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Stem cell therapy for ischemic heart disease: Where are we?
Abstract
Stem cell transplantation is currently generating a great deal of interest in the treatment of ischemic heart disease (IHD) as the replacement of akinetic scar tissue by viable myocardium should improve cardiac function, impede progressive left ventricular remodeling, and revascularize ischemic areas. Substantial work in stem cell therapy for ischemic heart disease has recently been reported.
Stem cell populations have been expanding. Most recently, induced pluripotent stem (iPS) cells have been discovered that have the potential to revolutionize stem cell therapy. Many of the efforts in stem cell therapy for ischemic heart disease have been inconclusive and often contradicting. Transdifferentiation of stem cells into cardiomyocytes remains controversial. The therapeutic effect of the stem cell seems consistent with paracrine function rather than transdifferentiation. Systemic and micromilieu factors appear to dictate the fate of implanted stem cells.
Although animal studies produce controversial results, and many basic questions remain unanswered, more and more clinical trials are underway. Consequently, researchers must begin to focus upon a few basic critical issues: the modulation of the systemic and microenvironment for stem cells in order to augment stem cell survival and transdifferentiation; the underlying mechanisms of stem cell therapy and the fate of stem cells; differentiation into myocytes or other terminal cell populations with favorable paracrine functions.
... The heart is one of the least regenerative organs and irreversible damage to a large amount of cardiomyocytes may result in heart failure. There have been numerous works on cardiac stem cell therapy for the regeneration of damaged myocardium, however the results have been controversial [1,2]. Various types of stem cells originating from both embryonic and adult human tissue sources were used for cardiac cell regeneration therapy. ...
... For example, stem cell populations such as human embryonic stem cells (hESCs), cardiac progenitor cells (CPCs), bone marrow-derived stem cells, tissue specific stem cells, and induced pluripotent stem cells have been studied for cardiac repair [1]. Each cell type had its own strengths and weaknesses and no single cell type has been proven to meet the criteria for clinical applications. ...
Background:
Anesthetic preconditioning can improve survival of cardiac progenitor cells exposed to oxidative stress. We investigated the role of protein kinase C and isoform protein kinase C-ε in isoflurane-induced preconditioning of cardiac progenitor cells exposed to oxidative stress.
Methods:
Cardiac progenitor cells were obtained from undifferentiated human embryonic stem cells. Immunostaining with anti-Nkx2.5 was used to confirm the differentiated cardiac progenitor cells. Oxidative stress was induced by H2O2 and FeSO4. For anesthetic preconditioning, cardiac progenitor cells were exposed to 0.25, 0.5, and 1.0 mM of isoflurane. PMA and chelerythrine were used for protein kinase C activation and inhibition, while εψRACK and εV1-2 were used for protein kinase C -ε activation and inhibition, respectively.
Results:
Isoflurane-preconditioning decreased the death rate of Cardiac progenitor cells exposed to oxidative stress (death rates isoflurane 0.5 mM 12.7 ± 9.3 %, 1.0 mM 12.0 ± 7.7 % vs. control 31.4 ± 10.2 %). Inhibitors of both protein kinase C and protein kinase C -ε abolished the preconditioning effect of isoflurane 0.5 mM (death rates 27.6 ± 13.5 % and 25.9 ± 8.7 % respectively), and activators of both protein kinase C and protein kinase C - ε had protective effects from oxidative stress (death rates 16.0 ± 3.2 % and 10.6 ± 3.8 % respectively).
Conclusions:
Both PKC and PKC-ε are involved in isoflurane-induced preconditioning of human embryonic stem cells -derived Nkx2.5(+) Cardiac progenitor cells under oxidative stress.
... In general, a shortage of reliable information on the survival, migration, and differentiation of SCs is pointed out (Li et al., 2010). In more fundamental studies, alternative mechanisms of cellular therapy are discussed, i.e., immunomodulating (Uccelli et al., 2008; Tyndall, Gratwohl, 2009), paracrine (LaPar et al., 2009), etc. The principal concepts vary correspondingly; in commercially oriented publications, the central topic is the replacement of damaged cells by SCs or their progeny, rejuvenation and remodeling of tissues, reconstruction of the brain (Borisov 2006), softening of myocardial scars and their involvement in cardiac contractions (Borisov 2006), etc. Conversely, in articles of a higher scientific level, a sometimes questionable therapeutic effect is explained by paracrine or immunomodulating mechanisms (Joggerst, Hatzopoulos, 2009), a stimulating action of the products of cell disintegration (Parlyuk et al., 2009), etc. ...
... The benefit from such vascularization, if it really occurs, is doubtful because ischemia is usually caused by the obstruction of larger vessels situated in the epicardium. Accordingly, ischemia can be alleviated by functioning collaterals, but not by a locally enhanced microcirculation (), more discussion is in the Chapter 7. The therapeutic effect of SC is also explained by their paracrine function and an activation of precursor cells in the microenvironment (LaPar et al., 2009; Mishra, 2008). However, as discussed above, there is no reason to assume a selective ability of SCs to influence their microenvironment through the paracrine or another mechanism. ...
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... Nevertheless, the discovery of stem cells within the mammalian heart has renewed the interest for cardiac regeneration, based on the idea that recruiting resident cells inherently programmed to reconstitute the damaged myocardium may achieve better results than forcing extra-cardiac stem cells to differentiate into contractile myocytes [10,11]. Indeed, experimental models and clinical trials performed using different exogenous stem cell lineages transplanted into the post-ischemic heart have only demonstrated a modest cell engraftment and myocardial regenerative capacities, being the observed beneficial effects mainly attributable to the paracrine improvement of tissue remodeling and preservation of the residual myocardium by the engrafted cells [12][13][14][15][16]. ...
... Preclinical studies on stem cell therapy for cardiac regeneration have reported significant improvements of ventricular pump function, ventricular remodeling, and myocardial perfusion after MSC transplantation in the post-ischemic heart [39,40]. Several lines of evidence suggest that the therapeutic potential of these cells in myocardial repair is related to their ability to improve myocardial function establishing paracrine interactions with the resident cardiac cells [14,35]. Once at the site of injury, in fact, the implanted cells secrete bioactive factors that promote neo-angiogenesis, inhibit apoptosis and decrease the stiffness of the scarred ventricular wall, thus acting as an active graft which dynamically contributes to improve the myocardial performance [41,42]. ...
The possibility to induce myocardial regeneration by the activation of resident cardiac stem cells (CSCs) has raised great interest. However, to propose endogenous CSCs as therapeutic options, a better understanding of the complex mechanisms controlling heart morphogenesis is needed, including the cellular and molecular interactions that cardiomyocyte precursors establish with cells of the stromal compartment. In the present study, we co-cultured immature cardiomyocytes from neonatal mouse hearts with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte growth in vitro. We found that cardiomyocyte proliferation was enhanced by direct co-culture with MSCs compared with the single cultures. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch-1 receptor by its ligand Jagged-1 expressed by the adjacent MSCs. In fact, the cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notch-intracellular domain (Notch-ICD) as compared with those cultured alone and this response was significantly attenuated when MSCs were silenced for Jagged-1. The presence of various cardiotropic cytokines and growth factors in the conditioned medium of MSCs underscored the contribution of paracrine mechanisms to Notch-1 up-regulation by the cardiomyocytes. In conclusions these findings unveil a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation through Notch-1/Jagged-1 pathway and suggest that stromal-myocardial cell juxtacrine and paracrine interactions may contribute to the development of new and more efficient cell-based myocardial repair strategies.
... iPS cell based cell replacement therapy is currently generating a great deal of interest in the treatment of ischemic heart diseases since iPS cells are capable of differentiating into patient-specific functional cardiomyocytes. The replacement of akinetic scar tissue by viable myocardium should improve cardiac function, impede progressive left ventricular remodeling, and revascularize ischemic areas [32]. But still, much has to be studied about the formation of electrical coupling between endogenous and transplanted cardiomyocytes. ...
... Theoretically fibroblasts which can be easily obtained from the patient's skin could be converted into cardiomyocytes without passing through the stage of pluripotency. A successful example for transdifferentiation is the conversion of mouse B lymphocytes to macrophages by the transcription factor CEBP [32]. More recently, exocrine pancreas cells were converted into endocrine b-cells in mice in vivo using three pancreatic transcription factors [33]. ...
Human induced pluripotent stem (iPS) cells hold great promise for therapy of a number of degenerative diseases such as ischemic heart failure, Parkinson's disease, Alzheimer's disease, diabetes mellitus, sickle cell anemia and Huntington disease. They also have the potential to accelerate drug discovery in 3 ways. The first involves the delineation of chemical components for efficient reprogramming of patient's blood cells or cells from biopsies, obviating the need for cellular delivery of reprogramming exogenous transgenes, thereby converting hope into reality for patients suffering from degenerative diseases. Patients worldwide stand to benefit from the clinical applicability of iPS cell-based cell replacement therapy for a number of degenerative diseases. The second is the potential for discovering novel drugs in a high throughput manner using patient-specific iPS cell-derived somatic cells possessing the etiology of the specific disease. The third is their suitability for toxicological testing of drugs and environmental factors. This review focuses on these potential applications of iPS cells with special emphasis on recent updates of iPS cell research contributing to the accelerated drug discovery.
... Atualmente uma perspectiva com possibilidade para tratamento de doenças crônicodegenerativas são as células-tronco. Vários tipos de células-tronco estão em investigação e o potencial terapêutico está presente nas células embrionárias do cordão umbilical e na medula óssea, denominadas células-tronco adultas [17,18]. ...
