| PART I Identification of cardiac stem cells(cardiosphere-derived cells)expanded human heart tissueBackgroundThe present therapies for ischemic heart disease just include cause-dependent interventions, which are not able to reduce myocardial necrosis and optimize cardiac repair following infarction.Now cell therapy is a major new strategy in the treatment of the public health challenge. While ideal graft cells should be non-immunogenic, easy to collect and expand in vitro. Most recently, compelling evidence shows us that there is the resident stem cells in heart. Stem cells, which are from recipient’s heart, are naturally non-immunogenic and adapt to the cardiac microenvironment. Resident cardiac stem cells may have a bright future. Cardiosphere and cardiosphere-derived cells are the mixture of cardiac stem cell and supporting cells, and it is an important choice for ischemic heart disease.ObjectivesThe chapter mainly introduced how to culture and expand CDCs from heart tissue in vitro. We investigated the composition of cardiosphere-derived cells from different population with different age and original diseases, and explored the composition of CDCs in different phase. We aimed to demonstrated CDCs is a reliable type of stem cells.MethodsCardiospheres and cardiosphere-derived cells were isolated from atrail and ventricular specimens belonging to surgical patients.The morphology of CSp and CDCs were observed by inverted phase contrast microscope. The expression of c-Kit, Sca-1, CD105, CD90, CD31, CD34, CD104b, CD45and lineage cocktail in different patients and in different generation of CDCs were characterized by flow cytometry.ResultsCDCs collected from heart had strong viability and ability of proliferation in the whole process of culture in vitro.Flow cytometry analysis shows CDCs includes6%c-Kit+cells,2.5%Sca-1+cells,100%CD105,31%CD90,6%CD31,5.8%CD34,2.2%CD104b,1%CD45and1%lineage.Compared to healthy group and AMI group, CDCs from cardiac transplantation had more c-Kit+、Sca-1+and CD104b+cells, and they were higher concentration in AMI group than in healty group(P<0.05). CDCs from different passage had the same composition.ConclusionThis is a suitable method to obtain CSp and CDCs effectively. CSp and CDCs have the same proportion in different patients with different original diseases and in different phase. CSp and CDCs are the mixture of cardiac stem cells and supporting cells, and they are the reliable origin of cardiac stem cell. Part Ⅱ Active vascular expulsion:a novel mechanism of cardiac stem cells migration across vessel wallsBackgroundStem cell therapy has been explored as a promising therapeutic strategy for cardiovascular disease.While many factors limit the utility of cell transplantation, such as cell type, cell dose and delivery route. Good tranplantation method should be minimally invasive for patients. Intravenous infusion is not selected because of trapping of cells in the lungs, resulting in acute retention rates of<1%. Intracoronary delivery of cells is safe and convenient and has the inherent advantage that cells can be homogeneously infused into myocardial area where there is enough oxygen and nutrient supply, of course without increasing the rate of restenosis in stent and arrythmia.While we should acknowledge the cell retention in heart is so low because almost cells delivered by intracoronay appear in body circulation again. If we want to use the intracoronary delivery, we will try to find the way to increase the retention or efficiency of stem cell in coronary artery. It should be learned about the whole process about how stem cells work in body delivery. Stem cells in tissue aroud blood vessel perhaps are the real hero for regeneration. So it is necessary for us to explore the mechanism about cell transmigrating outside of capillary.AimIn the present study, we invistigated the process of stem cells transmigration in coronay artery by IHC. We sought to understand the biological determinants about it, we observed the function of the integrins inhibitor and MMPs inhibitor and illustrated the related mechanism by IHC, Masson trichrome stain, echo and Elisa.MethodsCardiac-derived single cells and multi-cellular spheres were cultured as described from Wistar-Kyoto rats and infused into the coronary arteries of rats of the same strain after labeled with DiO. Fluorescent polymer spheres were infused as control. Quantification of extravasation efficiency was performed on confocal images of fixed heart sections with vessels labeled by anti-vWF antibodies. Perivascular hypoxia was detected by the administration and IHC detection of Hypoxyprobe TM. Cardiac function was assessed by echocardiography.Infarct size