepatitis. There is now increasing evidence to suggest that liver injuryinduces the expression and secretion of signaling mediators, such as SDF-1, IL-8,MMPs, HGF, and SCF, which facilitate the homing and engraftment of HSCs to the liver(Dalakas, Newsome et al. 2005). Migration secondary to extrinsic factorsHSCs or haematopoietic progenitor cells can be mobilized from bone marrow toperipheral blood and other organs in large numbers in response to external stimuli suchas the administration of a variety of drugs and cytokines (Velders and Fibbe 2005).There are extensive lists of mobilizing agents that can induce stem cells to migrate frombone marrow to peripheral blood, such as G-CSF (Molineux, Pojda et al. 1990), GM-CSF (Socinski, Cannistra et al. 1988), SIF (Gianni, Siena et al. 1989), IL-I (102) (Fibbe,Hamilton et al. 1992), IL-3 (Brugger, Bross et al. 1992), IL-7 (Grzegorzewski,Komschlies et al. 1995), IL-II (Mauch, Lamont et al. 1995), IL-12 (Jackson, Yan et al.1995), IL-8 (Laterveer, Lindley et al. 1995) and MIP-la (Lord 1995).The kinetics of mobilization by these diverse agents varies widely. For example,administration of some agents, such as IL-8 or SIF + G-CSF (Bodine, Seidel et al.1996), results in rapid (within 15-30 minutes) release of some HSCs into the periphery,whereas for most agents the response takes several days (Morrison, Wright et al.1997). When the most commonly used mobilizing agent, G-CSF, is given to mice, HSCsexpand greatly in the bone marrow prior to mobilization. After the initial expansionphase, HSCs are released abruptly into the blood. Substantially higher numbers ofHSCs are released following G-CSF treatment compared to IL-8 treatment (Morrison,Wright et al. 1997).Chemokines are also important in HSC mobilization (Premack and Schall 1996).For instance, the chemokine stromal cell-derived factor (SDF-I) plays an important rolein the mobilization process. Mobilized human CD34 progenitors express reduced levelsof the SDF-1 receptor CXCR4, which correlates with improved mobilization, suggestingthe involvement of SDF-1/CXCR4 interactions in the mobilization process. Over-18expression of SDF-1 in murine circulation leads to stem cell mobilization (Dar, Kollet etal. 2006).2.3.3 Clinical importance of stem cell mobilizationMobilization is used as a clinical strategy to increase the source of haematopoietic stemcells for cell transplantation. For example, we can mobilize HSCs from the bone marrowfor subsequent collection and transplantation (To, Haylock et al. 1997), as well as fordirect seeding of stem cells to injured tissue for repair. Mobilized circulated stem cellsare the preferable source of stem and progenitor cells harvested for transplantations toaccelerate haematopoietic recovery in patients with acute leukemia because of thehigher yield of these mobilized cells, leading to faster engraftment and decreasedprocedural risks compared with harvested BM cells (Henon, Liang et al. 1992).193 RATIONALE AND HYPOTHESIS3.1 HypothesisHaematopoietic stem cells either derived from the bone marrow or from other nichesparticipate in tissue repair under certain physiological and pathological conditions(Wagers, Christensen et al. 2002).It has been suggested that following tissue injury there is a mobilization of stemcells into injured tissue from other parts of the body, such as the bone marrow, andwhich appears to be triggered by tissue injury. A recent study has shown dynamicchanges in stem cell concentration in spleen and peripheral blood circulation followingacute renal ischemia, most likely secondary to the mobilization of stem cells from thebone marrow (Patschan, Krupincza et al. 2006). To the best of our knowledge, therehave been no publications that report dynamic changes in specific sub-groups of Adultstem cells in bone marrow and peripheral organs following acute myocardial infarction.We hypothesized that acute myocardial infarction results in significant dynamicchanges in some specific sub-groups of Adult stem cells in the bone marrow and otherorgans. The objective was to investigate the dynamic changes in c-Kit+/Lin- cells andKLS cells in the bone marrow, spleen, and circulating blood following AMI, as well asthe relationship of the changes in HSCs between these systems.3.2 Significance of the studyCongestive heart failure secondary to myocardial ischemia shows an increasing numberof cases with no