ObjetivoAvaliar a percepção da qualidade de vida de 60 pacientes com cardiopatia chagásica que participaram do Estudo Multicêntrico Randomizado de Terapia Celular em Cardiopatia no Hospital das Clínicas da Universidade Federal de Goiás.MétodosTrata-se de uma pesquisa de coorte retrospectivo de um estudo transversal anterior. Os participantes foram escolhidos randomicamente e foi utilizado o desenho duplo-cego para quem recebeu células tronco grupo experimental ou não recebeu grupo controle. Os dados foram retirados dos prontuários de 60 pacientes que responderam o questionário sócio demográfico, o Minnesota Living with Heart Failure Questionnaire, a classificação funcional e o teste de caminhada de seis minutos, no tempo basal, 2, 6 e 12 meses de segmento. Foram analisados os dados referentes a qualidade de vida, meio dos testes estatísticos descritivos (porcentagem, média, desvio-padrão) e comparativos (teste t de Student e de correlação de Pearson, para p 0,05). ResultadosForam incluídos os 60 pacientes chagásicos, a maioria do sexo masculino (70%), idade média 50,98 anos. O grupo que recebeu células-tronco e o grupo que não recebeu permaneceu semelhante, com boa qualidade de vida durante o desenvolvimento da pesquisa. Observa-se que, dois meses após o procedimento houve melhora da qualidade de vida em ambos os grupos, quando comparada aos demais tempos. A correlação da classificação funcional e do teste de caminhada com a qualidade de vida mostrou que quanto melhor a condição clínica do participante, melhor era o escore de qualidade de vida. ConclusãoNão houve alteração na qualidade de vida dos participantes do grupo experimental em comparação ao grupo controle. Assim, a qualidade de vida dos participantes que receberam células-tronco não foi impactada.
... Ischemic cardiomyopathy (ICM) involving damage to cardiac structure and function induced by irreversible myocardial cell necrosis is a leading cause of morbidity and mortality worldwide as a result of modern lifestyle (1). The currently available primary treatments include drug interventions, percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG) and heart transplantation (2). ...
In the present study, subcutaneous fat was obtained from adult women that had undergone conventional liposuction surgery. A comparative study was performed to investigate the effect of transparent and white poly‑β‑hydroxyethyl methacrylate (PHEMA) stents, which have different surface and cross‑sectional morphological characteristics, on the differentiation of adipose‑derived stem cells (ASCs) into myocardial cells. The cell counting kit‑8 assay revealed that cell growth increased at varying rates among the different treatment groups. The absorbance of the experimental transparent PHEMA treated group increased in a time‑dependent manner with the duration of incubation. The highest levels of proliferation were observed in the transparent PHEMA group. In addition, the transparent PHEMA treated group exhibited the strongest cell adhesion ability, which was significantly different to that of the white PHEMA group (P<0.01 and P<0.05 for Matrigel and fibronectin assay, respectively). Comparisons between the two stent materials with the inducer control group revealed statistically significant differences in the rate of ASC differentiation (P<0.05). The level of differentiation was the greatest in the transparent PHEMA group, and was significantly different to the white PHEMA group (P<0.05) and the blank control group (P<0.01). The results suggest that the inducers 5-aza-2-deoxycytidin and laminin, and material microstructure stents effectively promote the proliferation, growth and adhesion of ASCs. However, the transparent material microstructure may be a more suitable candidate for ASC‑associated injections. The present study provides further evidence that a PHEMA stent structure, comprised of a high number of matrixes and a low water content, induces a high level of ASC differentiation to myocardial cells.
... Due to their advantages in various fields, ASCs have a strong potential for application in future stem cell treatments and are beneficial for allogenic transplantation treatments. ASCs may emerge as the ideal seed stem cells in cell transplantation and tissue engineering clinical treatments (2)(3)(4)(5). ...
The aim of the present study was to improve methods for the isolation and identification of adipose-derived stem cells (ASCs). Human subcutaneous adipose tissue was collected during liposuction surgery, without ultrasound-assisted liposuction and other assisted techniques, and digested with 0.075% collagenase I. First (P1) and second (P2) passage ASCs were applied to the subsequent experiments. ASCs were observed under a microscope, the growth curves of the cells were assessed using a cell counting kit-8 assay and the membrane expression of cell surface antigens, including cluster of differentiation (CD)44, CD105 and CD45, were detected by flow cytometry. In addition, ASCs were induced to differentiate into lipocytes and osteocytes. Oil red staining was applied to examine adipogenic induction, whereas alkaline phosphatase (ALP) staining was used to assess osteogenic induction. Primary ASCs adhered to the culture vessel wall after 72 h, were fusiform in appearance at 5 days and exhibited stable growth with active proliferation. In total, 1x10⁵ stem cells were gained per 50 ml of lipo-aspirate. ASCs were plated in a 25 cm² culture flask at a density of 5x10⁴/ml; the cells underwent the first logarithmic growth period after 72 h and grew to 90% confluence within 3 days. Flow cytometry demonstrated that the cells were highly positive for CD105 and CD44, and weakly positive for CD45; 18.6% of P1 cells and 90.7% of P2 cells were CD44⁺CD45⁻CD105⁺. Oil red and ALP staining were positive. The results of the present study suggested that ASCs may be considered a promising cell type for tissue engineering. Furthermore, the present study established an effective method for the isolation and identification of ASCs, which reduced damage to the stem cells and simplified the identification procedure.
... , , , (Mishra, 2008;LaPar et al., 2009). , , ...
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... The numbers of proliferating myocytes were also increased at both 1 and 4 weeks after MI, as assessed by Ki67 [25,26]. Myocyte regeneration and proliferation are controversial topics [27][28][29][30][31][32]. On one hand several investigators report that this mechanism is important to repairing the infarcted heart [33][34][35][36][37][38]. ...
Promoting angiogenesis is a key therapeutic target for protection from chronic ischemic cardiac injury. Endothelial-Monocyte-Activating-Polypeptide-II (EMAP II) protein, a tumor-derived cytokine having anti-angiogenic properties in cancer, is markedly elevated following myocardial ischemia. We examined whether neutralization of EMAP II induces angiogenesis and has beneficial effects on myocardial function and structure after chronic myocardial infarction (MI). EMAP II antibody (EMAP II AB), vehicle, or non-specific IgG (IgG) was injected ip at 30min and 3, 6, and 9days after permanent coronary artery occlusion in mice. EMAP II AB, compared with vehicle or non-specific antibody, significantly, p<0.05, improved the survival rate after MI, reduced scar size and attenuated the development of heart failure, i.e., left ventricular ejection fraction was significantly higher in EMAP II AB group, fibrosis was reduced by 24%, and importantly, more myocytes were alive in EMAP II AB group in the infarct area. In support of an angiogenic mechanism, capillary density (193/HPF vs. 172/HPF), doubling of the number of proliferating endothelial cells, and angiogenesis related biomarkers were upregulated in mice receiving EMAP II AB treatment as compared to IgG. Furthermore, EMAP II AB prevented EMAP II protein inhibition of in vitro tube formation in HUVECs. We conclude that blockade of EMAP II induces angiogenesis and improves cardiac function following chronic MI, resulting in reduced myocardial fibrosis and scar formation and increased capillary density and preserved viable myocytes in the infarct area.
Copyright © 2014. Published by Elsevier Ltd.
... It is of great interest to devise novel treatment options for those IHD patients who do not respond sufficiently to conventional care. One relatively new approach is the use of stem cell therapy with the potential to support regeneration of the damaged myocardium [3,4]. ...
Adipose-derived stromal cells (ASCs) stimulated with vascular endothelial growth factor (VEGF) and serum-deprived, are applied in the first in-man double-blind placebo-controlled MyStromalCell Trial, as a novel therapeutic option for treatment of ischemic heart disease (IHD). This in vitro study explored the effect of VEGF and serum deprivation on endothelial differentiation capacity of ASCs from healthy donors and IHD patients.
ASCs stimulated with rhVEGFA165 in serum-deprived medium for one to three weeks were compared with ASCs in serum-deprived (2% fetal bovine serum) or complete medium (10% fetal bovine serum). Expression of VEGF receptors, endothelial and stem cell markers was measured using qPCR, flow cytometry and immunocytochemistry. In vitro tube formation and proliferation was also measured.
ASCs from VEGF-stimulated and serum-deprived medium significantly increased transcription of transcription factor FOXF1, endothelial marker vWF and receptor VEGFR1 compared with ASCs from complete medium. ASCs maintained stem cell characteristics in all conditions. Tube formation of ASCs occurred in VEGF-stimulated and serum-deprived medium. The only difference between healthy and patient ASCs was a variation in proliferation rate.
ASCs from IHD patients and healthy donors proved equally inclined to differentiate in endothelial direction by serum-deprivation, however with no visible additive effect of VEGF stimulation. The treatment did not result in complete endothelial differentiation, but priming towards endothelial lineage.
... Accord ingly, ischemia can be alleviated by functioning collat erals, but not by locally enhanced microcirculation (Schaper, Buschmann, 1999;Nagy et al., 2003). The therapeutic action of SCs is also explained by their paracrine function and the activation of precursor cells in the microenvironment (LaPar et al., 2009;Mishra, 2008). However, as discussed above, there is no reason to assume the selective ability of SCs to influence their microenvironment by means of a para crine or another mechanism. ...
The title was changed by the editors (in the original it was "On the eve of scientific approach"). The copyediting performed by the editors made the text awkward in some places.
ABSTRACT:
The stem cells and cell therapies have become a popular last time. The theme is cluttered with publications of questionable reliability. Not all stem cell therapies applied in practice are based on reliable research. In the abundant literature, there is a gap between supposed healing properties of stem cells associated with their capability to migrate to injured tissues, and lack of clear morphological evidence thereof. Accordingly, there is a gap between advertising and the better part of professional literature: the former speaks about rejuvenation of tissues, and the latter explains the questionable therapeutic effects by paracrine or immunomodulating mechanisms, secretion of cytokines and growth factors. However, stem cells are undifferentiated, and a specific and efficient paracrine function can hardly be awaited from them compared to other, more differentiated cells.
