Left ventricular heart aneurism--a new source of resident cardiac stem cells

In the past few years it has been established that the heart contains a reservoir of stem and progenitor cells that have the ability to differentiate in vitro and in vivo toward vascular and cardiac lineages and that show cardiac regeneration potential in vivo following injection into the infracted myocardium. The aim of the present study was to characterize cardiac stem cells in the tissue of chronic left ventricular aneurism. It was shown that human c-kit positive cells were scattered in fibrous, muscle and adipose parts of aneurism tissue. C-kit positive cells localized mainly in fibrous tissue nearby large vessels, however, c-kit positive cells did not express endothelial, smooth muscle or cardiomyocyte cell markers. Co-localization experiments demonstrated that all c-kit positive cells were of non-hematopoietic origin, since they did not express markers such as CD34 and CD45. Majority of c-kit positive cells expressed MDR1, but showed no proliferation activity (Ki67). It thus appears that aneurism tissue could be an alternative source of autologous cardiac stem cells. However, their regeneration capacity should be further explored.     

Stem cell‐mediated natural tissue engineering

Recently, we demonstrated that a fully differentiated tissue developed on a ventricular septal occluder that had been implanted due to infarct‐related septum rupture. We suggested that this tissue originated from circulating stem cells. The aim of the present study was to evaluate this hypothesis and to investigate the physiological differentiation and transdifferentiation potential of circulating stem cells. We developed an animal model in which a freely floating membrane was inserted into each the left ventricle and the descending aorta. Membranes were removed after pre‐specified intervals of 3 days, and 2, 6 and 12 weeks; the newly developed tissue was evaluated using quantitative RT‐PCR, immunohistochemistry and in situ hybridization. The contribution of stem cells was directly evaluated in another group of animals that were by treated with granulocyte macrophage colony‐stimulating factor (GM‐CSF) early after implantation. We demonstrated the time‐dependent generation of a fully differentiated tissue composed of fibroblasts, myofibroblasts, smooth muscle cells, endothelial cells and new blood vessels. Cells differentiated into early cardiomyocytes on membranes implanted in the left ventricles but not on those implanted in the aortas. Stem cell mobilization with GM‐CSF led to more rapid tissue growth and differentiation. The GM‐CSF effect on cell proliferation outlasted the treat ment period by several weeks. Circulating stem cells contributed to the development of a fully differentiated tissue on membranes placed within the left ventricle or descending aorta under physiological conditions. Early cardiomyocyte generation was identified only on membranes positioned within the left ventricle.     

Main-chain length control of conformation, membrane activity, and antibiotic properties of lipopeptaibol sequential analogues

To investigate the effect of backbone length and amphiphilicity on the 3D structure, membrane permeability, and antibacterial properties of trichogins, a subclass of lipopeptaibols, we prepared, by the segment condensation approach in solution and chemically characterized, a set of N(alpha)-1-octanoylated -X-(GLUG)(n)-I-L- ( X=G or U where U=Aib; n=1-4) sequential peptide esters. In parallel, the 12-mer (UGGL)(3) aneurism peptide, an analogue of the 11-mer sequential peptide (n=2) with an amino acid insertion was also synthesized and studied. By FT-IR absorption technique, we clearly showed that, in CDCl(3) solution, all peptides essentially populate intramolecularly H-bonded, helical conformations. Moreover, CD spectroscopy indicates that all peptides, with the single exception of the shortest oligomer (the heptamer), adopt mixed 3(10)-/alpha-helical structures, to an extent approximately correlating with main-chain length, in MeOH solution and sodium dodecylsulfate (SDS) micelles. Significant membrane permeability properties were found only for the three longest GLUG-based peptides, with the 15-mer oligomer (n=4) resulting the most active. The lack of activity exhibited by the aneurism peptide in this experiment strongly suggests a relevant role for the sequence amphiphilicity. In addition, antibacterial activity and selectivity were highlighted and demonstrated to be dependent on peptide main-chain length and amphiphilicity, in the sense that the two shortest GLUG-based homologues are active against Gram-positive strains, whereas the two longest homologues are able to penetrate the membranes of the Gram-negative strains, and the UGGL-based aneurism peptide is inactive.     