satisfactory treatment. Potential alternative treatments for CHF, suchas adult stem cells for cardiac repair and gene therapy, are promising. Adult stem cells20are available in many types of tissue, especially in bone marrow (Ozturk, Guven et al.2004).Adult stem cells can be harvested from BM and then injected directly into injuredtissue, or they can be induced to enter the peripheral blood by mobilizing factors andsubsequently moved into injured tissue. Improvement in heart function has beenobserved following local injection or mobilization of adult stem cells from BM, howeverthe results are quite controversial (Caplice and Gersh 2003).The major disadvantage of local injection of BMSCs is that the majority of cellscannot survive in the new environment, and the number of surviving cells is notadequate for tissue repair (van Laake, Hassink et al. 2006).The purpose of the study was to better understand the dynamic changes in HSCsfollowing acute myocardial infarction, which would provide insight into the potentialoptimal time and sources for harvesting of mobilizing stem cells after acute myocardialinfarction.214 MATERIALS & METHODS4.1 AnimalsThirty female mice (C57BL/6J, Jackson Laboratory, Maine, USA) approximately 12weeks old were used in the experiment and were housed in the animal facility at theUniversity of British Columbia (UBC) under specific pathogen free conditions with freeaccess to foods and acidified drinking water. All animals received humane care inaccordance with the guidelines of the Canadian Council of Animal Care (CCAC). Theexperimental protocol was approved by the Animal Care and Use Committee of UBC.4.2 Animal modal and surgical procedures4.2.1 Animal modelAcute myocardial infarction in the mice was created by ligation of the left anteriordescending artery (LAD) through a left thoracotomy. The animals were allowed torecover for up to 12 days following the acute myocardial infarction. Tissue samples forstem cell measurements were collected according to the experimental protocol andgroups.4.2.2 Anesthesia and mechanical ventilationThe mouse was weighed and pre-anesthetized with intraperitoneal (IP) administration ofa mixture of xylazine /Ketamine in 0.9 % saline. (10 pg/10 mg of body weight) (Figure1). General anesthesia was induced by inhalation of 3% isoflurane with 30% 02 for 3-5minutes in an induction chamber (Figure 2). The hair on the left chest was shaved andophthalmic ointment was applied to both eyes to protect the cornea during the surgery.After general anesthesia, the mouse was placed in a supine position on acustom-made device with 45-degree slope (Figure 3). The larynx was exposed by a22curved forceps, and an external light source was placed directly in the animal's neck inorder to illuminate the glottis during intubation. A 22-gauge i.v. catheter was carefullyinserted into the trachea. After confirming the correct position of the intubation byobserving changes in breath patterns by blocking the catheter, the catheter wasconnected to the ventilator. The animal was ventilated at 120bpm with the tide volumeof 200p1. General anesthesia was maintained by inhalation of 1.5-2% isofluranethrough a small rodent ventilator (HARVARD Inspira AVS, USA)(Figure 4).4.2.3 Surgical ProcedureThe mouse was fixed in a supine position by taping its legs with the left side elevated(Figure 5). A 1-1.5 cm skin incision was made at the middle-clavicle line of the left chestand parallel to the sternum. After separating the chest muscles, the 3 rd and 4th ribs wereexposed and cut with extreme caution so as to avoid injury to the internal mammaryartery and lung and to allow access to the chest cavity.The heart was exposed using a small retractor and the pericardium was openedcarefully to expose the heart (Figure 6). The left coronary artery and its branches couldbe identified under a 10X surgical microscope. The left anterior descending artery (LAD)was carefully ligated with a stitch of an 8-0 Nylon suture. A drop of 2% xylocaine wasapplied to the heart surface to prevent cardiac arrhythmia. Complete occlusion of theLAD was confirmed by the development of an area of pale color with lessmovement/contraction. After re-expanding the lung, the chest was closed in layers using6/0 absorbable Vicryl sutures (Figure 7). After spontaneous breath recovered, themouse was extubated and kept in a recovery cage with a supply of oxygen for about 