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... Each transplant route attempts to maximize not only the ease of administration, but the targeted homing, retention, stem cell engraftment to damaged myocardium and improved cardiac function. 28 The therapeutic effect(s) of MSCs relies on their capacity to engraft and survive in distinctive target tissue, and poor stem cell survival is responsible for unsatisfactory cell transplantation prognosis post MI. 29 Compared to IV infusion, cell delivery by IM injection increased the engraftment and survival of MSCs in infarcted hearts. These results might explain why cell delivery by the IM route could ameliorate the cardiac function and infarct size post MI, rather than by IV infusion. ...
Purpose
Results for implantation efficiency and effective improvement of cardiac function in the field of mesenchymal stem cells (MSCs) are controversial. To attempt to clarify this debate, we utilized magnetic resonance imaging (MRI) and near-infrared optical imaging (OI) to explore the effects of different delivery modes of mesenchymal stem cells on cell retention time and cardiac function after myocardial infarction (MI).
Methods
Rat MSCs were labeled with superparamagnetic iron oxide nanoparticles and 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) for noninvasive cell tracking in a rat MI model. Rats underwent coronary artery ligation and were randomized into three experimental groups: intravenous (IV), intramyocardial (IM), and a control group. The first two groups referred to the route of delivery of the transplanted dual-labeled MSCs; whereas the control group was given an IV injection of serum-free medium one day post-MI. Cellular engraftment was determined 1 day and 7 days post cell delivery by measuring the iron and optical signals in explanted organs. Prussian blue staining and fluorescent microscopy were performed on histological sections for iron and DiD, respectively. Cardiac function was measured by echocardiography on day 7.
Results
The cardiac function of the IM group increased significantly compared to the IV and control groups at day 7. In the IM group, labeled cells were visualized in the infracted heart by serial MRI, and the intensity by OI was significantly higher on day 1. In the IV group, the heart signals were significantly attenuated by dual-modal tracking at two time points, but the lung signals in OI were significantly stronger than the IM group at both time points.
Conclusion
IM injection of MSCs increased cell engraftment within infarcted hearts and improved cardiac function after MI. However, IV infusion has a low efficacy due to the cell trapping in the lung. Therefore, direct injection may provide an advantage over IV, with regard to retention of stem cells and protection of cardiac function.
... I n recent years, cell therapy has emerged as a strategy for improving contractility of the diseased heart (1,2). Restorative therapies consider the use of exogenous multipotent stem cells capable of differentiating into cardiomyocytes. ...
Arrhythmia is of concern after cardiac stem cell transplantation in repairing infarcted myocardium. However, whether transplantation improved the ventricular fibrillation threshold and whether severe malignant ventricular arrhythmia is induced in the myocardial infarction model are still unclear. We sought to investigate the electrophysiologic characteristics and ventricular fibrillation threshold in rats with myocardial infarction by treatment with allogeneic cardiac stem cells.
Prospective, randomized, controlled study.
University-affiliated hospital.
Male Sprague-Dawley rats.
Myocardial infarction was induced in 20 male Sprague-Dawley rats. Two weeks later, animals were randomized to receive 5 × 10(6) cardiac stem cells labeled with PKH26 in phosphate buffer solution or a phosphate buffer solution-alone injection into the infarcted anterior ventricular-free wall.
Six weeks after the cardiac stem cell or phosphate buffer solution injection, electrophysiologic characteristics and ventricular fibrillation threshold were measured at the infarct area, infarct marginal zone, and noninfarct zone. Labeled cardiac stem cells were observed in 5-μm cryostat sections from each harvested heart. The unipolar electrogram activation recovery time dispersions were shorter in the cardiac stem cell group compared with those at the phosphate buffer solution group (15.5 ± 4.4 vs. 38.6 ± 14.9 msecs, p = .000177). Malignant ventricular arrhythmias were significantly (p = .00108) less inducible in the cardiac stem cell group (one of ten) than the phosphate buffer solution group (nine of ten). The ventricular fibrillation thresholds were greatly improved in the cardiac stem cell group compared with the phosphate buffer solution group. Labeled cardiac stem cells were identified in the infarct zone and infarct marginal zone and expressed Connexin-43, von Willebrand factor, α-smooth muscle actin, and α-sarcomeric actin.
Cardiac stem cells may modulate the electrophysiologic abnormality and improve the ventricular fibrillation threshold in rats with myocardial infarction treated with allogeneic cardiac stem cells and cardiac stem cell express markers that suggest muscle, endothelium, and vascular smooth muscle phenotypes in vivo.
The role of stem cells in augmenting reparative processes in the heart after ischemic injury has been successfully demonstrated in small and large animal models. However, the outcomes of cell therapy in clinical trials have been somewhat variable, with overall effects of autologous stem cell therapies demonstrating a modest improvement in cardiac structure and function. How stem cells repair the heart after cardiac injury is still not well understood. Most recent studies suggest that adult derived stem cells act primarily through paracrine signaling to exert beneficial effects, including modulation of immune response, stimulation of new blood vessel formation or by inducing mature myocytes to transiently reenter the cell cycle, rather than robust direct differentiation of the transplanted cells into myocytes. Additionally, data from multiple labs confirmed clearance of stem cells themselves within a few days still leading to functional benefits further confirming the role of paracrine signaling in augmenting cardiac reparative processes rather than direct differentiation of cells. These findings rapidly evolved the field of extracellular vesicles specifically microvesicles as they are active hubs of autocrine, paracrine and endocrine signaling targeting different biological processes. The beneficial effects seen after stem cell transplantation could be linked to the cardioprotective factors packaged in the microvesicles secreted from stem cells. Therefore, stem cell microvesicles provide a new avenue for the treatment of cardiovascular disease through a multitude of mechanisms including cellular communication within the stem cell niches, delivery of genetic information, regulation of the immune system in the heart, and stimulation of angiogenesis which will be discussed in this review.
Cell therapy has the potential to improve healing of ischemic heart, repopulate injured myocardium and restore cardiac function. The tremendous hope and potential of stem cell therapy is well understood, yet recent trials involving cell therapy for cardiovascular diseases have yielded mixed results with inconsistent data thereby readdressing controversies and unresolved questions regarding stem cell efficacy for ischemic cardiac disease treatment. These controversies are believed to arise by the lack of uniformity of the clinical trial methodologies, uncertainty regarding the underlying reparative mechanisms of stem cells, questions concerning the most appropriate cell population to use, the proper delivery method and timing in relation to the moment of infarction, as well as the poor stem cell survival and engraftment especially in a diseased microenvironment which is collectively acknowledged as a major hindrance to any form of cell therapy. Indeed, the microenvironment of the failing heart exhibits pathological hypoxic, oxidative and inflammatory stressors impairing the survival of transplanted cells. Therefore, in order to observe any significant therapeutic benefit there is a need to increase resilience of stem cells to death in the transplant microenvironment while preserving or better yet improving their reparative functionality. Although stem cell differentiation into cardiomyocytes has been observed in some instance, the prevailing reparative benefits are afforded through paracrine mechanisms that promote angiogenesis, cell survival, transdifferentiate host cells and modulate immune responses. Therefore, to maximize their reparative functionality, ex vivo manipulation of stem cells through physical, genetic and pharmacological means have shown promise to enable cells to thrive in the post-ischemic transplant microenvironment. In the present work, we will overview the current status of stem cell therapy for ischemic heart disease, discuss the most recurring cell populations employed, the mechanisms by which stem cells deliver a therapeutic benefit and strategies that have been used to optimize and increase survival and functionality of stem cells including ex vivo preconditioning with drugs and a novel “pharmaco-optimizer” as well as genetic modifications.
The heart failure is becoming a growing problem in public health and a leading cause of hospitalization worldwide, thus generating a large economic impact on society, implying in terms of mortality and quality of life. Even with today's technological advances, mortality may exceed 50% in five years. The impairment of cardiac function, high rate of complications and reduced life expectancy is a challenge due to the numerous health problems consecutive aging.
Background: Whether transplanted cardiac stem cells (CSCs) can improve ventricular fibrillation threshold (VFT) and whether severe malignant ventricular arrhythmia is induced in the myocardial infarction model are still unclear. Objective: To clarity the electrophysiological characteristics and VFT in rats with myocardial infarction by treatment with allogenic CSCs. Methods: Left anterior descending coronary arteries of SD rats were ligated to induce myocardial infarction. Two weeks later, animals were randomized to receive 5×106 CSCs labeled with PKH26 in phosphate buffer solution (PBS) or PBS alone as a placebo by injection into the ischemic zone in the anterior ventricular free wall. Six weeks after the CSCs or PBS injection, electrophysiological characteristics and VFT were measured at the infarct area. Labeled CSCs were observed in 5 μm cryostat sections from each harvested heart. Results and Conclusion: Compared with the PBS group, the unipolar electrograms activation recovery time dispersions were shorter in the CSCs group, malignant ventricular arrhythmias were significantly less inducible in the CSCs group, the VFTs were greatly improved in the CSCs group. Labeled CSCs were identified in the infarct marginal zone and expressed Connexin-43 and α-sarcomeric actin. CSCs can modulate the electrophysiological abnormality and improve the VFT in rats with myocardial infarction treated with allogenic CSCs and CSCs express muscle and connexin markers in vivo.