Severely damaged kidneys possess multipotent renoprotective stem cells

Background aimsTissue-specific stem cells are a promising target for kidney regeneration, because it has been shown that they play a primary role in kidney repair. Several methods have been developed for the isolation of stem/progenitor cells from healthy kidneys but the existence of these cells in chronically damaged kidneys has not been noticed so far.MethodsA mouse model of chronic kidney failure was developed by ligation of the left ureter for 5 months, and then isolation of stem cells from this tissue as well as normal kidneys was attempted.ResultsWe found that multipotent stem cells could be isolated from both types of tissue. In addition, the cells isolated from damaged kidneys showed potential for homing to the site of injury and a renoprotective effect in an animal model of cisplatin-induced nephropathy.ConclusionsThese results show that multipotent renoprotective stem cells exist in severely damaged kidneys, which could be a target for designing new therapies.     

人脐带间充质干细胞在急性肾小管坏死模型犬的体内分布及趋化性

目的 观察人脐带间充质干细胞在家犬急性肾小管坏死模型的体内分布及归巢.方法 健康家犬18 只随机分为3 组.模型1 组:肌注新鲜配制的0.2﹪二氯化汞溶液7 ml/kg建立急性肾小管坏死模型,采用经外周静脉注射法输注体外分离培养并用4',6- 二脒基-2- 苯基吲哚(DAPI)标记的人脐带间充质干细胞.模型2 组:造模...     

Neonatal mouse testis-derived multipotent germline stem cells improve the cardiac function of acute ischemic heart mouse model

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. We previously reported that undifferentiated mGS cells are phenotypically similar to embryonic stem cells and that fetal liver kinase 1 (Flk1)⁺ mGS cells have a similar potential to differentiate into cardiomyocytes and endothelial cells compared with Flk1⁺ embryonic stem cells. Here, we transplanted these Flk1⁺ mGS cells into an ischemic heart failure mouse model to evaluate the improvement in cardiac function. Significant increase in left ventricular wall thickness of the infarct area, left ventricular ejection fraction and left ventricular maximum systolic velocity was observed 4 weeks after when sorted Flk1⁺ mGS cells were transplanted directly into the hearts of the acute ischemic model mice. Although the number of cardiomyocytes derived from Flk1⁺ mGS cells were too small to account for the improvement in cardiac function but angiogenesis around ischemic area was enhanced in the Flk1⁺ mGS cells transplanted group than the control group and senescence was also remarkably diminished in the early phase of ischemia according to β-galactosidase staining assay. In conclusion, Flk1⁺ mGS cell transplantation can improve the cardiac function of ischemic hearts by promoting angiogenesis and by delaying host cell death via senescence.     

Regeneration of infarcted myocardium with resveratrol-modified cardiac stem cells

The major problem in stem cell therapy includes viability and engraftment efficacy of stem cells after transplantation. Indeed, the vast majority of host-transfused cells do not survive beyond 24-72 hrs. To increase the survival and engraftment of implanted cardiac stem cells in the host, we developed a technique of treating these cells with resveratrol, and tested it in a rat model of left anterior descending (LAD) occlusion. Multi-potent clonogenic cardiac stem cells isolated from rat heart and stably transfected with EGFP were pre-treated with 2.5 μM resveratrol for 60 min. Rats were anaesthetized, hearts opened and the LAD occluded to induce heart attack. One week later, the cardiac reduced environment was confirmed in resveratrol treated rat hearts by the enhanced expression of nuclear factor-E2-related factor-2 (Nrf2) and redox effector factor-1 (Ref-1). M-mode echocardiography after stem cell therapy, showed improvement in cardiac function (left ventricular ejection fraction, fractional shortening and cardiac output) in both, the treated and control group after 7 days, but only resveratrol-modified stem cell group revealed improvement in cardiac function at the end of 1, 2 and 4 months time. The improvement of cardiac function was accompanied by enhanced stem cell survival and engraftment as demonstrated by the expression of cell proliferation marker Ki67 and differentiation of stem cells towards the regeneration of the myocardium as demonstrated by the expression of EGFP up to 4 months after LAD occlusion in the resveratrol-treated stem cell group. Expression of stromal cell-derived factor and myosin conclusively demonstrated homing of stem cells in the infarcted myocardium, its regeneration leading to improvement of cardiac function.     