30minutes. Analgesia was achieved with subcutaneous injection of buprenorphine (0.025mg/kg) during the follow-up according to our postoperative care protocol.234.2.4 Postoperative careAfter the surgery, the mice were checked every half hour for the first four hours. Closemonitoring was carried out during the early postoperative phase and the health of theanimals was assessed daily by monitoring of the surgical scar, body weight, eating,drinking, defecating, urinating, socializing and grooming, according to the monitoringscoring sheet (Figure 8). The mortality rate following AMI in this experiment was around11% (3 out of 27 mice died or were euthanized before the end of the experiment).4.3 Animal euthanasia and sample collectionAt the scheduled time points according to the protocol and groups, the mice wereeuthanized for sample collection. Fifteen minutes before euthanization, intraperitonealheparin (250u/kg, hepalean®, Organon Canada Ltd., Toronto, ON) was given. The micewere deeply anesthetized by intraperitoneal injection of ketamine (100mg/kg) andXylocain (xylazine 10mg/kg) and inhalation of isoflurane. They were then fixed in asupine position. After opening the abdominal cavity, blood collected slowly from theinferior vena cava and the spleen was then harvested. The femora and tibiae weredissected out and collected in a 60 mm petri dish placed on ice. The heart washarvested and flushed with PBS, followed by staining with 1% 2,3,5-Triphenyltetrazolium chloride (TTC) at room temperature for 20 minutes to identify theinfarction. The infarcted area can be easily identified (Figures 9, 10)4.4 Experimental groupsThe animals underwent ligation of the LAD and were sacrificed to collect samples onday-1(n=7) ,-3(n=5), -6(n=6), -12(n=6) after creating acute myocardial infarction.24Another 6 mice that did not undergo the LAD occlusion procedure were used as normalcontrol4.5 Sample preparation for flowcytometry analysis4.5.1 Blood sampleThe heparinized blood sample was placed in a 15 ml conical sterile centrifuge tube andmixed with an equal volume of PBS at room-temperature. The Histopaque 1083(Sigma) solution was carefully placed in a layer underneath the blood/PBS mixture byplacing the tip of the pipet containing the Histopaque at the bottom of the sample tube(3 ml Histopaque per 10 ml blood/PBS mixture). It was then centrifuged for 30 minutesin a GH-3.7 rotor at 440 x g at 18.°to 20°C, without a brake. The upper layer containingthe plasma and most of the platelets, was removed. The mononuclear cell layer wastransferred to another centrifuge tube and the mononuclear cells washed with PBS (thevolume was the same as the amount of the mononuclear cell layer) and centrifuged for5 minutes at 440 x g at18°to 20°C, repeated three times to remove the platelets ascompletely as possible.4.5.2 SpleenThe spleen was placed in a 60 mm petri dish containing 3m1 of staining media (SM,PBS with 5% Fetal Bovine Serum and 1mM EDTA) and ground with a pair of glassslides. The cell suspension was filtered through a 70pm cell strainer and thesplenocytes were collected in a 15 ml centrifuge tube. Single cell suspension was thenobtained.4.5.3 Bone marrowBoth ends of each bone shaft were cut and the bone marrow was flushed out with 3m125of PBS using a 5-ml syringe with a 25-G needle. The bone marrow tissue wasdisaggregated by several passages though the same needle. The supernatant wastransferred to a new 15 ml centrifuge tube, which was centrifuged for five minutes at440 x g at 4°C. The supernatant was then discarded.4.5.4 Lysis of red blood cells (RBCs)The collected cell pellets from the blood, spleen, and bone marrow were centrifugedagain. The collected cells were then resuspended in 1m1 RBC lysing solution andincubated for 2-4 minutes at room temperature to lyse the RBCs. The collected cellpellets were washed twice by no more than 5 ml of staining media solution, andresuspended with staining media to achieve a final concentration of 1 x 107 cells/ml.4.5.5 Immunofluorescence stainingAppropriate controls were prepared to obtain correct results, such as unstained control,isotype antibody stained control, and positive control etc. Briefly, cell suspension in thestaining media at a concentration of 1x107 cells/ml was achieved after the lysis ofRBCs. 