Myocardial infarction (MI) leads to the loss of cardiomyocytes followed by left ventricular (LV) remodeling and cardiac dysfunction. We hypothesize that an elastic, biodegradable nanofibrous cardiac patch loaded with mesenchymal stem cells (MSCs) could restrain LV remodeling and improve cardiac function after MI. Poly(ε-caprolactone) (PCL)/gelatin (PG) nanofibers were fabricated by electrospinning and the nanofibers displayed porous and uniform nanofibrous structure with a diameter of 244 ± 51 nm. An MI model was established by ligation of the left anterior descending coronary artery (LADCA) of female Sprague-Dawley rats. The PG nanofibrous patch seeded with MSCs, isolated from rat bone marrow was further implanted on the epicardium of the infarcted region of the LV wall of the heart. After transplantation, the PG-cell patch restricted the expansion of the LV wall effectively, reduced the scar size and the density of the microvessels increased. Cells within the patch were able to migrate towards the scar tissue, and promoted in new blood vessel formation at the infarct site. Angiogenesis and the cardiac functions improved significantly after four weeks of implantation. The MSC-seeded PG nanofibrous patches are demonstrated to provide sufficient mechanical support, to induce angiogenesis and to accelerate cardiac repair in a rat model of MI. Our studies highlight the positive impact of implantation of an MSC seeded PG nanofibrous patch as a novel constituent for MI repair.
The in vitro basic biological characteristics and directed differentiation potential towards cardiomyocytes of adult adipose-derived stem cells (ADSCs) induced by angiotensin II were both investigated. ADSCs were isolated from adult adipose tissue and cultured in vitro, and were subsequently induced into adipocytes, chondrocytes, and osteoblasts for assays of multipotential differentiation. The morphological characteristics of ADSCs were observed under an inverted microscope in bright field and phase-contrast ways and a confocal laser scanning microscopy. Moreover, the directional differentiation potential was observed by Oil Red, alkaline phosphatase, von Kossa, and toluidine blue stainings, respectively. The expressions of CD34, CD44, CD45, CD105, and HLA-DR were also detected via flow cytometry. Following to this, ADSCs were induced by angiotensin II and basic fibroblast growth factor for the purpose of directional differentiation towards cardiomyocyte-like cells, and the cells treated with 5-azacytidine were regarded as the control. The results showed that the isolated and cultured ADSCs presented a typical morphology of fusiform shape and also expressed CD44, CD105, but not CD34, CD45, and HLA-DR with assays of flow cytometry. The multi-differentiations to adipocytes, chondrocytes, and osteoblasts confirmed that the isolated cells maintained the stem characteristics generating from adipose tissues. After 4 weeks of induction by angiotensin II, the cells expressed myosin heavy chain, troponin I, and connexin43 by immunocytochemistry staining, but without beating of the cells. This current study indicated that ADSCs possessed the characteristics of mesenchymal stem cells and angiotensin II could induce ADSCs into cardiomyocyte-like cells.
A tissue-specific stem cell niche functions to direct either self-renewal or differentiation. The niche comprises all local
cues that can be sensed by the cell including soluble and insoluble signals, physical forces and cell–cell contacts. Approximating
the stem cell niche through the utilization of biomaterials may give rise to a greater understanding of the biology of the
stem cell niche as well as potential in vitro culture systems and translatable avenues for stem cell therapy, tissue engineering
and regenerative medicine. Stem cell niches within the cardiovascular system have been described within the heart, the bone
marrow compartment and in vascular beds within various tissues. Progenitor cell populations have been characterized which
can give rise to all the major cells of the cardiovascular system including cardiomyocytes, endothelial cells, mural cells
and fibroblasts. The extent to which these progenitor populations can be identified and isolated; however, is variable. Biomaterials
have an important role in the development of artificial stem cell niches for in vitro culture or in vivo therapy. Biomaterials
can be controlled in order to provide insoluble matrix signals, present soluble signals, control cell–cell contacts and transmit
or augment physical signals all of which can contribute towards enhancing cell function or directing cell phenotype. This
chapter focuses specifically on how biomaterials can be used within the context of a stem cell niche to direct and maintain
differentiation towards cardiovascular cell types.
Cardiomyocytes are terminally differentiated cells with limited regenerative capacity in the adult heart, making cell replacement therapy an attractive option to repair injured hearts. Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are pluripotent and capable of infinite expansion in vitro, implicating them as ideal cell types for cell replacement therapy. During the past several years, significant advances in iPS cell generation technology, cardiac differentiation, and cell purification protocols were achieved for the development of stem cell-based heart therapies. The discovery of iPS cells has also sparked the novel idea of direct conversion of mature cell types into another cell type without passing through a pluripotent stem cell state. Functional cardiomyocytes could therefore be directly reprogrammed from differentiated somatic cells by transduction of the three cardiac transcription factors, Gata4, Mef2c, and Tbx5. Herein, we review the recent research achievements and discuss future challenges in stem cell-based cardiac generation and direct cardiac reprogramming technology for heart regeneration.
Introduction:
Whether transplanted cardiac stem cells (CSCs) and mesenchymal stem cells (MSCs) improved ventricular fibrillation threshold (VFT) similarly is still unclear. We sought to compare the effects of the CSC and MSC transplantation on the electrophysiological characteristics and VFT in rats with myocardial infarction (MI).
Methods:
MI was induced in 30 male Sprague-Dawley rats. Two weeks later, animals were randomized to receive 5 × 10(6) CSCs labeled with PKH26 in PBS or 5 × 10(6) MSCs labeled with PKH26 in phosphate buffer solution(PBS) or PBS alone injection into the infarcted anterior ventricular free wall. Six weeks after the injection, electrophysiological characteristics and VFT were measured. Labeled CSCs and MSCs were observed in 5 μm cryostat sections from each heart.
Results:
Malignant ventricular arrhythmias were significantly (P = 0.0055) less inducible in the CSC group than the MSC group. The VFTs were improved in the CSC group compared with the MSC group. Labeled CSCs and MSCs were identified in the infarct zone and infarct marginal zone. Labeled CSCs expressed Connexin-43, von Willebrand factor, α-smooth muscle actin and α-sarcomeric actin,while the Labeled MSCs expressed von Willebrand factor, α-smooth muscle actin and α-sarcomeric actin in vivo.
Conclusions:
After 6 weeks of cell transplantation, CSCs are superior to MSCs in modulating the electrophysiological abnormality and improving the VFT in rats with MI. CSCs and MSCs express markers that suggest muscle, endothelium and vascular smooth muscle phenotypes in vivo, but MSCs rarely express Connexin-43.
Cell therapy and the stem cells (SC) have become a popular topic during last time. The theme is cluttered with numerous publications of questionable reliability. Not all methods applied in praxis are founded on evidence-based research. In the abundant literature, there is a gap between the supposed SC's healing properties associated with their capability to migrate to and engraft in injured tissue, and lack of clear morphological evidence thereof. Accordingly, there is a gap between advertizing and the better part of professional literature: the former speaks about rejuvenation of tissues, and the latter explains sometimes questionable therapeutic effects by paracrine or immunomodulating mechanisms, secretion of cytokines and growth factors. However, a SC is an undifferentiated cell, and a specific and efficient paracrine function can hardly be awaited from it as compared to others, more differentiated cells. It should be noted in conclusion that the main problem with the SC and cell therapy is commercial influence. Probably, experience of some foreign countries should be studied, where moves have been made to stop the use of unproven treatments, including some stem cell therapy.
UPDATE: https://www.researchgate.net/publication/350515988_Stem_cells_and_cell_therapy_Sober_view
RUSSIAN:
https://www.researchgate.net/publication/349794754_Problemy_kletocnoj_terapii_i_stvolovyh_kletok_Some_aspects_of_cell_therapy_and_stem_cells
https://www.researchgate.net/publication/333756573_Nekotorye_aspekty_primenenia_stvolovyh_kletok_v_kardiologii
https://www.researchgate.net/publication/51447514_Stem_cells_and_cell_therapy_On_the_eve_of_scientific_approach
https://www.researchgate.net/publication/276027862_Stvolovye_kletki_i_kletocnaa_terapia_na_puti_k_dokazatelnoj_medicine
A growing body of preclinical evidence suggests that mesenchymal stem cells (MSCs) are effective for the structural and functional recovery of the infracted heart. Accordingly, clinical trials are underway to determine the benefit of MSC-based therapies. While systemic administration of MSCs is an attractive strategy, and is the route currently used for the administration of MSCs in clinical studies for myocardial infarction, the majority of infused cells do not appear to localize to infracted myocardium in animal studies. Recently, important progress has been made in identifying chemokine receptors critical for the migration and homing of MSCs. Here, we review recent literature regarding mechanisms of MSC homing and recruitment to the ischemic myocardium, and discuss potential influences of low engraftment rates of systemically administered MSCs to the infracted heart tissue on the effects of MSC-based therapies on myocardial infarction.
Recent studies have shown that treatments involving injection of stem cells into animals with damaged cardiac tissue result in improved cardiac functionality. Clinical trials have reported conflicting results concerning the recellularization of post-infarct collagen scars. No clear mechanism has so far emerged to fully explain how injected stem cells, specifically the commonly used mesenchymal stem cells (MSC) and endothelial precursor cells (EPC), help heal a damaged heart. Clearly, these injected stem cells must survive and thrive in the hypoxic environment that results after injury for any significant repair to occur. Here we discuss how ischemic preconditioning may lead to increased tolerance of stem cells to these harsh conditions and increase their survival and clinical potential after injection. As injected cells must reach the site in numbers large enough for repair to be functionally significant, homing mechanisms involved in stem cell migration are also discussed. We review the mechanisms of action stem cells may employ once they arrive at their target destination. These possible mechanisms include that the injected stem cells (1) secrete growth factors, (2) differentiate into cardiomyocytes to recellularize damaged tissue and strengthen the post-infarct scar, (3) transdifferentiate the host cells into cardiomyocytes, and (4) induce neovascularization. Finally, we discuss that tissue engineering may provide a standardized platform technology to produce clinically applicable stem cell products with these desired mechanistic capacities.