was measured from Masson trichrome-stained heart sections.Micro-embolic injury was assessed by measuring the concentrations of troponin I in the serum. To reveal the mediators in the active vascular expulsion process, a subset of animals were intraperitoneally injected with integrin inhibitor RGDS peptide and MMP inhibitor GM6001, or the scrambled versions of the two. For in vitro pocketing experiments, CSp were plated on HUVEC monolayers (on fibronectin-coated surface) and HUVEC networks (on Matrigel TM).Results1. Infused single cells (SC) or multicellular spheres (CSp) lodged within the Micro-vasculature shortly after infusion (T=10min), occupying the full luminal diameter. By24hr, SC and CSp remained within the blood vessels, but they were now sidelined and surrounded by endothelial projections, with vessel patency restored in some cases. By72hr, the infused cells had been expelled into the extravascular space, and all vessels were patent. Endothelial pocketing and extravasation required biorecognition:inert polystyrene microspheres infused into coronary arteries embolized and occluded capillaries, but they did not undergo encapsulation or cross the vascular barrier Pooled data revealed that extravasation and vessel patency progress inexorably (over72hr) with SC or CSp; meanwhile, PSPs simply remained lodged within vessels. As a consequence of PSPs vessel microembolization, the myocardial tissue became hypoxic, and remained so over the72hr period of observation; in contrast, CSp-infused tissue was modestly hypoxic at24hr, but recovered completely by72hr, consistent with the observed time course of vessel recanalization We found the process was different from all mechenisms reported. So we name the new one:active vascular expulsion.2. Multicellular spheres (CSp) lodged within human vein endothelial cells. We found endothelial pocketing, which was similar with the model in vivo. The cell pocket would be formed within6hours.3. In vivo administration of RGDS peptide, a potent integrin inhibitor, decreased acute CSp retention within infused tissue (P<0.05), indicating that initial cell adhesion was at least partially integrin-dependent; the RGDS-insensitive component of cell retention presumably reflected physical micro-embolization, as CSp were larger in diameter than the typical capillary. Given that inert microspheres were not surrounded by endothelial projections, we conjectured that integrins might also play a role in endothelial pocketing. RGDS prevented endothelial pocketing of CSp (P<0.05), leading to suppression of extravasation (P <0.05) and potentiation of tissue hypoxia (P<0.05).4. Endothelial pocketing was recreated in vitro when CSp were plated upon monolayers of human vein endothelial cells; the pocketing process plays out over several hours. PSPs did not elicit responses from the HUVEC, a null phenotype which could be mimicked by co-applying RGDS peptide with CSp. Thus, integrins are required for endothelial pocketing.5. We examined MMPs histologically and found that they indeed concentrated in the adjacent vascular wall during active vascular expulsion. MMPs increased markedly at24hr, then declined, in response to CSp infusion; in contrast, MMPs were activated by PSPs weakly and monotonically (P<0.01). Functionally, the extravasation of CSp was largely blocked by the broad-spectrum MMP inhibitor GM6001, but not by a negative control compound (GM6001-NC, P<0.01). Interestingly, GM6001did not affect endothelial pocketing (P=0.77).6. To model pocketing and extravasation in vitro, we plated CSp upon HUVEC networks formed on Matrigel. Some CSp landed far from the endothelial tubes, but those that happened to fall upon a tubular nexus were quickly surrounded by HUVEC projectionsand eventually penetrated into the underlying Matrigel, simulating the extravasation process seen in vivo. Time course analysis revealed that endothelial pocketing preceded CSp penetration. Consistent with the in vivo data on vascular wall breakdown, the MMP inhibitor GM6001blocked CSp penetration into the underlying Matrigel (P<0.05) but did not prevent the formation of HUVEC pockets around the CSp (P=0.69). These findings supported a sequential model of active expulsion in which pocketing was an obligatory precursor to vascular wall breakdown; inhibition of either blocks extravasation, but suppression of vascular wall breakdown left endothelial pocketing unaffected.7. Integrative implications of active vascular expulsion. We detected the rat’s cardiac function and morphology by echo and Masson stain.Myocardial scarring, ischemic biomarker elevation (cTnI), and global heart dysfunction (LVEF) did not occur with CSp, whereas PSPs infusion