100p1 of the cell suspension was added to 96-well V-bottom plates, then mixedwith 14p1 of the pre-diluted labeled antibody and incubated at 4 °C in darkness. Lineage-PECy7 (1:400, eBioscience Inc., San Diego, USA), Ckit-PE (1:400, StemCell,Vancouver, Canada), Sca-APC (1:200, eBioscience Inc., San Diego, USA) antibodieswere used for the immunofluorescence staining. The cell plates were washed with 2m1staining media and centrifuged at 440 x g, 4°C. Finally, the cell pellets wereresuspended in 400u1 staining media at 4°C for flowcytometry analysis. Data wascollected with CELLQuest software on BD FASCalibus.264.6 Quantification of hematopoietic stem cells by flowcytometryA FACS instrument is usually used to sort out the rare stem cells from the millions ofother cells. The principle of the methodology has been described previously. Using thistechnique a suspension of mononucluer cells (MNC), for example, is sent underpressure through a nozzle so narrow that cells can only pass through it one at a time.Upon exiting the nozzle, cells then pass, one by one, through a light source, usually alaser. The system is adjusted so that there is a low probability of more than one cellbeing present in a droplet.Just before the stream breaks into droplets, the flow passes through afluorescence measuring station where the fluorescent character of interest of each cellis measured. An electrical charging ring is placed at the point where the stream breaksinto droplets. A charge is placed on the ring-based fluorescence intensity measurementand the opposite charge is trapped on the droplet as it breaks from the stream. Thecharged droplets then fall through an electrostatic deflection system that diverts thedroplets into containers based upon their charge. The fluorescent cells becomenegatively charged, while non-fluorescent cells become positively charged.In some systems the charge is applied directly to the stream and the dropletbreaking off retains the same charge as the stream. The stream is then returned toneutral after the droplet breaks off. The charge difference allows stem cells to beseparated from other cells.Cells passing through the beam will scatter light which is detected as forward andside scatter (Figure 11). The combination of scattered and fluorescent light is thendetected and analyzed. Forward scatter correlates with the cell size, with side scatter27depending on the density of the cells. In this manner, cell populations can often bedistinguished based on their difference in size and density.In this study, quantitative evaluation of c-Kit+/Lin- cells and cKit+/Lineage-/Sca-1+(KLS) cells was performed using FACS analysis. Representative FACS data are shownin (Figure 11).4.7 Statistical analysesResults are expressed as mean ± standard error of the mean (SEM). The means of thegroups were compared using one-way repeated measures Analysis of Variance(ANOVA) followed by the Tukey-Kramer post hoc test. Differences were consideredsignificant at P< 0.05.28Figure 1. Anesthesia system and induction chamber29Figure 2. Induction of anesthesia in the induction chamberprior to intubation of the mouse.30Figure 3. Setting up for intubation.31Figure 4. Harvard ventilator for mice.32Figure 5. Animal preparation for surgery after intubation.33Figure 6. Exposure of the heart and identification of the LAD anatomy.Figure 7. Closure of the thoracotomy after ligation of the LAD.34Figure 8. Recovered mouse placed in a new cage.35Figure 9. Sections of the heart with myocardial infarction showing pale color.36Figure 10. Mouse heart showing the infarcted area (pale color) in the left ventricular.37Bone MarrowA1024 81.1986%6_ 768-6c/i 5128° 256256^512^768^1024Forward ScatterB10246 768SpleenC1024.6 768retiBlood82.9676% 74.6708%512 vi 512a256 2 124;lc0oytes u) 256 ;:,1;Monocytes00^256^512^768^1024^0^256^512^768^1024Forward Scatter Forward Scatter101^10'^10°^10°Scat +KLSr x.0483 'cKit+'  4 0.1488%10110°100GcKrtPEH10.10o.10FtKit PE cKrtPEFigure 11. Quantitative evaluation of c-kit+ Lineage-/Sca-1+ (KLS) mononuclear cells byFACS analysis, and representative FACS data obtained from bone marrow, spleen, andblood. Middle penal represents Lineage negative mononuclear cells selected as the lower5% Lineage staining gated from mononuclear cell region (upper penal). Lower penalrepresents Lineage negative mononuclear cells with both c-Kit and Sca-1 positivestaining that were judged as KLS cells.38*—1— -7-2-1-15 RESULTSDynamic changes in the concentration of c-kit+/Lin- cells and KLS cells