Cardiac Repair With Intramyocardial Combined Injection Of Mesenchymal Stem Cells And Growth Factors After Myocardial Infarction

The traditional belief is that the cardiac muscle is terminal differentiation tissue, and can only be replaced by scar tissue after myocardial infarction.In order to maintain the cardiac pump function,a degenerative left ventricular remodeling occurs in the body,causing the cardiac function decrease and leading to heart failure eventually. Bone mesenchymal stem cells(BMSCs)have the potential to differentiate into cardiomyocyte-like cells in the cardiac microenvironment and appear as functional phenotypes of myocardial cells.Whose special gene can be expressed into the structure proteins.It has been a great interest in myocardial infarction treatment to use stem cells to prevent from the deterioration process of heart failure,and even to repair the damaged myocardium.Growth factor(GF)plays an important role in the cell migration, survival,and differentiation.It has been reported that the growth factors applied in the local microenvironment can improve the survival rate and functions of the transplanted cells.Unfortunately,the mechanism has not been well documented.In this study,the hepatocyte growth factor(HGF)and insulin-like growth factor-1(IGF-1)were used in the culture of BMSCs.The purpose of this study was to observe the effects of growth factors on the GATA-4 expression of BMSCs and the effects of cardiac function after myocardial infarction by the intramyocardial combined injection. 1.Co-culture of mesenchymal stem cells and myocardial cellsA total of 2-3 ml rabbit femur bone marrow was collected from healthy adult rabbits(about 5 months old and 2.0-2.5 kg weight,Animal Testing Center,China Medical University),and was then inoculated in the culture bottle after washing in 5% CO₂ incubator at the temperature of 37℃.Two days later,BMSCs adhered to the bottom of culture flask,while blood cells and others were suspended in culture medium. The culture medium was changed every 3 to 4 days.After 4 times of medium change, suspended cells were removed completely.The myocardial cells were taken from the ventricle of newborn rabbits(1-3 days).The ventricular tissue of a beating-heart was cut with scissors into a small piece of 0.5 to 1 mm³.After isolated using trypsin,the cells were cultured in DMEM culture containing 15%FBS,and then put in CO₂ incubator with 5%CO₂ at a temperature of 37℃.The medium was changed once a day for 2 days.The myocardial cells were mixed with BMSCs in a ratio of 1:1.In addition,150 ng/ml HGF(Peptech,UK)and 200 ng/ml IGF-1(Peptech)were added into the experimental group.2.Animal Model.All healthy adult rabbits(about 5 months old and 2.0-2.5 kg weight)studies were approved by the Animal Testing Center,China Medical University.Four different studies(n=7)were conducted.During surgery,MI was induced by a 60-min occlusion of the LAD,followed by reperfusion.At 3 d after MI,animals were randomized to receive intramyocardial injections of either allogeneic MSCs 40ul(5×10⁶/10ul),the hepatocyte growth factor(HGF)and insulin-like growth factor-1(IGF-1)in 8ul DMEM (150ng/ml HGF.200ng/ml IGF-1)and MSCs with GF or placebo.MSC injections were guided by the presence and location of the surgical markers.3.Immunocytochemical stainingThe animals were followed for an 6-week period and were then humanely killed. Myocardial tissue was prepared for immunohistochemistry.Tissue samples were obtained from three specific areas:infarct zone,infarct border zone,and remote tissue. Antibodies examined included-actinin(Santa),connexin 43(Boster).4.Transmission electron microscopyThe cultured cells were directly fixed in Eppendorf tubes.After gradient ethanol dehydration and embedded in epon resin,the slices were made with ULTRACUT E-type ultramicrotome.The slides were examined under Hitachi H-600-4 transmission electron microscope and their photos were taken with camera after the slides were double stained with uranyl acetate and lead citrate.5.Capture of mesenchymal stem cells by laser capture microdissection systemBMSCs and myocardial cells were co-cultureed for 1 day,3 days,1 week,2 weeks until 6 weeks,and were double-stainied for nuclear and muscle fibers by immunocytochemical staining in both groups.The IgG antibody was used to stain muscle fibers by the combination of both rabbit antiα-actin monoclonal body and goat anti-rabbit peroxidase,and the hematoxylin was used to label cell nuclei.We used laser capture microdissection system(Arcturus)and chose the cells with expression ofαactin-positive and containing mitotic cell at the same time as myocardial cells being differentiated from BMSCs.A total of 500 cells was captured in each group.6.RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)Total RNA was isolated from the captured stern cells using PicoPureRNA Isolation Kit(Arcturus).RNA pellet was resuspended in 1 ml of 75%ethanol, centrifuged again,and the supernatant was discarded.The pellet was resolved in 2μl of DEPC treated water,and cDNA was synthesized via reverse transcription.PCR was performed using cDNA template in reaction buffer containing 6.25 pmol/L of each primer with Taq HS DNA polymerase(0.1μl)(Invitrogen),Primers(GeneCore)are as followings:rabbit GATA-4(188 bp):upstream 5′-GCCCCTCATcAAGCCTCAG-3′, and downstream 5′-TCCCCTCTTTCCGCATCG-3′;β-actin(315 bp):upstream 5′-TTCCAGCCCTCCTTCCTG-3′,and downstream 5′-GCCCGACTCGTCAT ACTCC-3′.PCR condition:denaturing at 94℃for 40 seconds,annealing at 60℃for 40 seconds,elongation at 72℃for 60 second,and final extension at 72℃for 5 minutes after 45 cycles.