Induced pluripotent stem cells (iPSCs) can be derived from diverse different somatic cells and share many of the characteristics of embryonic stem cells (ESCs). Because iPSCs avoid many of the ethical concerns associated with the use of embryonic or fetal material, iPSCs have great potential in cell-based regenerative medicine. However, several hurdles will need to be surmounted before their potential can be realized in therapeutic applications. For example, the use of viral vectors, some of which are oncogenes raises the risk of tumor formation in patients, the differentiation of iPSCs into required functional cells in vivo remains to be established, the obtaining of pure populations of target cells from iPSCs is still difficult. Of these, some are shared by both iPSCs and ESCs, others are unique to iPSCs. We will describe these stumbling blocks in detail and discuss possible ways to overcome them. Despite many significant advances, there is as yet no technological framework that would allow the exploitation of iPSCs in a clinical setting in the immediate future. Further research will be required before directed reprogramming can provide a source of cells suitable for application in regenerative medicine.
The notion of the adult heart as terminally differentiated organ without self-renewal potential has been undermined by the existence of a subpopulation of replicating myocytes in normal and pathological states. The origin and significance of these cells has remained obscure for lack of a proper biological context. We report the existence of Lin(-) c-kit(POS) cells with the properties of cardiac stem cells. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing approximately 70% of the ventricle. Thus, the adult heart, like the brain, is mainly composed of terminally differentiated cells, but is not a terminally differentiated organ because it contains stem cells supporting its regeneration. The existence of these cells opens new opportunities for myocardial repair.
Survival and proliferation of skeletal myoblasts within the cardiac environment are crucial to the therapeutic efficacy of myoblast transplantation to the heart. We have analyzed the early dynamics of myoblasts implanted into the myocardium and investigated the mechanisms underlying graft attrition. At 10 min after implantation of [14C]thymidine-labeled male myoblasts into female mice hearts, 14C measurement showed that 39.2 +/- 3.0% of the grafted cells survived, and this steadily decreased to 16.0 +/- 1.7% by 24 h and to 7.4 +/- 0.9% by 72 h. PCR of male-specific Smcy gene calculated that the total (surviving plus proliferated) number of donor-derived cells was 18.3 +/- 1.6 and 23.3 +/- 1.3% at 24 and 72 h, respectively, indicating that proliferation of the surviving cells began after 24 h. Acute inflammation became prominent by 24 h and was reduced by 72 h as indicated by myeloperoxidase activity and histological findings. Multiplex RT-PCR revealed corresponding changes in IL-1beta, TGF-beta, IL-6, and TNF-alpha expression. Treatment with CuZn-superoxide dismutase attenuated the initial rapid death and resulted in enhanced cell numbers afterward, giving a twofold increased total number at 72 h compared with the nontreatment. This effect was associated with reduced inflammatory response, suggesting a causative role for superoxide in the initial rapid graft death and subsequent inflammation. These data describe the early dynamics of myoblasts implanted into the myocardium and suggest that initial oxidative stress and following inflammatory response may be important mechanisms contributing to acute graft attrition, both of which could be potential therapeutic targets to improve the efficiency of cell transplantation to the heart.
Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.
The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease.
A task force has been established by the European Society of Cardiology to investigate the role of progenitor/stem cell therapy in the treatment of cardiovascular disease. This article is the consensus of this group, of what clinical studies are needed in this field, and the challenges to be addressed in the translation of progenitor/stem cell biology to repair of the heart.
Pilot studies suggest that intracoronary transplantation of progenitor cells derived from bone marrow (BMC) or circulating blood (CPC) may improve left ventricular function after acute myocardial infarction. The effects of cell transplantation in patients with healed myocardial infarction are unknown.
After an initial pilot trial involving 17 patients, we randomly assigned, in a controlled crossover study, 75 patients with stable ischemic heart disease who had had a myocardial infarction at least 3 months previously to receive either no cell infusion (23 patients) or infusion of CPC (24 patients) or BMC (28 patients) into the patent coronary artery supplying the most dyskinetic left ventricular area. The patients in the control group were subsequently randomly assigned to receive CPC or BMC, and the patients who initially received BMC or CPC crossed over to receive CPC or BMC, respectively, at 3 months' follow-up.
The absolute change in left ventricular ejection fraction was significantly greater among patients receiving BMC (+2.9 percentage points) than among those receiving CPC (-0.4 percentage point, P=0.003) or no infusion (-1.2 percentage points, P<0.001). The increase in global cardiac function was related to significantly enhanced regional contractility in the area targeted by intracoronary infusion of BMC. The crossover phase of the study revealed that intracoronary infusion of BMC was associated with a significant increase in global and regional left ventricular function, regardless of whether patients crossed over from control to BMC or from CPC to BMC.
Intracoronary infusion of progenitor cells is safe and feasible in patients with healed myocardial infarction. Transplantation of BMC is associated with moderate but significant improvement in the left ventricular ejection fraction after 3 months.
Stem cells show promise for repair of damaged cardiac tissue. Little is known with certainty, however, about the distribution of these cells once introduced in vivo. Previous attempts at tracking delivered stem cells have been hampered by the autofluorescence of host tissue and limitations of existing labeling techniques. We have developed a novel loading approach to stably label human mesenchymal stem cells with quantum dot (QD) nanoparticles. We report the optimization and validation of this long-term tracking technique and highlight several important biological applications by delivering labeled cells to the mammalian heart. The bright QD crystals illuminate exogenous stem cells in histologic sections for at least 8 weeks following delivery and permit, for the first time, the complete three-dimensional reconstruction of the locations of all stem cells following injection into the heart.
Disclosure of potential conflicts of interest is found at the end of this article.
To test the hypothesis that human embryonic stem cells (hESCs) can be guided to form new myocardium by transplantation into the normal or infarcted heart, and to assess the influence of hESC-derived cardiomyocytes (hESCMs) on cardiac function in a rat model of myocardial infarction (MI).
Undifferentiated hESCs (0.5-1x10(6)), human embryoid bodies (hEBs) (4-8 days; 0.5-1x10(6)), 0.1 mm pieces of embryonic stem-derived beating myocardial tissue, and phosphate-buffered saline (control) were injected into the normal or infarcted myocardium of athymic nude rats (n = 58) by direct injection into the muscle or into preimplanted three-dimensional alginate scaffold. By 2-4 weeks after transplantation, heart sections were examined to detect the human cells and differentiation with fluorescent in situ hybridisation, using DNA probes specific for human sex chromosomes and HLA-DR or HLA-ABC immunostaining.
Microscopic examination showed transplanted human cells in the normal, and to a lesser extent in the infarcted myocardium (7/7 vs 2/6; p<0.05). The transplanted hESCs and hEBs rarely created new vessels and did not form new myocardium. Transplantation of hESCM tissue into normal heart produced islands of disorganised myofibres, fibrosis and, in a single case, a teratoma. However, transplantation of hESCMs into the infarcted myocardium did prevent post-MI dysfunction and scar thinning.
Undifferentiated hESCs and hEBs are not directed to form new myocardium after transplantation into normal or infarcted heart and may create teratoma. Nevertheless, this study shows that hESC-derived cardiomyocyte transplantation can attenuate post-MI scar thinning and left ventricular dysfunction.
Mesenchymal stem cells (MSCs) from healthy donors improve cardiac function in experimental acute myocardial infarction (AMI) models. However, little is known about the therapeutic capacity of human MSCs (hMSCs) from patients with ischemic heart disease (IHD). Therefore, the behavior of hMSCs from IHD patients in an immune-compromised mouse AMI model was studied. Enhanced green fluorescent protein-labeled hMSCs from IHD patients (hMSC group: 2 x 10(5) cells in 20 microl, n = 12) or vehicle only (medium group: n = 14) were injected into infarcted myocardium of NOD/scid mice. Sham-operated mice were used as the control (n = 10). Cardiac anatomy and function were serially assessed using 9.4-T magnetic resonance imaging (MRI); 2 wk after cell transplantation, immunohistological analysis was performed. At day 2, delayed-enhancement MRI showed no difference in myocardial infarction (MI) size between the hMSC and medium groups (33 +/- 2% vs. 36 +/- 2%; P = not significant). A comparable increase in left ventricular (LV) volume and decrease in ejection fraction (EF) was observed in both MI groups. However, at day 14, EF was higher in the hMSC than in the medium group (24 +/- 3% vs. 16 +/- 2%; P < 0.05). This was accompanied by increased vascularity and reduced thinning of the infarct scar. Engrafted hMSCs (4.1 +/- 0.3% of injected cells) expressed von Willebrand factor (16.9 +/- 2.7%) but no stringent cardiac or smooth muscle markers. hMSCs from patients with IHD engraft in infarcted mouse myocardium and preserve LV function 2 wk after AMI, potentially through an enhancement of scar vascularity and a reduction of wall thinning.
The possibility that adult bone marrow cells (BMCs) retain a remarkable degree of developmental plasticity and acquire the cardiomyocyte lineage after infarction has been challenged, and the notion of BMC transdifferentiation has been questioned. The center of the controversy is the lack of unequivocal evidence in favor of myocardial regeneration by the injection of BMCs in the infarcted heart. Because of the interest in cell-based therapy for heart failure, several approaches including gene reporter assay, genetic tagging, cell genotyping, PCR-based detection of donor genes, and direct immunofluorescence with quantum dots were used to prove or disprove BMC transdifferentiation. Our results indicate that BMCs engraft, survive, and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes. BMCs and myocytes express at their interface connexin 43 and N-cadherin, and this interaction may be critical for BMCs to adopt the cardiomyogenic fate. With time, a large number of myocytes and coronary vessels are generated. Myocytes show a diploid DNA content and carry, at most, two sex chromosomes. Old and new myocytes show synchronicity in calcium transients, providing strong evidence in favor of the functional coupling of these two cell populations. Thus, BMCs transdifferentiate and acquire the cardiomyogenic and vascular phenotypes restoring the infarcted heart. Together, our studies reveal that locally delivered BMCs generate de novo myocardium composed of integrated cardiomyocytes and coronary vessels. This process occurs independently of cell fusion and ameliorates structurally and functionally the outcome of the heart after infarction.