in the spleen,bone marrow, and blood were measured by FACS analysis on day-1, -3, -6 and -12after ligation of the LAD in the mice. Results showed significant changes in these cellsin the three systems following AMI.5.1 Effects of myocardial infarction on c-kit+/Lin- cells5.1.1 Bone marrow c-kit+/Lin- cellsThe percentage of the c-kit+/Lin- cells in the bone marrow significantly increased on day-3 following AMI compared to the baseline level in normal control mice (1.47 ± 0.094%vs 1.127± 0.019%, P < 0.05). After day-3 following AMI, the percentage of the c-kit+Lin-cells decreased gradually, and returned to the baseline level on day-12 (Figure 12).Ctrl POD1 POD3 POD6 POD12GroupsFigure 12. Changes in percentage of c-kit+/Lin- cells in bone marrow at different timepoints following AMI. * p < 0.5 vs control (Ctrl).39.350.30N▪ 0.10C.) 0.050.00Mit**5.1.2 Splenic c-kit+/Lin- cellsThe percentage of the c-kit+/Lin- cells in the spleen increased gradually following AMIand was significantly higher than the baseline level of normal control mice on day-3, -6,and -12 following AMI. It reached the maximal level (approximately 3 times the baselinelevel) on day-6 (Figure 13).Ctrl POD1 POD3 POD6 POD12GroupsFigure 13. Changes in percentage of c-kit+/Lin- cells in spleen at different time pointsfollowing MI. * p < 0.5 vs control (Ctrl).5.1.3 Blood c-kit+/Lin- cellsThere were no significant changes in the percentage of the c-kit+/Lin- cells in thecirculating blood at any time point following AMI in ischemic mice relative to the baselinelevel in the normal control mice. However, there was a trend towards an increase in thepercentage of c-kit+Lin- cells only on day-1 following MI (Figure 14).400.000^- —Ctrl POD1 POD3 PO136 POD12Groups0.125—e 0.100-u)c.) 0.075-=I.0 050-+ (i) 0.025-Figure 14. Change in percentage of c-kit+lin- cells in blood at different time pointsfollowing AMI.5.2 Effects of myocardial infarction on KLS cells5.2.1 Bone marrow KLS cellsThe baseline level of KLS cells in bone marrow of the normal control mice was near0.2% of c-Kit+/Lin- cells. The percentage of KLS cells in the bone marrow slightlydecreased on day-1 following AMI, and then significantly increased on day-3 after AMI,reaching more than two times the baseline level of the normal control group (0.3653 ±0.012 % vs 0.1848 ± 0.019% , P < 0.05). 6 to 12 days following AMI, the percentage ofKLS cells slowly decreased, but remained higher than the control group. (Figure 15)41*10.4071' 0.2U)_10.10.0Ctrl POD1 POD3 POD6 POD12GroupsFigure 15. Changes in percentage of KLS cells in BM at different time points followingAMI. * p < 0.5 vs control (Ctrl).5.2.2 Splenic KLS cellsThe baseline level of the percentage of KLS cells in the spleen was five times less thanthat in the bone marrow in the normal control mice, which was about 0.04% of the c-kit+/Lin- cells. The percentage of KLS cells in the spleen gradually and continuouslyincreased during 12 days following AMI, and was significantly higher on the 6 th (0.1078± 0.0076 %) and 12th day (0.1174 ± 0.035 %) following AMI than in the normal controlmice (0.0425 ± 0.0064 % )(p < 0.05) (Figure 16).420.20.0 **Ctrl POD1 POD3 POD6 POD12GroupsFigure 16. Changes in percentage of KLS cells in spleen at different time points followingAMI. * p < 0.5 vs control (Ctrl).5.2.3 Blood KLS cellsThe baseline level of the KLS cells in the blood was similar to that in the spleen in thenormal mice. In contrast to the changes in the spleen and bone marrow, the percentageof the KLS cells in the blood decreased gradually during the 12 days following AMI,although there was no statistical significance in the percentage of the KLS cells at anytime point compared with the normal control level. On the third day following MI, thepercentage of the KLS cells reached the lowest level (Figure 17).430.06-0.05-o 0.04-NTS) 0.03-tn21 0.02-0.01-0.00^ I^I^ICtrl POD1 POD3 POD6 POD12GroupsFigure 17. Changes in percentage of KLS cells in blood at different time points followingAMI.446 DISCUSSION AND CONCLUSIONSA small number of stem/progenitor cells remain in the circulating blood to keep thebalance of the stem cell pool in tissue niches that are located in different organs(Fliedner 1998).The number of stem cells may increase in peripheral blood after so-calledpharmacological mobilization (Ratajczak, Majka et al. 2003) (Pituch-Noworolska, Majkaet al. 2003) or tissue injury (li, Nishimura