produced gross tissue injury (P<0.05). The findings gave good reason to hypothesize that active vascular expulsion served as an effective repair mechanism against damage.Conclusions1. we report a new mechanism of cell transmigration, active vascular expulsion, which underly the extravasation of infused heart-derived stem cells. In this mechanism, the vascular barrier undergo extensive remodeling, while the cells themselves are relatively passive.2. Endothelial cells and stem cells talk with each other by integrins,entothelial cells will form pocket.3. Matrix metalloproteinases play the critical roles in vascular wall breakdown.4. Active vascular expulsion may be the key role in the extravasation of larger stem cell or stem cell mixture.We will enhance the efficiency of transvascular stem cell migration by utilizing manipulable steps in our finding. Part III Dose-Dependent Functional Benefit of Human Cardiosphere Transplantation in Mice with Acute Myocardial InfarctionBackgroundCardiovascular diseases, especial acute myocardial infarction, remain the number one which threaten human health in the world.Current treatments at best only slow the progression of disease leading to heart failure and death; once scar has developed, the injury to heart muscle is generally cumulative and irreversible. Stem cell therapy represents a promising strategy to mitigate remodeling and regenerate the damaged myocardium. While optimal cell dosing remains elusive. Without optimized cell dose, the analysis on the clinical trials will be not all around, and the result of preclinical trials can not guide the clinical trial very well. So the reseach on cell dose is a very important project.Different cell types should have different cell dose.Cardiospheres(CSp) was used in this study. CSp and cardiosphere-derived cells (CDC) are natural mixtures of resident cardiac stem cells and supporting cell types. Li had conducted a direct head-to-head comparison of CDCs, bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and bone marrow mononuclear cells. CDCs exhibited a balanced profile of paracrine factor production and, among various comparator cell types/subpopulations, provided the greatest functional benefit in experimental myocardial infarction. The phase I clinical trial about CDCs have done, showed us CDCs were safe for patients and had strong regeration ablility. Another trial demonstrated that CSp, compared to CDCs, increased the expression of ECM and beneficial factors and reinforced resistance to oxidative stress, improved cell engraftment and functional benefit in vivo. CSp may be an important choice in future clinical trials. So the research on CSp dose should be significant.AimThe present study was designed to examine the dose-dependent effects of intramyocardial injection of human CSps in a mouse model of acute myocardial infarction (MI). In addition, we sought to determine, on the basis of histological analysis, the mechanisms for this dose-dependent benefit. The hope was to systematically optimize dose in mice and then used these results to guide dosing in large animals and humans trials.MethodsHuman right ventricle biopsies from male donors were minced into small fragments. After collagenase digestion, the tissue fragments were cultured as "explants", harvested cardiosphere-forming cells and then plated on poly-D-Lysine-coated plates. After3～5days, the cardiosphere-forming cells spontaneously aggregated into cardiospheres. Acute MI was created in adult male SCID-beige mice. All mices were then subjected to intramyocardial injections with a30-gauge needle at four points in the infarct border zone, with one of the following5randomly-assigned treatments:1)20μl PBS (Control group);2)1×104cells-formed CSps;3)5×104cells-formed CSps;4)1×105cells-formed CSps;5)5×105cells-formed CSps. Cardiac functions were assessed by echocardiography at baseline (4hour post-MI) and3weeks afterwards. A sub-population of animals were excised for determining the numbers of engrafted cells by quantitative PCR. All other animals were sacrificed at3weeks and hearts were excised for morphometry, immunohistochemisty and Western Blot.Results1. Quantitive PCR quantitation of human cells in the mouse hearts revealed a-10%cell retention rate24hours after injection into the injured hearts,11.42±2.24%、9.97±4.24%.11.70±4.73%.10.23±3.95%, P>0.05.2. Echocardiography revealed that baseline left ventricular ejection fraction (LVEF) after surgery did not differ among groups. Three weeks after treatment, LVEFs in mice receiving the3highest doses (5×10,1x105, and5×105) outperformed the Saline group. No significant difference was seen between the Saline and the1×104group. 