β-actin was used as the intemal standard.The band density of PCR products was quantified.7.Measure on Cardiac FunctionWe measured the effect of MSC therapy on post-MI 6 weeks cardiac Function in animals by IE33 model ultraphonic with 3D detecting head on PHILIP Corporation. Whole left ventricle volume graph were obtained in.Analyze these volume graph with QLAB software.Results1.Morphological changes of mesenchymal stem cells and myocardial cells in vitroAt first,the morphology and size of these BMSCs varied.About 1 week later,the adherent cells became separated colonies of relatively homogeneous morphology:most appearing as fibroblasts with spindle and shuttle shape.The colonies continuously grew, reaching 80%-90%confluences in 2 weeks.Under the inverted microscope, not-adherent subculture cells without adherence appeared in a round shape,while those after adherence gradually became in polygon with uniform cell size.24 hours later the single cells became beat,having extended parapodium that became interconnected with each other.2.Trans-differentiation of mesenchymal stem cells to myocardial cellsCardiomyocyte-like ceils derived from BMSCs demonstrated the immuno-staining characteristics of myocardial cells as well as the myocardial cell-like ultrafine structure under electron microscope.All the myocardial cells showedα-actin-positive and connexin 43 staining positive.The electron microscopy images showed the homogeneous in the central nucleus without nuclear fission observed.The BMSCs differentiated into myocardial cells exhibiting myocardium-like ultrastructure,and these cells contained nucleus with multiple nucleoli,indicating that these myocardial cells differentiated from BMSCs.Simple BMSCsα-actin immuno-staining showed negative by electron microscopy and had a nucleus with multiple nucleoli.After being co-cultured with myocardial cells in a ratio of 1:1,some BMSCs differentiated into myocardial cells whose nucleus occurred in mitosis,and whoseα-actin immuno-staining is positive.And as well in intramyocardial BMSCs marked with DAPI showed connexin 43 staining positive.3.Expression of transcription factor GATA-4Cardiac transcription factor GATA-4 mediates the complex physiological process of a heart through regulating the expression of cardiac-specific genes.Native cardiac myocyte can express transcription factor GATA-4,but the BMSCs not.After being cultured with myocardial cells,some BMSCs became to express the transcription factor GATA-4.Analysis of RT-PCR has confirmed the differentiation of BMSCs,that is to say,after co-culture of BMSCs with myocardial cells,the captured BMSCs by laser can be detected in the expression of GATA-4,while no expression of GATA-4 for BMSCs cultured alone.At the same time the combined delivery of HGF and IGF-1 can increase the expression of GATA-4 in BMSCs.The result suggests that the myocardial cells differentiating from BMSCs can express cardiac transcription factor GATA-4,i.e. possessing myocardial cell function in the regulation of heart gene,and particularly the combined delivery of HGF and IGF-1 in this process plays an important role. Compared with the control group,the combined delivery of hepatocyte growth factor (HGF)and insulin-like growth factor-1(IGF-1)in co-culture of BMSCs with myocardial cells can greatly increase the differentiation of GATA-4 expression of BMSCs in experimental group.After adding of growth factor,the GATA-4 expression of the captured BMSCs kept increasing since the first day,and reached the maximal level after two weeks,and decreased slightly thereafter.4.MSC Therapy Effect on Cardiac Function.Table 1 depicts indices of ventricular function creation and at 6 weeks after MI in the three treatment groups(MSC,GF,MSC+GF injections).As shown,there is a sizable reduction in systolic and diastolic function(increased LVEF and P＜0.05vs.control groups values).In four groups,indicative of equivalent degrees of injury.In the placebo-treated group(n=7),impaired cardiac function evident showed either no sign of recovery or a tendency to worsen over 6 weeks of follow-up:Indices of myocardial contraction fell,and end-diastolic volume rose,as did(Table 1)in marked contrast, animals receiving MSCs(n=7)and MSCs+GF(n=7)exhibite drecovery to essentially normal levels of both systolic anddiastolic function.To further evaluate the effect of MSC and GF therapy on global cardiac function,Whole left ventricle volume graph determination of LV ejection fraction(LVEF)was performed.6 weeks after MI,there was a marked recovery in LVEF:56%for MSC and 59%for MSC+GF respectively. However,there was minimal change in LVEF between MSCs groups and MSCs+GF groups.LVEF increased from 34 to 56%in MSC-treated animals and from 34%to 59% in MSC+GF-treated animals during the 6-week follow-up period.ConclusionsBMSCs can differentiate into cardiomyocytes-like cells after being co-cultured with myocardial cells,and express the cardiac transcription factor GATA-4.HGF