Passaging HuES human embryonic stem cell-lines with trypsin

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings. Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture. First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA. Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette. The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation. The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media. An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells. The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter. It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.     

Autologous stem cell transplantation for myocardial repair

Current therapies for heart failure due to transmural left ventricular (LV) infarction are limited. We have developed a novel patch method for delivering autologous bone marrow stem cells to sites of myocardial infarction for the purpose of improving LV function and preventing LV aneurysm formation. The patch consisted of a fibrin matrix seeded with autologous porcine mesenchymal stem cells labeled with lacZ. We applied this patch to a swine model of postinfarction LV remodeling. Myocardial infarction was produced by using a 60-min occlusion of the left anterior descending coronary artery distal to the first diagonal branch followed by reperfusion. Results were compared between eight pigs with stem cell patch transplantation, six pigs with the patch but no stem cells (P), and six pigs with left anterior descending coronary artery ligation alone (L). Magnetic resonance imaging data collected 19 +/- 1 days after the myocardial infarction indicated a significant increase of LV systolic wall thickening fraction in the infarct zone of transplanted hearts compared with P or L hearts. Blue X-gal staining was observed in the infarcted area of transplanted hearts. PCR amplification of specimens from the X-gal-positive area revealed the Ad5 RSV-lacZ vector fragment DNA sequence. Light microscopy demonstrated that transplanted cells had differentiated into cells with myocyte-like characteristics and a robust increase of neovascularization as evidenced by von Willebrand factor-positive angioblasts and capillaries in transplanted hearts. Thus this patch-based autologous stem cell procedure may serve as a therapeutic modality for myocardial repair.     

Red wine antioxidant resveratrol‐modified cardiac stem cells regenerate infarcted myocardium

To study the efficiency of maintaining the reduced tissue environment via pre-treatment with natural antioxidant resveratrol in stem cell therapy, we pre-treated male Sprague-Dawley rats with resveratrol (2.5 mg/kg/day gavaged for 2 weeks). After occlusion of the left anterior descending coronary artery (LAD), adult cardiac stem cells stably expressing EGFP were injected into the border zone of the myocardium. One week after the LAD occlusion, the cardiac reduced environment was confirmed in resveratrol-treated rat hearts by the enhanced expression of nuclear factor-E2-related factor-2 (Nrf2) and redox effector factor-1 (Ref-1). In concert, cardiac functional parameters (left ventricular ejection fraction and fractional shortening) were significantly improved. The improvement of cardiac function was accompanied by the enhanced stem cell survival and proliferation as demonstrated by the expression of cell proliferation marker Ki67 and differentiation of stem cells towards the regeneration of the myocardium as demonstrated by the enhanced expression of EGFP 28 days after LAD occlusion in the resveratrol-treated hearts. Our results demonstrate that resveratrol maintained a reduced tissue environment by overexpressing Nrf2 and Ref-1 in rats resulting in an enhancement of the cardiac regeneration of the adult cardiac stem cells as demonstrated by increased cell survival and differentiation leading to cardiac function.     

Bone marrow and bone marrow derived mononuclear stem cells therapy for the chronically ischemic myocardium

Bone marrow stem cells have been shown to differentiate into various phenotypes including cardiomyocytes, vascular endothelial cells and smooth muscle. Bone marrow stem cells are mobilized and home in to areas of injured myocardium where they are involved in tissue repair. In addition, bone marrow secretes multiple growth factors, which are essential for angiogenesis and arteriogenesis. In some patients, these processes are not enough to avert clinical symptoms of ischemic disease. Therefore, in vivo administration of an adequate number of stem cells would be a significant therapeutic advance. Unfractionated bone marrow derived mononuclear stem cells, which contain both hematopoietic and nonhematopoietic cells may be more appropriate for cell therapy. Studies in animal models suggest that implantation of different types of stem cells improve angiogenesis and arteriogenesis, tissue perfusion as well as left ventricular function. Several unanswered questions remain. For example, the optimal delivery approach, dosage and timing of the administration of cell therapy as well as durability of improvements need to be studied. Early clinical studies have demonstrated safety and feasibility of various cell therapies in ischemic disease. Randomized, double blind and placebo-controlled clinical trials need to be completed to determine the effectiveness of stem cell.     