et al. 2005) (Patschan, Krupincza et al. 2006).Increases in the number of CD34+ stem cells and progenitor cells (EPCs) in the bloodand the infarcted area after AMI is a documented phenomenon potentially influencingleft ventricular function in the post-infarction setting (Leone, Rutella et al. 2005) and incongestive heart failure (Valgimigli, Rigolin et al. 2004).However, no study has been reported on the dynamic change in, or mobilizationof, more specific sub-groups of HSCs, such as KLS cells, following AMI. The KLS cellsare those with c-kit+, Lin-, and Sca-1+. C-kit is a stem cell factor receptor, Sca-1 is astem cell antigen specifically expressed in various stem cells (only in mice) and Lin is amixture of antibodies against lineage markers for blood adult cells (mouse: Gra-1, Mac-1, B220, CD3 and Ter119; human: CD3, CD4, CD8, CD19, CD33 and Glycophylin A).Previous studies have confirmed that the presence of the c-kit+, sca-1+ and Lin-markers on a particular stem cell indicates that the cell is BM derived HSCs. Lanza , forexample, reported that 70% of BM derived HSCs express the c-kit receptor (Lanza,Moore et al. 2004). Osawa reported that the cells with c-kit+, sca-1 + and Lin- are alwayspurified from murine BM that have long-term multilinge repopulating and contain veryprimitive HSCs. The data in the study by Osawa et al. clearly indicates that c-kit+, sca-I+ cells are the only cells in the Lin- fraction with HSC activity (Osawa, Nakamura et al.451996) (Nishi, Osawa et al. 1995). Therefore, KLS cells represent a pure population ofHSCs, which are found correlating with very primitive bone marrow activity (Surdez,Kunz et al. 2005; Tadokoro, Ema et al. 2007). KLS cells are considered to be the mostcommon active population of bone marrow HSCs. This study attempted to answer thequestion of whether acute myocardial infarction related stress also triggers dynamicchanges or mobilization of KLS cells and c-kit+/Lin- cells from the BM into the peripheralblood and spleen.This study presents for the first time a chronological observation of the dynamicchanges in specific sub-groups of HSCs in the bone marrow, spleen, and circulatingblood in the same animal following acute myocardial infarction. Significant dynamicchanges in the percentage of c-kit+/Lin- cells and KLS cells in the bone marrow, spleen,and circulating blood during the first 12 days after AMI by ligation of the LAD wereclearly demonstrated.The most interesting findings show that:1) in the bone marrow these cells slightly decrease in number on the first dayafter AMI, peak on the third day, and remain elevated between day-6 and -12;2) there is a continuous and significant increase in the number of these cells inthe spleen during the first 12 days following AMI;3) there is a trend towards a decrease in cell numbers, particularly KLS cells, inthe circulating blood during the first 12 days following AMI;4) the most significant increase in the number of these cells was observed in thespleen; and in BM on the 3rd day following AMI (supplementary Figures 1, 2).The decrease in HSCs in the bone marrow at the very early stage following AMIis probably due to initial release of existing stem cells from bone marrow to the46circulating blood and organs. After this stage a significant amount of new stem cells areproduced in the bone marrow due to a stimulation of some undetermined factors thatare most likely activated by AMI or released from injured tissue. This stimulationappears more significant during the first three days following AMI as the HSC levelreaches maximal in the bone marrow on day-3 after AMI. The HSC level thendecreases gradually and almost returns to the baseline level on day-12 following AMI,which may indicate a gradual decline in the stimulation of stem cell production in bonemarrow and/or an increase in the release of stem cells from bone marrow to circulatingblood and spleen.Although the mechanism of stem cell mobilization following AMI has not beencompletely confirmed, it has been reported that ischemia induces the liberation of stemcells from the bone marrow pool by multiple mechanisms; many mobilizingfactors/cytokines, such as G-CSF, SDF-1, CSF, or VEGF, are involved in the stem cellmobilization and homing.Leone (Leone, Rutella et al. 2006) has demonstrated that the spontaneousmobilization of CD34+ cells into the peripheral blood of patients with AMI is