and IGF-1 can stimulate the differentiation of BMSCs into cardiac phenotype,and enhance the expression of GATA-4.These indicate that growth factors have a great potential in clinical cellular therapy.And as well combined injection of mesenchymal stem cells and growth factors into regions of damaged myocardium could tremendous enhancement in both systolic and diastolic cardiac function.However GF in combination with MSCs transplantation did not enhance the cardiac repair effects of MSCs transplantation alone.In general,as a result of trans-differentiation,BMSCs have the ability to differentiate into cardiomyocyte-like cells and show the myocardial cells phenotype in the appropriate micro-environment.Our results have proved that,in addition to the differentiation of local cells,the combined HGF and IGF-1 indeed promote the cell survival and the elevated expression level of GATA-4 and enhances the cardiac function after myocardial infarction.All these results indicate that the growth factor has a great potential in the clinical cell therapy.DiscussionAlthough there is great enthusiasm to repair the heart by using cell therapy,the simple cell implantation cannot revive the cell survival and differentiation.Furthermore, experimental data indicates that only a small number of cells can be implanted in the host tissue with normal function.Therefore,attempts of using all kinds of approaches to enhance the effectiveness of cell therapy has become a challenge.Utilizing growth factors in a local tissue environment to increase the survival function of transplanted cells has been considered as a practical.Hepatocyte growth factor and insulin-like growth factor both are potent cardiomyocyte growth and survival factors.HGF is a multifunctional factor to promote cell mitosis,which activates the c-Met receptor,leading to the formation of matrix metalloproteinases that digest collagen and other extracellular components of the interstitium,facilitating cell migration and homing in the organs.In addition,HGF can promote the cell survival and regeneration,inhibit the apoptosis of MSC,increase the MSC transplantation survival rate.More importantly,HGF enhances the vessel growth and favors cell-extracellular matrix interaction,the critical steps during myocardial regeneration after infarction.IGF-1 can promote angiogenesis in infarcted myocardium,reduce the degree of myocardial necrosis,maintain the myocardial structure,stimulate proliferation of cardiac fibroblast,inhibit matrix degradation,as a result prevent ventricular dilatation and reduce load capacity of the heart.HGF and IGF-1 have played an important role in proliferation and differentiation of stem cell.Chedrawy and others have shown that the physical barrier between BMSCs and cardiomyocytes is nonconducive for BMSCs to transdifferentiate,despite the presence of a myocardial micro-environment.Rangappa also proved that in addition to soluble signaling molecules,direct cell-to-cell contact is necessary for relaying the external cues of the microenvironment controlling the differentiation of adult stem cells to cardiomyocytes.Therefore,we designed that the Current experiment in which BMSCs and myocytes were cocultured,use laser microdissection system to capture the differentiated stem cells,quantify the expression of GATA-4 in captured BMSCs by semi-quantitative RT-PCR.In this study,we proved that rabbit BMSCs can differentiate into cardiomyocytes in vitro in the presence of myocytes,while the growth factor played an important role in this process,Immuno-staining showed that the cultured BMSCs are positive forα-actinin and connexin 43,and the electron microscope data demonstrated the existence of muscle fiber,mitochondria and endoplasmic reticulum.The various shapes and multiple nucleoli of cells provide solid evident to support that these cells were BMSCs.The result is consistent with other in vivo and in vitro studies.Additionally, RT-PCR data also proved that the cardiomyocyte-like cells derived from bone marrow cells could express GATA-4.The combined delivery of HGF and IGF-1 took a significant effect in promoting the differentiation process of BMSCs to myocardial cells,compared with the control group,which could greatly increase the expression of GATA-4 in differentiation of BMSCs,and such expression began to increase since the first day,reached the maximal level after two weeks,and decreased slightly thereafter. And as to cardiac function combined injection of mesenchymal stem cells and growth factors into regions of damaged myocardium could tremendous enhancement in both systolic and diastolic cardiac function.However GF in combination with MSCs transplantation did not enhance the cardiac repair effects of MSCs transplantation alone. In general,as a result of trans-differentiation,BMSCs have the ability to differentiate into cardiomyocyte-like cells and show the myocardial cells phenotype in the appropriate micro-environment.Our results have proved that,in addition to the differentiation of local cells,the combined HGF and IGF-1 indeed promote the cell survival and the elevated expression level of GATA-4 and enhances the cardiac function after myocardial infarction.All these results indicate that the growth factor has a great potential in the clinical cell therapy.