Adropin-based dual treatment enhances the therapeutic potential of mesenchymal stem cells in rat myocardial infarction

Both weak survival ability of stem cells and hostile microenvironment are dual dilemma for cell therapy. Adropin, a bioactive substance, has been demonstrated to be cytoprotective. We therefore hypothesized that adropin may produce dual protective effects on the therapeutic potential of stem cells in myocardial infarction by employing an adropin-based dual treatment of promoting stem cell survival in vitro and modifying microenvironment in vivo. In the current study, adropin (25 ng/ml) in vitro reduced hydrogen peroxide-induced apoptosis in rat bone marrow mesenchymal stem cells (MSCs) and improved MSCs survival with increased phosphorylation of Akt and extracellular regulated protein kinases (ERK) l/2. Adropin-induced cytoprotection was blocked by the inhibitors of Akt and ERK1/2. The left main coronary artery of rats was ligated for 3 or 28 days to induce myocardial infarction. Bromodeoxyuridine (BrdU)-labeled MSCs, which were in vitro pretreated with adropin, were in vivo intramyocardially injected after ischemia, following an intravenous injection of 0.2 mg/kg adropin (dual treatment). Compared with MSCs transplantation alone, the dual treatment with adropin reported a higher level of interleukin-10, a lower level of tumor necrosis factor-α and interleukin-1β in plasma at day 3, and higher left ventricular ejection fraction and expression of paracrine factors at day 28, with less myocardial fibrosis and higher capillary density, and produced more surviving BrdU-positive cells at day 3 and 28. In conclusion, our data evidence that adropin-based dual treatment may enhance the therapeutic potential of MSCs to repair myocardium through paracrine mechanism via the pro-survival pathways.Subject terms: Cell death, Heart stem cells     

Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.     

Nonviral vector gene modification of stem cells for myocardial repair

Therapeutic angiogenesis and myogenesis restore perfusion of ischemic myocardium and improve left ventricular contractility. These therapeutic modalities must be considered as complementary rather than competing to exploit their advantages for optimal beneficial effects. The resistant nature of cardiomyocytes to gene transfection can be overcome by ex vivo delivery of therapeutic genes to the heart using genetically modified stem cells. This review article gives an overview of different vectors and delivery systems in general used for therapeutic gene delivery to the heart and provides a critical appreciation of the ex vivo gene delivery approach using genetically modified stem cells to achieve angiomyogenesis for the treatment of infarcted heart.     

Rediscovering pluripotency: from teratocarcinomas to embryonic stem cells. Cardiff, 10-12 October 2011

The pluripotent potential of embryonic stem cells has often seen them touted as the future of regenerative medicine. The road to any therapeutic success however, must stretch back to teratocarcinoma, the tumour from which pluripotent stem cells (embryonal carcinoma cells) were first derived. This 2011 meeting in Cardiff acted as a historical perspective from which the impact of embryonal carcinoma cell research on the present pluripotent stem cell landscape could be observed, with many of the early luminaries in this field still very active. The meeting addressed the genetic and epigenetic make-up of pluripotent stem cells, the mechanisms which control their fate, and their relationship to the early embryo proper. With each speaker tasked with revisiting previous questions, this meeting demonstrated how far has been travelled, yet how far is left to go.     

How do resident stem cells repair the damaged myocardium?