3. Masson’s trichrome staining clearly distinguished scar tissue (blue) from normal myocardium (pink). Quantitative morphometry at3weeks showed severe LV chamber dilatation and infarct wall thinning in the Saline-injected hearts. In contrast, high dose cardiosphere-treated hearts exhibited attenuated LV remodeling and less abnormal heart morphology. Magnification of the infarct region revealed the preservation and/or regeneration of normal myocardium tissue in the infarct. Further quantitation indicated thicker infarcted walls and more viable tissue in the infarct area from the hearts received the3high doses of cardiospheres. The measures from the low-dose group (1×104cells) were somewhat higher than in the Saline group, although not significantly so.4. Myocardial neovascularization assessed by vascular density at3weeks was dose-dependently enhanced in mice receiving CSp transplantation. Blood vessels were stained with alpha smooth muscle actin. Hearts receiving high doses of CSps exhibited higher vascular densities than those in the low dose (1×104cells) or Saline groups. The vascular density in the low dose group was similar to that in the Saline group. A similar dose-dependent enhancement was found in promoting cell cycle re-entry of cardiomyocytes. More Ki67+cardiomyocytes were evident in hearts from the3high dose groups, as compared to the low dose or the Saline group. Again, no difference was seen between the low dose and Saline group. In addition to tissue regeneration, tissue preservation might be a salutary mechanism of cell therapy for acute MI. To quantify tissue preservation, apoptotic cells in the hearts were TUNEL-stained. Fewer apoptotic cells were detected in the hearts from the3high dose groups. No difference was found between the Saline and the low dose group. It should be noted that, at3weeks, the numbers of TUNEL-positive cells were a cumulative reflection of maladaptive LV remodeling rather than acute cell death due to ischemia.5. VEGF and SDF-1α protein were assessed by Western Blot at3weeks was dose-dependently enhanced in mice receiving CSp transplantation. Both protein compared to GAPDH exhibited higher expression than those in the low dose (1×104cells) or Saline groups. There was no significant difference between the low dose and Saline group or among another three higher dose groups. ConclusionsOur study demonstrate dose-dependent functional benefit of cardiosphere trans-plantation in mice with acute myocardial infarction. Taking together these considerations and manufacturing feasibility, we anticipate using～150×106CSp cell-equivalent dosage in large animals and humans. Part IV Intramyocardial injection of platelet gel promotes endogenous repair and augments cardiac function in rats with myocardial infarctionBackgroundcardiovascular diseases, except of the infectious disease, represent the main cause of death. Coronary atherosclerotic heart disease is one of most common cardiovascular disease. Myocardial infarction is most likely to lead to cardiac failure. A lot of cardiomyocytes loss play a key role in progress of heart failure. At present the conventional treatments mainly relieve symptoms, the treatment effect is limited. Though the cardiac transplantation is the gold standard for end-stage heart, but donor hearts far outstrip the number of people who could benefit for transplantation.It is necessary to find a new method to take place of so many dead cells. The emerging field of tissue engineering and biomaterials has begun to provide promising alternatives or adjuncts to cellular cardiomyoplasty. Various injectable biomaterials have been studied for cardiac regeneration, including fibrin glue, collagen, alginate, matrigel, self-assembling peptides, extracellular matrix emulsion and synthetic polymer hydrogel or microspheres, with generally positive effects. An ideal biomaterial for cardiac regeneration should be biodegradable, biocompatible, cause little or no foreign body reaction, and provide both mechanical and functional support to the injured heart. AimThe study aimed to explore therapeutic potential of platelet gel for the treatment of rat with myocardial infarction. We would illustrate the function and mechanisms of platelet gels, and explore it how to reverse cardiac remodeling and improve the cardiac function after myocardial infarction by IHC, ELISA, ECHO, Masson stain etc. We hope to provide some new method for cardiovascular disease.MethodsPlatelet gel was derived from the vein blood of syngeneic rats and its structure morphology, biocompatibility with myocytes, secretion of beneficial factors, and degradation in post-MI hearts were characterized. Platelet gel or the same volume of control (vehicle) was