• myocardial infarction
• myocardial regeneration
• stem cells
• transdifferentiation
We thank Drs Robert Adams, Gary Friday, Philip Gorelick, and Sylvia Wasserthiel-Smoller, members of Stroke Statistics Subcommittee; Drs Joe Broderick, Brian Eigel, Kimberlee Gauveau, Jane Khoury, Jerry Potts, Jane Newburger, and Kathryn Taubert; and Sean Coady and Michael Wolz for their valuable comments and contributions. We acknowledge Tim Anderson and Tom Schneider for their editorial contributions and Karen Modesitt for her administrative assistance.
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Writing Group Disclosures
# Summary {#article-title-2}
Each year the American Heart Association, in conjunction with the Centers for Disease Control and Prevention, the National Institutes of Health, and other government agencies, brings together the most up-to-date statistics on heart disease, stroke, and their risk factors and presents them in its Heart Disease and Stroke Statistical Update. The Statistical Update is a valuable resource for researchers, clinicians, healthcare policy makers, media, the lay public, and many others who seek the …
Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.
After myocardial infarction (MI), adverse remodeling with left ventricular (LV) dilatation is a major determinant of poor outcome. Skeletal myoblast (SkM) implantation improves cardiac function post-MI, although the mechanism is unclear. IL-1 influences post-MI hypertrophy and collagen turnover and is implicated in SkM death after grafting. We hypothesized that SkM expressing secretory IL-1 receptor antagonist (sIL-1ra) at MI border zones would specifically attenuate adverse remodeling and exhibit improved graft cell number. Stable murine male SkM lines (5 x 10(5) cells), expressing or nonexpressing (cont) for sIL-1ra, were implanted into infarct border zones of female nude mice immediately after left coronary artery occlusion. LV ejection fraction (LVEF), end-diastolic diameter, and transmitral peak early/late (E/A) flow velocity ratio were determined by echocardiography. Cardiac myocyte hypertrophy and fibrosis were assessed by morphometry, picrosirius red staining, and hydroxyproline assay. At 3 weeks, cont-SkM-engrafted hearts showed reduced hypertrophy, improved LVEF (55.7 +/- 1.2% vs. MI-only: 40.3 +/- 2.9%), and preserved E/A ratios. sIL-1ra-SkM implantation enhanced these effects (LVEF, 67.0 +/- 2.3%) and significantly attenuated LV dilatation (LV end-diastolic diameter, 4.0 +/- 1.1 mm vs. cont-SkM, 4.5 +/- 1.2 mm vs. MI-only, 4.8 +/- 1.8 mm); this was associated with greater graft numbers, as shown by PCR for male-specific smcy gene. Enzyme zymography showed attenuated matrix metalloproteinase-2 and -9 up-regulation post-MI by either donor SkM type, although infarct-remote zone collagen was reduced only with sIL-1ra-SkM. These results suggest that SkM implantation improves cardiac function post-MI by modulation of adverse remodeling, and that this effect can be significantly enhanced by targeting IL-1 as a key upstream regulator of both adverse remodeling and graft cell death.
We hypothesized that mesenchymal stem cells (MSCs) overexpressing insulin-like growth factor (IGF)-1 showed improved survival and engraftment in the infarcted heart and promoted stem cell recruitment through paracrine release of stromal cell–derived factor (SDF)-1α. Rat bone marrow–derived MSCs were used as nontransduced (NormMSCs) or transduced with adenoviral-null vector (NullMSCs) or vector encoding for IGF-1 (IGF-1MSCs). IGF-1MSCs secreted higher IGF-1 until 12 days of observation (P< 0.001 versus NullMSCs). Molecular studies revealed activation of phosphoinositide 3-kinase, Akt, and Bcl. xL and inhibition of glycogen synthase kinase 3β besides release of SDF-1α in parallel with IGF-1 expression in IGF-1MSCs. For in vivo studies, 70 μL of DMEM without cells (group 1) or containing 1.5× 106 NullMSCs (group 2) or IGF-1MSCs (group 3) were implanted intramyocardially in a female rat model of permanent coronary artery occlusion. One week later, immunoblot on rat heart tissue (n= 4 …
Clinical trials in cardiac cell-based therapy (CBT) have demonstrated the immense potential of stem progenitor cells (SPCs) to repair the injured myocardium. The bulk of evidence so far has shown that CBT can lead to structural and functional improvements. Unresolved issues remain, however, including gaps in the understanding of mechanisms and mixed results from CBT trials. To try to provide answers for these issues, assessment of the biological fate of SPCs once delivered to the injured heart has been called for. Advances in contrast agents and imaging modalities have made feasible the objective assessment of the in vivo molecular and cellular evolution of transplanted SPCs. In vivo imaging can target fundamental processes related to SPCs to gain information on their biological activities and outcomes within specific authentic microenvironments. Advantages and inherent drawbacks of imaging techniques, such as reporter-gene systems, optical imaging, radionuclide imaging, and MRI, are discussed in this Review. More than ever, it has become clear to scientists and clinicians that parallel developments in cell-based therapies and in vivo imaging modalities will strengthen this blossoming field.
Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infarcted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16(INK), p21 and p19(ARF). VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs or VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.
Bone marrow stromal cells are capable of differentiating into cardiomyogenic cells. We tested the hypothesis that transcoronary implantation of bone marrow stromal cells may regenerate infarcted myocardium and reduce cardiac dysfunction.
Isolated bone marrow stromal cells from the isogenic donor rats were transfected with LacZ reporter gene for cell labeling. To induce cardiomyogenic differentiation, the bone marrow stromal cells were treated with 5-azacytidine before implantation. Two weeks after left coronary ligation, these cells (1 x 10(6) in 150 microL) were infused into the briefly distally occluded ascending aorta of the recipient rats (n = 15) to simulate direct coronary infusion clinically. Control animals were infused with cell-free medium (n = 14). Cardiac function was evaluated by echocardiography at preimplantation and 4 and 8 weeks postimplantation. The hearts were then immunohistochemically studied to identify phenotypic changes of implanted bone marrow stromal cells.
Immediately after cell infusion, the bone marrow stromal cells were trapped within coronary vessels in both infarcted and noninfarcted areas. However, after 8 weeks, most of the cells were identified in the scar and periscar tissue, expressing sarcomeric myosin heavy chain and cardiomyocyte-specific protein troponin I-C. Some bone marrow stromal cells were found to be connected to the adjacent host cardiomyocytes with gap junction. Two-way repeated-measures analysis of variance revealed significant improvement in fractional shortening and end-diastolic and end-systolic diameter of the left ventricle (P =.0465,.002,.0004, respectively) in the bone marrow stromal cell group.
Although bone marrow stromal cells had been reported to improve cardiac function when injected directly into the myocardial scar, this study demonstrated for the first time that bone marrow stromal cells can be delivered via the coronary artery, as they are capable of targeted migration and differentiation into cardiomyocytes in the scar tissue to improve cardiac function.
Systemic delivery of bone marrow-derived mesenchymal stem cells (BM-MSCs) is an attractive approach for myocardial repair. We aimed to test this strategy in a rat model after myocardial infarction (MI).
BM-MSCs were obtained from rat bone marrow, expanded in vitro to a purity of >50%, and labeled with 99mTc exametazime, fluorescent dye, LacZ marker gene, or bromodeoxyuridine. Rats were subjected to MI by transient coronary artery occlusion or to sham MI. 99mTc-labeled cells (4x10(6)) were transfused into the left ventricular cavity of MI rats either at 2 or 10 to 14 days after MI and were compared with sham-MI rats or MI rats treated with intravenous infusion. Gamma camera imaging and isolated organ counting 4 hours after intravenous infusion revealed uptake of the 99mTc-labeled cells mainly in the lungs, with significantly smaller amounts in the liver, heart, and spleen. Delivery by left ventricular cavity infusion resulted in drastically lower lung uptake, better uptake in the heart, and specifically higher uptake in infarcted compared with sham-MI hearts. Histological examination at 1 week after infusion identified labeled cells either in the infarcted or border zone but not in remote viable myocardium or sham-MI hearts. Labeled cells were also identified in the lung, liver, spleen, and bone marrow.
Systemic intravenous delivery of BM-MSCs to rats after MI, although feasible, is limited by entrapment of the donor cells in the lungs. Direct left ventricular cavity infusion enhances migration and colonization of the cells preferentially to the ischemic myocardium.
Experimental and initial clinical studies suggest that transplantation of circulating blood- (CPC) or bone marrow-derived (BMC) progenitor cells may beneficially affect postinfarction remodeling processes after acute myocardial infarction (AMI). To relate functional characteristics of the infused cells to quantitative measures of outcome at 4-month follow-up, we performed serial contrast-enhanced MRI and assessed the migratory capacity of the transplanted progenitor cells immediately before intracoronary infusion.
In 28 patients with reperfused AMI receiving either BMCs or CPCs into the infarct artery 4.7+/-1.7 days after AMI, serial contrast-enhanced MRI performed initially and after 4 months revealed a significant increase in global ejection fraction (from 44+/-10% to 49+/-10%; P=0.003), a decrease in end-systolic volume (from 69+/-26 to 60+/-28 mL; P=0.003), and unchanged end-diastolic volumes (122+/-34 versus 117+/-37 mL; P=NS). Infarct size, measured as late enhancement (LE) volume, decreased significantly, from 46+/-32 to 37+/-28 mL (P<0.05). There was a significant correlation between the reduction in LE volume and global ejection fraction improvement. The migratory capacity of transplanted cells as assessed ex vivo toward a gradient of vascular endothelial growth factor for CPCs and stromal cell derived factor-1 for BMCs was closely correlated with the reduction of LE volume. By multivariate analysis, migratory capacity remained the most important independent predictor of infarct remodeling.