It has been a decade since the monumental discovery of resident stem cells in the mammalian heart, and the following studies witnessed the continuous turnover of cardiomyocytes and vascular cells, maintaining the homeostasis of the organ. Recently, the autologous administration of c-kit-positive cardiac stem cells in patients with ischemic heart failure has led to an incredible outcome; the left ventricular ejection fraction of the celltreated group improved from 30% at the baseline to 38% after one year and to 42% after two years of cell injection. The potential underlying mechanisms, before and after cell infusion, are explored and discussed in this article. Some of them are related to the intrinsic property of the resident stem cells, such as direct differentiation, paracrine action, and immunomodulatory function, whereas others involve environmental factors, leading to cellular reverse remodeling and to the natural selection of "juvenile" cells. It has now been demonstrated that cardiac stem cells for therapeutic purposes can be prepared from tiny biopsied specimens of the failing heart as well as from frozen tissues, which may remarkably expand the repertoire of the strategy against various cardiovascular disorders, including non-ischemic cardiomyopathy and congenital heart diseases. Further translational investigations are needed to explore these possibilities.     

Clinical trials using autologous bone marrow and peripheral blood-derived progenitor cells in patients with acute myocardial infarction

This paper discusses the current data concerning the results of major clinical trials using bone marrow-derived and peripheral blood-derived stem/progenitor cells in treatment of patients with acute myocardial infarction (AMI) and depressed left ventricular ejection fraction. In all major trials (TOPCARE-AMI, BOOST), the primary outcome measure was increase in left ventricular systolic function (LVEF) and left ventricle remodeling. The most consistent finding is the significant increase in LVEF. Some trials suggest also reduction of left ventricular remodeling. Although the absolute LVEF increase is small (6-9%), it may substantially contribute to the improvement of global LV contractility. None of the studies in AMI patients treated with intracoronary infusion of progenitor cells revealed excess risk of arrythmia, restenosis or other adverse effects attributable to the therapy. The exact mechanism of improved myocardial contractile function remains unknown, however, there are several possible explanations: therapeutic angiogenesis improving the blood supply to the infarct border zone, paracrine modulation of myocardial fibrosis and remodeling (e.g. inhibition of myocyte apoptosis) and transdifferentiation of stem/progenitor cells into functional cardiomyocytes. No study showed the superiority of the particular subpopulation of autologous progenitor cells in terms of left ventricular function improvement in AMI. In fact, most of the clinical trials used the whole population of mononuclear bone marrow-derived progenitor cells, peripheral blood derived progenitor cells (endothelial progenitors).     

Physical model of water in a split-root system

Summary A physical model, based on Darcy's law and an Ohm's-law analogy, was developed to show that water can move from a wetter side of a root system to a drier side or vice versa. In the model, a wick in the form of an inverted Y was used, with the two ends of the Y in separate beakers and the third end (stem) of the Y extending into the air. The left root, right root, and stem were about 6,5, and 4 cm long, respectively. The difference in total head (potential) between the left root and the right root was varied for different potentials applied to the stem. Experiments were done either in a darkened laboratory or with a sunlamp shining on the stem. The stem was thus exposed to low-evaporation (in the dark) or high-evaporation (with the sunlamp) conditions. Total heads (sum of head due to gravity and head due to pressure-other heads were negligible) and flows of water were calculated or measured for each part of the split-root system (left root, right root, crown, stem). The results showed that the direction and quantity of water flowing in each part of the system depended upon the total head for the stem, crown, and each half of the root (the flow could be up, down, left, or right), and that the gravity component of the total head was important in moving water down the plant when light intensity was low.     

Study of the impact of intramyocardial implantation of stem cells on myocardial perfusion and contractility in patients with coronary heart disease concurrent with postinfarct cardiosclerosis and chronic heart failure

OBJECTIVE: to study of intramyocardial implantation of cultured bone marrow stem cells on myocardial perfusion and contractility in the surgical treatment of patients with coronary heart disease (CHD) and chronic heart failure (CHF), by synchronized single-photon emission computed tomography (SSPECT) of the myocardium. SUBJECTS AND METHODS: The study included 11 patients. Intramyocardial injection of cell injections into the myocardial periscarring areas was made at coronary bypass surgery. All the patients underwent 99mTc myocardial SSPECT MIBI before and 3, 6, 12 months after surgery. RESULTS AND CONCLUSIONS: Implantation of bone marrow stem cells into the left ventricular myocardium favorably affects left ventricular remodeling and contributes to the improvement of myocardial perfusion and contractility, as evidenced by 99mTc.     

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.