injected intramyocardially into rat hearts with myocardial infarction. The animals were followed for three weeks and the treatment effects from the two groups were compared. We would observe the following phenomenan: characterization of platelet gel in vitro; degradation of injected platelet gel in infracted rat hearts; the number of cardiomyocyte, endothelial cell, de novo angiogenesis, apoptosis nuclei, c-Kit-positive stem cell, CD68-positive macrophages in the infarct at the7th day; myocyte morphology, capillary density at the third week; morphometry and cardiac function.ResultsResults in vitro1. In vivo, the Gel area was degraded with time.We found the area in the first day, the seventh day and fourteenth day is100%.41.5±7.89%and10.47±5.45%.2. ELISA revealed robust and sustained release of VEGF, IGF-1and HGF from the platelet gel (without cells) for at least14days3. In vitro, neonatal rat cardiomyocytes exhibited normal morphology, unaffected viability and function (beating) after14days of culture in the gel compared to TCP group(P>0.05). Results in vivo4. The areas of platelet gel could be readily identified with the detection of Texas Red fluorescence.The area at the1st day,7th day and14th day separately was100%、41.5±7.89%and10.47±5.45%.5. After delivery into infarcted rat hearts, the gel was efficiently infiltrated by cardiomyocytes and endothelial cells. As compared to control-injected hearts, gel-treated hearts exhibited greater number of cardiomyocytes (P<0.0001), endothelial cells (P<0.05) and c-Kit positive cells (P<0.01), but less apoptotic nuclei (P<0.01) and CD68-positive macrophages (P<0.01) in the infarct7days after treatment. All results meant platelet gel could enhance the recruitment of endogenous cardiomyocytes and endothelial cells and stem cells, penetrating into the gel in infarction area,and had the anti-inflammation ability.6. At the3week time point, the platelet gel-treated hearts exhibited higher capillary density (P<0.001) and less compensatory myocyte hypertrophy (P<0.05).7. Masson trichrome staining followed by quantitative morphometry revealed attenuated LV remodeling and less abnormal heart morphology in the gel-treated hearts, which exhibited thicker infarcted walls (P<0.05), smaller LV cavities (P<0.05) and shorter infarct perimeters (P<0.05). Echocardiography was performed at baseline (4hours after MI) and3weeks after injection of platelet gel and control. The cardiac function of control-injected animals deteriorated over the3-week time course, while that of platelet gel-injected animals did not. At3weeks, the gel-treated hearts exhibited greater LVEF than the control-injected hearts (P<0.05). This meant platelet gel could reverse the remodeling and improve the cardiac function after myocardial infarction.ConclusionsIntramyocardial injection of autologus platelet gel ameliorate cardiac dysfunction after myocardial infarction. The striking functional benefits, the simplicity of manufacturing and the potentially autologous nature of this biomaterial provide impetus for further translation. Part Ⅴ Cardiac regeneration from co-transplantation of cardiac-derived stem cells with blood-derived hydrogel in a rat model of myocardial infarctionBackgroundInjectable biomaterials with stem cell, such as bone marrow cells or adipose-derived stem cells or bone marrow-derived cardiac stem cells can produce functional preservation after myocardial infarction. Whereas many studies demonstrate that injectable biomaterials with or without stem cell have the same improvement on cardiac function.Good biomaterials plus good stem cells,which can promote each other,should have the more benefits.Over the last10years, people recognize the resident cardiac stem cell deeply. A good many researches show the cardiac stem cell’s ability of regenerating cardiomyocytes, endothelial cells and so on. Cardiosphere-derived cells exist in heart, and they are the mixture of cardiac stem cells and supporting cells. The recent study demonstate CDCs have a balanced profile of paracrine factor production and, among bone marrow-derived mesenchymal stem cell, adipose-derived mesenchymal stem cell and bone marrow mononuclear cells, provided the best function in mice with myocardial infarction. And our previous project showed that platelet gel alone attenuates adverse LV remodeling and preserves cardiac function in experimental myocardial infarction. The gel can improve the ability cardiomyocytes. So we guess the platelet gel with CDCs will have more therapeutic benefits.AimIn the present study, we focused on the interaction between platelet gel and CDCs, then we compared the therapeutic benefits of intramyocardial injection of