Analysis of serial contrast-enhanced MRI suggests that intracoronary infusion of adult progenitor cells in patients with AMI beneficially affects postinfarction remodeling processes. The migratory capacity of the infused cells is a major determinant of infarct remodeling, disclosing a causal effect of progenitor cell therapy on regeneration enhancement.
Cardiomyocyte regeneration is limited in adult life. Thus, the identification of a putative source of cardiomyocyte progenitors is of great interest to provide a usable model in vitro and new perspective in regenerative therapy. As adipose tissues were recently demonstrated to contain pluripotent stem cells, the emergence of cardiomyocyte phenotype from adipose-derived cells was investigated. We demonstrated that rare beating cells with cardiomyocyte features could be identified after culture of adipose stroma cells without addition of 5-azacytidine. The cardiomyocyte phenotype was first identified by morphological observation, confirmed with expression of specific cardiac markers, immunocytochemistry staining, and ultrastructural analysis, revealing the presence of ventricle- and atrial-like cells. Electrophysiological studies performed on early culture revealed a pacemaker activity of the cells. Finally, functional studies showed that adrenergic agonist stimulated the beating rate whereas cholinergic agonist decreased it. Taken together, this study demonstrated that functional cardiomyocyte-like cells could be directly obtained from adipose tissue. According to the large amount of this tissue in adult mammal, it could represent a useful source of cardiomyocyte progenitors.
Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy. However, in the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential. Recent studies have even surmised the existence of resident cardiac stem cells, endothelial cells generating cardiomyocytes by cell contact or extracardiac progenitors for cardiomyocytes, but these findings are still controversial. We describe the isolation of undifferentiated cells that grow as self-adherent clusters (that we have termed "cardiospheres") from subcultures of postnatal atrial or ventricular human biopsy specimens and from murine hearts. These cells are clonogenic, express stem and endothelial progenitor cell antigens/markers, and appear to have the properties of adult cardiac stem cells. They are capable of long-term self-renewal and can differentiate in vitro and after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse to yield the major specialized cell types of the heart: myocytes (ie, cells demonstrating contractile activity and/or showing cardiomyocyte markers) and vascular cells (ie, cells with endothelial or smooth muscle markers).
Current stem cell protocols for ischemic heart disease are limited by the small numbers of cells that can be obtained by bone marrow aspirate. To increase myocardial delivery of bone marrow stem cells in patients with chronic ischemic heart disease (CIHD), we used granulocyte colony stimulating factor (G-CSF) for bone marrow mobilization of CD34+ cells, enabling intracoronary infusion of large numbers of CD34+ stem cells. Patients with CIHD (n = 5) demonstrated significantly reduced numbers of CD34+ cells mobilized by G-CSF in comparison to age-matched controls. Sustained reduction in anginal symptoms and improvement in quality of life scores was seen in all patients following infusion of cells. Moreover, mean collateral flow grade at 12-month follow-up angiography significantly improved, indicating sustained myocardial neovascularization. No proliferative retinopathy was induced and no in-stent restenosis seen. However, in two patients with documented increase in collateral flow, complications arose, one developing an acute coronary syndrome and the other a lentigo maligna. These results demonstrate the feasibility of G-CSF mobilization, leukapheresis and intracoronary transfer of CD34+ stem cells in patients with CIHD, but longer-term studies are required to ensure that this protocol is safe and effective.
Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities.
Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, 1x10(7) of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7x10(7)+/-5.8x10(6) photons.s(-1).cm(-2) per steradian (sr) and 0.08+/-0.03% injected dose/g, respectively (P<0.05 versus control). Both signals increased progressively from week 1 to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (n=4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (n=4) treated with saline (1 mL/kg).
This is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. This versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.
Mesenchymal stem cells (MSCs), rare bone marrow-derived stem cell precursors of non-haematopoietic tissues, have shown promise in potentially repairing infarcted myocardium. These and similar cell types are being tested clinically, but understanding of delivery and subsequent biodistribution is lacking. This study was designed to quantitatively compare MSC engraftment rates after intravenous (IV), intracoronary (IC), or endocardial (EC) delivery in a porcine myocardial infarction (MI) model.
Allogeneic, male MSCs were cultured from porcine bone marrow aspirates. Iridium nanoparticles were added during culturing and internalized by the MSCs. An MI was induced in female swine (27-40 kg in size) by prolonged balloon occlusion of the mid-left anterior descending artery. Animals (n = 6 per group) were randomized to one of three delivery methods. Cellular engraftment was determined 14+/-3 days post-delivery by measuring ex-vivo the iridium nanoparticle concentration in the infarct. Confirmation of cellular engraftment utilized both DiI and fluorescence in situ hybridization (FISH) labelling techniques. During MSC infusion, no adverse events were noted. However, following IC infusion, half of the pigs exhibited decreased blood flow distal to the infusion site. At 14 days, the mean number of engrafted cells within the infarct zone was significantly greater (P< or =0.01) following IC infusion than either EC injection or IV infusion and EC engraftment was greater than IV engraftment (P< or =0.01). There was less systemic delivery to the lungs following [EC vs. IV (P = 0.02), EC vs. IC (P = 0.06)]. Both DiI and FISH labelling demonstrated the presence of engrafted male MSCs within the female infarcted tissue.
IC and EC injection of MSCs post-MI resulted in increased engraftment within infarcted tissue when compared with IV infusion, and IC was more efficient than EC. However, IC delivery was also associated with a higher incidence of decreased coronary blood flow. EC delivery into acutely infarcted myocardial tissue was safe and well tolerated and was associated with decreased remote organ engraftment with compared with IC and IV deliveries.
Regeneration of infarcted myocardium by injecting stem cells has been proposed to prevent heart failure. We studied the i.c. administration of human umbilical cord blood stem cells (USSC) in a porcine model of myocardial infarction (MI) and reperfusion. In 15 swine, MI was induced by balloon-occlusion of the left circumflex coronary artery (LCX) for 2 h followed by reperfusion. Five swine served as healthy controls. One week later, magnetic resonance imaging (MRI) was performed to assess left ventricular (LV) function and infarct size. Then, under immune suppression, 6 of the 12 surviving MI swine received intracoronary injection of approximately 10(8) human USSC in the LCX while the other MI-swine received medium. Four weeks later all swine underwent follow-up MRI, and were sacrificed for histology. One week after MI, end-diastolic volume (92+/-3 mL) and LV mass (75+/-2 g) were larger, while ejection fraction (42+/-2%) was smaller than in healthy control (68+/-3 mL, 66+/-3 g and 55+/-3%, all P<0.05). Regional wall thickening (-7+/-2%) in the LCX area became akinetic. No difference in global and regional LV function at 5 weeks was observed between MI animals receiving USSC or medium. Infarct size after USSC treatment was significantly larger (20+/-3 g vs. 8+/-2 g, P<0.05). USSC survived only in the infarct border zone at 5 weeks and did not express cardiomyocyte or endothelial markers. Histology showed that intracoronary injection of USSC caused micro infarctions by obstructing blood vessels. In swine with a 1 week old MI, injection of USSC via the intracoronary route does not improve LV function 4 weeks later.
Therapeutic efficacy of bone marrow (BM) cell injection for treating ischemic chronic heart failure has not been established. In addition, experimental data are lacking on arrhythmia occurrence after BM cell injection. We hypothesized that therapeutic efficacy and arrhythmia occurrence induced by BM cell injection may be affected by the cell delivery route.
Three weeks after left coronary artery ligation, wild-type female rats were injected with 1x10(7) mononuclear BM cells derived from green fluorescent protein-transgenic male rats through either a direct intramyocardial or a retrograde intracoronary route. Both intramyocardial and intracoronary injection of BM cells demonstrated similar improvement in left ventricular ejection fraction measured by echocardiography and a similar graft size analyzed by real-time polymerase chain reaction for the Y chromosome-specific Sry gene. Noticeably, intramyocardial injection of BM cells induced frequent ventricular premature contractions (108+/-73 per hour at 7 days after BM cell injection), including multiform, consecutive ventricular premature contractions and ventricular tachycardia for the initial 14 days; intracoronary injection of BM cells and intramyocardial injection of phosphate-buffered saline rarely induced arrhythmias. Immunohistochemistry demonstrated that intramyocardial BM cell injection formed distinct cell clusters containing donor-derived cells and accumulated host-derived inflammatory cells in the infarct border zone, whereas intracoronary BM cell injection provided more homogeneous donor cell dissemination with less inflammation and without disrupting the native myocardial structure.
BM cell injection is able to improve cardiac function in ischemic chronic heart failure but has a risk of arrhythmia occurrence when the intramyocardial route is used. Such arrhythmias may be prevented by using the intracoronary route.
We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.
Heart disease remains the leading cause of death in the industrialized world. Stem cell therapy is a promising treatment modality for injured cardiac tissue. A novel mechanism for this cardioprotection may include paracrine actions. Cardiac surgery represents the unique situation where preischemia and postischemia treatment modalities exist that may use stem cell paracrine protection. This review (1) recalls the history of stem cells in cardiac disease and the unraveling of its mechanistic basis for protection, (2) outlines the pathways for stem cell-mediated paracrine protection, (3) highlights the signaling factors expressed, (4) explores the potential of using stem cells clinically in cardiac surgery, and (5) summarizes all human stem cell studies in cardiac disease to date.