significantlycorrelated to endogenous G-CSF. G-CSF is a well-known potent mobilizer of CD34+cells into peripheral blood and is currently widely used for transplantation ofhaematopoietic progenitor cells, instead of the whole bone marrow as in current clinicalpractice.More attention has been devoted recently to the role of the chemokine stromalcell-derived factor-1 alpha (SDF-1a) and its receptor CXCR4 in the trafficking andhoming of human stem/progenitor cells. Interestingly, SDF-1 alpha is transiently up-regulated at the mRNA level early after myocardial infarction and might contribute to47intralesional stem/progenitor cells homing (Askari, Unzek et al. 2003). Cumulativeevidence has suggested the pivotal role of the interplay between the systemicmobilizing effect of G-CSF and the local contribution to engraftment of SDF1-alpha.It was shown in this study that the percentage of HSC cells in the bone marrowreaches a maximal level around day-3 following AMI, which is consistent with thefindings of Wright (Wright 1985; Wright, Cheshier et al. 2001), who found that theuptake of BrdUr by BM usually occurs 2-3 days following the administration of GrowthFactors (G-F). HSCs incorporate BrdUr into its DNA, which reaches the maximum levelthree days after administration of G-F. Observations made in this study also agree withBarcew's finding that the percentage of HSCs in BM peaks on day-2, but the absolutecell number reaches its peak on day-3 following activation by G-F (Barcew, Kacinska etal. 2004).The decreased level of HSCs in the BM on day-6 following AMI is probably dueto a significantly increased release of stem cells from the bone marrow to circulatingblood and other organs. Levesque has observed that the stem cell release from thebone marrow is probably activated by increased numbers of enzymes called serineproteases (Levesque, Liu et al. 2004).Serine proteases, including enzymes such as casthepsin and elastin, arereleased upon the activation of neutrophili in the BM after stimulation of G-F and otherinflammatory agents. The enzymes have been noted to reach the maximum level ofconcentration around day-5 or -6. They disrupt the cell surface interaction between stemcells and the microenvironment of the BM, which allows the stem cells to be released totheir respective niches and transmigrate across the endothelium layer into theperipheral blood and other organs (Levesque, Hendy et al. 2003).48The decreased HSC level in bone marrow on day-6 corresponds to thesignificant increase in HSCs in the spleen observed from day-6 to day-12 following AMI,which further supports the probability of significantly increased release of stem cellsfrom BM on day-5 to -6 following AMI. Although the subsequent decrease in HSC levelin the BM from its peak level on day-6 is most likely attributed to the mobilization fromBM to the circulating blood and other organs, it could also be due to the subsequentdevelopment of these stem cells and early progenitors to mature blood cell lineages tohelp in the recovery of the whole population of bone marrow cells (Nienaber, Petzsch etal. 2006).Another interesting finding of this study is that the percentage of HSCs (KLS andc-kit+/Lin- cells) fails to increase in circulating blood from day-1 to day-12 following AMI,but also shows a trend towards decrease during the first 12 days. In contrast to thechange in the blood, the percentage of these cells in the spleen increases continuouslyand significantly during the first 12 days, particularly after day-3, following AMI. Thesefindings indicate that the majority of mobilized HSCs in the circulating blood from thebone marrow are either actively or passively removed by the spleen and/or possiblymigrate into the injured tissue for tissue repair.It is unclear whether the spleen plays a role simply as a potential transientreservoir of mobilized HSCs or as an important organ actively participating in stem cellmobilization, trafficking, and activation. The accumulation of the mobilized stem cells inthe spleen may play an important role in the late phase of stem cell migration into theinjured tissue for the process of continuous tissue repair. Early tissue-injury-inducedmobilization of stem cells from the bone marrow decreases after the acute phase of MI.49The phenomenon of a lack of the increase in HSCs in circulating blood followingAMI is consistent with findings of Patschan on EPCs