platelet gel along and platelet gel plus CDCs in a rat model of acute myocardial infarction. We also exploited the mechanisms underlying the benefit increment from the cells. MethodsPlatelet gel and cardiosphere-derived cells (CDCs) were derived from the vein blood and heart biopsies of syngeneic rats, respectively. In vitro, the viability, growth, and morphology of CDCs cultured in platelet gel and NRCMs cultured in platelet gel with CDCs were characterized. Myocardial infarction rats were randomly divided into three groups:DMEM group, CDCs group and Gel+CDCs group. In vivo, Cardiac function was assessed by echocardiography. Masson’s trichrome staining was performed to detect morphometric parameters; IHC was used to examine the the number of cardiomyocyte, endothelial cell, the number of c-Kit cells.ResultsResults in vitro1. To compare the production of cytokines and growth factors from platelet gel with and without CDCs, ELISA revealed robust and sustained release of those factors from platelet gel with CDCs for at least14days,and they rigorously increased the production of those factors compared to Gel group(P<0.05).2. After14days of culture in the platelet gel, CDCs elongated cell body and a "blood-vessel-like" network structure were seen from the CDCs cultured in the platelet gel as compared to cells on TCP. The proliferation rates of CDCs cultured in the platelet gel was comparable to that from the control cells (P>0.05). Long axis/short axis of CDCs in platelet gel is more than it in TCP, Live/Dead assay revealed favorable viability (less EthD-positive dead cells) from the CDCs cultured in platelet gel (P<0.05). All of them meant platelet gel was good for cell’s proliferation and viability.3. To evaluate the in vitro degradation of the platelet gel, we measured the thickness of the gel over time. We found that nearly two thirds of the gel degraded after14days’ of culture under physiological condition.4. The differentiation capacity of CDCs cultured in the platelet gel was confirmed with the expression of cardiovascular-specific makers(α-SA for cardiomyocyte, vWF for endothelial cells and α-SMA for smooth muscle cells). 5. Better spreading of NRCMs was confirmed in platelet gel containing CDCs than in platelet gel alone. Quantitative cell morphology analysis revealed more elongated cell body in the Gel+CDC group (P<0.05). More spontaneously-beating NRCMs were found in platelet gel with CDCs (P<0.05). These compound results suggested that CDCs acted as "matrix softeners" to aid cardiomyocytes maintaining their normal morphology and functionality in the hydrogel.Results in vivo6. To confirm our findings in vitro, we examined the impact of CDCs on the recruitment of cardiomyocytes and endothelial cells to the injected gel in the injured heart. Quantitation revealed that more endothelial cells, cardiomyocytes and c-Kit+cells penetrated into the gel in the Gel+CDC group than in the Gel only group (P<0.05). Consistent with our findings in vitro, some cardiomyocytes and endothelial cells in the Gel+CDC group (Fig.4D; empty arrows) exhibited mature phenotype These data suggested that co-transplantation of CDCs with the platelet gel augmented the recruitment of endogenous stem cells and local cardiovascular cells into the gel matrix and might promote de novo angiogenesis and cardiomyogenesis on site.7. Quantitative morphometry at3weeks showed severe LV chamber dilatation and infarct wall thinning in the control (vehicle-injected) hearts. platelet gel with CDCs-treated hearts exhibited attenuated LV remodeling and less abnormal heart morphology, with smallest infarct size and thickest infarcted walls (P<0.05).8. Left ventricular ejection fraction (LVEF) at baseline did not differ among the three treatment groups, indicating a comparable degree of initial injury. Over the next3weeks, LVEF declined progressively in the control group, but not in the platelet gel-treated hearts (P<0.05).9. The quantitation suggested that direct regeneration played a minor role, as the CM-Dil+cells in the Gel+CDC group only account for28.7%and27.3%of the total increment of cardiomyocytes and capillaries, respectively, from the Gel only group. Conclusions1. CDCs can grow very well in platelet gel.2. Pre-seeding platelet gel with CDCs enhance the functional benefit of biomaterial therapy for treating myocardial infarction.3. The mixture of platelet gel and CDCs mainly increase the regeration of cardiomyocytes, endothelial cells and the density of capillary, stimulate the recruiment of endogenous stem cells,then reverse the ventricular remodeling, improve the cardiac function after myocardial infarction. |
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