following renal ischemia(Patschan, Krupincza et al. 2006). They found that a 25-minute period of unilateral renalischemia resulted in no increase in the number of peripheral circulating EPCs at 10minutes, 3, 6, 24 hours, and 7 days following renal ischemia.The lack of increase in HSCs or EPCs in circulating blood may be due to theexistence of a dynamic equilibrium between circulating and tissue-residing HSCs orEPCs characterized by a slow and continuous cell migration from the bone marrow orother niches via peripheral blood to the spleen and later on to sites of ischemic tissue(Levesque, Hendy et al. 2003).Patschan demonstrated also a significant transient accumulation of EPCs in thespleen occurs only between 3-6 hours following renal ischemia, which is different fromthis study's findings, which showed that the significant accumulation of HSCs in thespleen continues after 3 days following AMI. This difference in findings could be due todifferent groups of cells studied and the ischemic model of different organs.Alternatively, in the study an early peak of accumulation of HSCs in the spleen duringthe first day following AMI could have been missed as the cells were not measuredduring the first day.Hematopoietic stem cell mobilization (migration) is a commonly used practice inthe clinical setting to increase the number of HSCs in the peripheral blood forsubsequent collection and transplantation (Mayhall, Paffett-Lugassy et al. 2004). HSCshave in particular been extensively used in the process of repair or regeneration ofdamaged tissue in the heart, kidney, brain and skin—mostly through transplantation ofcells harvested from the bone marrow. However, it has been found that the results of50direct transplantation of bone marrow stem cells are not satisfactory because thenumbers of harvested cells are inadequate (van Laake, Hassink et al. 2006), or themajority of transplanted stem cells die soon after transplantation. This study providesthe dynamics of HSCs following AMI in the circulating blood, spleen, and bone marrow,which may help in developing better therapeutic strategies that augments the naturallyoccurring repair process following AMI through mobilization of stem cells, and inidentifying better sources of HSCs and/or a better time for harvesting andtransplantation.In conclusion, our study provides for the first time the longest observation of thedynamics of specific sub-groups of adult stem cells (c-Kit+/Lin- cells and KLS cells) inmultiple systems following AMI. 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Proc Natl Acad Sci U S A 102(33):11728-33.580.4000% -0.3500% - —  0.3000% -V1H. " 0.2500% -ori4.0© 0.2000% -a.),S5 0.1500% -V= 0.1000% -4.10.0500% -0.0000% ^- Bone marrowSpleen- Blood F^Control^POD1^POD3^POD6^POD12GroupsAPPENDIXSupplementary Figure 1 KLS cells in three systemscon 1.8000%7.;c4  1.6000%• 1.4000%1.2000%••••1P--1 1.0000%44-1 0.8000%0t) 0.6000%0.4000%V▪ 0.2000%4.■C140.0000%- Bone marrow- -II-- SpleenA^BloodControl^POD1^POD3^POD6^POD 12GroupsSupplementary Figure 2 C-kit+ Lin- cells in three systems59ID0.4000%0.3500% -10.3000% -10.2500% -1Oti)0.2000% -0.1500% -CJ 0.1000% -0.0500% - 1---0.0000%Bone marrowSpleen^ BloodControl^POD1^POD3^POD6^POD12GroupsAPPENDIXSupplementary Figure 1 KLS cells in three systems1.8000%1.6000%1.4000%1.2000%1.0000%c4-1 0.8000%Oa 0.6000%0.4000%CJ 0.2000%6rNo- 0.0000% Bone marrowSpleene^ BloodControl^POD1^POD3^POD6^POD12GroupsSupplementary Figure 2 C-kit+ Lin- cells in three systems59 THE UNIVERSITY OF BRITISH COLUMBIAANIMAL CARE CERTIFICATEApplication Number: A07-0116Investigator or Course Director: Jian. YeDepartment: SurgeryAnimals:Mice wild-type C57 B16 96Mice GFP 180Start Date:^May 1, 2007 ApprovalDate: May 24, 2007Funding Sources:FundingAgency:Funding Title:Canadian Institutes of Health Research (CIHR)Near-Infrared, thermal and magnetic resonance imaging in diagonistics andtreatment of cardiac ischema and infarctionUnfunded title: N/AThe Animal Care Committee has examined and approved the use of animals for the above experimentalproject.This certificate is valid for one year from the above start or approval date (whichever is later) providedthere is no change in the experimental procedures. Annual review is required by the CCAC and somegranting agencies.A copy of this certificate must be displayed in your animal facility.60


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