Despite rapid advances in the stem cell field, the ability to identify and track transplanted or migrating stem cells in vivo is limited. To overcome this limitation, we used magnetic resonance imaging (MRI) to detect and follow transplanted stem cells over a period of 28 days in mice using an established myocardial infarction model. Pluripotent mouse embryonic stem (mES) cells were expanded and induced to differentiate into beating cardiomyocytes in vitro. The cardiac-differentiated mES cells were then loaded with superparamagnetic fluorescent microspheres (1.63 μm in diameter) and transplanted into ischemic myocardium immediately following ligation and subsequent reperfusion of the left anterior descending coronary artery. To identify the transplanted stem cells in vivo, MRI was performed using a Varian Inova 4.7 Tesla scanner. Our results show that (a) the cardiac-differentiated mES were effectively loaded with superparamagnetic microspheres in vitro, (b) the microsphere-loaded mES cells continued to beat in culture prior to transplantation, (c) the transplanted mES cells were readily detected in the heart in vivo using noninvasive MRI techniques, (d) the transplanted stem cells were detected in ischemic myocardium for the entire 28-day duration of the study as confirmed by MRI and post-mortem histological analyses, and (e) concurrent functional MRI indicated typical loss of cardiac function, although significant amelioration of remodeling was noted after 28 days in hearts that received transplanted stem cells. These results demonstrate that it is feasible to simultaneously track transplanted stem cells and monitor cardiac function in vivo over an extended period using noninvasive MRI techniques.
Disclosure of potential conflicts of interest is found at the end of this article.
Emerging evidence suggests that adipose tissue-derived stem cells (ASCs) can be used for the treatment of ischemic heart diseases. However, the mechanisms underlying their therapeutic effects have not been clearly defined. In this study cytokines released by ASCs were detected by ELISA and pro-angiogenic effects were assessed by tube formation assay. To define the anti-apoptotic effect of ASCs, neonatal rat cardiomyocytes were subjected to hypoxia condition in a co-culture system. Our data show that ASCs secrete significant amounts of VEGF (810.65+/-56.92 pg/microg DNA) and IGF-I (328.33+/-22.7 pg/microg DNA). Cardiomyocytes apoptosis was significantly prevented by ASCs and 62.5% of the anti-apoptotic effect was mediated by IGF-I and 34.2% by VEGF. ASCs promoted endothelial cell tube formation by secreting VEGF. In conclusion we demonstrated that ASCs have a marked impact on anti-apoptosis and angiogenesis and helps to explain data of stem cells benefit without transdifferentiation.
Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.
The aim of this study was to investigate the ability of adult human bone marrow mesenchymal stem cells to differentiate towards a cardiomyogenic phenotype IN VITRO.
Bone marrow samples were aspirated from 30 patients undergoing open heart surgery from the anterior iliac crest. Second passaged cells were treated with 10 microM 5-azacytidine. As control groups we used cells not expanded in culture and cells untreated with 5-azacytidine. Morphologic characteristics were analysed by confocal and electron microscopy. The expression of the cytoskeletal protein vimentin and muscle-specific myocin heavy chain was analysed by immunohistochemistry. The expression of the cardiomyocyte specific genes alpha-cardiac actin, beta-myocin heavy chain and cardiac troponin-T was detected by reverse transcriptase polymerase chain reaction.
Mesenchymal stem cells were spindle-shaped with irregular processes. Cells treated with 5-azacytidine assumed a stick-like morphology. They connected with adjoining cells to form myotube-like structures. Numerous myofilaments were detected in induced cells which were immunohistochemically positive for myosin heavy chain and vimentin. The mRNAs of all specific cardiac genes were expressed in both induced and uninduced cells.
These results indicate that adult human bone marrow mesenchymal stem cells treated with 5-aza can differentiate towards a cardiomyogenic lineage IN VITRO.
Phase I clinical studies have demonstrated the feasibility of implanting autologous skeletal myoblasts in postinfarction scars. However, they have failed to determine whether this procedure was functionally effective and arrhythmogenic.
This multicenter, randomized, placebo-controlled, double-blind study included patients with left ventricular (LV) dysfunction (ejection fraction < or = 35%), myocardial infarction, and indication for coronary surgery. Each patient received either cells grown from a skeletal muscle biopsy or a placebo solution injected in and around the scar. All patients received an implantable cardioverter-defibrillator. The primary efficacy end points were the 6-month changes in global and regional LV function assessed by echocardiography. The safety end points comprised a composite index of major cardiac adverse events and ventricular arrhythmias. Ninety-seven patients received myoblasts (400 or 800 million; n=33 and n=34, respectively) or the placebo (n=30). Myoblast transfer did not improve regional or global LV function beyond that seen in control patients. The absolute change in ejection fraction (median [interquartile range]) between 6 months and baseline was 4.4% (0.2; 7.3), 3.4% (-0.3; 12.4), and 5.2% (-4.4; 11.0) in the placebo, low-dose, and high-dose groups, respectively (P=0.95). However, the high-dose cell group demonstrated a significant decrease in LV volumes compared with the placebo group. Despite a higher number of arrhythmic events in the myoblast-treated patients, the 6-month rates of major cardiac adverse events and of ventricular arrhythmias did not differ significantly between the pooled treatment and placebo groups.
Myoblast injections combined with coronary surgery in patients with depressed LV function failed to improve echocardiographic heart function. The increased number of early postoperative arrhythmic events after myoblast transplantation, as well as the capability of high-dose injections to revert LV remodeling, warrants further investigation.
To determine the effect of transplantation of undifferentiated and cardiac pre-differentiated adipose stem cells compared with bone marrow mononuclear cells (BM-MNC) in a chronic model of myocardial infarction.
Ninety-five Sprague-Dawley rats underwent left coronary artery ligation and after 1 month received by direct intramyocardial injection either adipose derived stem cells (ADSC), cardiomyogenic cells (AD-CMG) or BM-MNC from enhanced-Green Fluorescent Protein (eGFP) mice. The control group was treated with culture medium. Heart function was assessed by echocardiography and 18F-FDG microPET. Cell engraftment, differentiation, angiogenesis and fibrosis in the scar tissue were also evaluated by (immuno)histochemistry and immunofluorescence.
One month after cell transplantation, ADSC induced a significant improvement in heart function (LVEF 46.3+/-9.6% versus 27.7+/-8% pre-transplant) and tissue viability (64.78+/-7.2% versus 55.89+/-6.3% pre-transplant). An increase in the degree of angiogenesis and a decrease in fibrosis were also detected. Although transplantation of AD-CMG or BM-MNC also had a positive, albeit smaller, effect on angiogenesis and fibrosis in the infarcted hearts, this benefit did not translate into a significant improvement in heart function or tissue viability.
These results indicate that transplantation of adipose derived cells in chronic infarct provides a superior benefit to cardiac pre-differentiated ADSC and BM-MNC.
Current efforts to direct differentiation of human embryonic stem cells (hESC) into a particular cell lineage usually lead to a heterogeneous cell population with only a fraction of the desired cell type present. We show the generation of an essentially pure population of human cardiomyocytes from hESC using lineage selection.
A construct comprising the murine alpha-myosin heavy chain (alpha-MHC) promoter driving the neomycin-resistance gene was introduced into hES3 cells to generate stable transgenic lines. Transgenic hESC lines were differentiated into cardiomyocytes and subjected to G418 selection. Both G418-selected and non-selected cardiomyocytes were characterized by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The teratoma-forming potential of differentiated cells was assessed by injection of about 2 million cells into the hind limb muscle of SCID mice. Results After cardiac differentiation and antibiotic selection in a suspension culture process, more than 99% of the transgenic cells showed immunoreactivity to alpha-MHC and alpha-actinin; this enrichment efficiency was observed for independent transgenic cell lines. Quantitative RT-PCR analysis revealed high levels of enrichment for cardiac-specific messages in the selected population. Importantly, injection of selected cells into six SCID mice resulted in no apparent teratoma formation, in contrast to differentiated but non-selected controls.
Our results represent a significant step toward scalable production of pure human cardiomyocytes from stable, expandable hESC lines that will facilitate the development of cell therapies, safety pharmacology and drug discovery.
Mesenchymal stem cells (MSC) transplantation has been proved to be promising strategy to treat the failing heart. The effect of MSC transplantation is thought to be mediated mainly in a paracrine manner. Recent reports have suggested that cardiac progenitor cells (CPC) reside in the heart. In this study, we investigated whether MSC had paracrine effects on CPC in vitro. CPC were isolated from the neonatal rat heart using an explant method. MSC were isolated from the adult rat bone marrow. MSC-derived conditioned medium promoted proliferation of CPC and inhibited apoptosis of CPC induced by hypoxia and serum starvation. Chemotaxis chamber assay demonstrated that MSC-derived conditioned medium enhanced migration of CPC. Furthermore, MSC-derived conditioned medium upregulated expression of cardiomyocyte-related genes in CPC such as beta-myosin heavy chain (beta-MHC) and atrial natriuretic peptide (ANP). In conclusion, MSC-derived conditioned medium had protective effects on CPC and enhanced their migration and differentiation.
Mesenchymal stem cells (MSC) have recently been shown to possess immunomodulatory properties in vitro and in vivo. The present study aimed to investigate the regulatory effect of MSC transplantation on the immuno-inflammatory response in myocardial infarction (MI).
MI was induced in Sprague-Dawley rats by left anterior descending coronary artery ligation, and the animals were randomly assigned into the following three groups: sham ( n=8); phosphate-buffered saline (PBS) injected (MI+PBS, n=8); and MSC transplantation (MI+MSC, n=8). BrdU-labeled MSC or PBS was transplanted into peri-infarct myocardium by direct myocardial injection. At 1 and 28 days post-transplantation, cardiac function was evaluated by echocardiography. Transplanted cells were investigated through immunohistochemistry. Lymphocyte cytotoxic activity was evaluated with the crystal violet method. The activity of NF-kappaB and protein expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-10 in myocardium were assessed by immunohistochemistry and Western blot.
Echocardiographic examination revealed that the MSC transplantation prevented left ventricular dilation and dysfunction at 28 days after the operation. BrdU-stained cells were found living in host heart 4 weeks after transplantation. MSC transplantation attenuated the cytotoxic activity of spleen lymphocytes. Transplantation of MSC inhibited the activity of NF-kappaB, attenuated the protein production of TNF-alpha and IL-6, and increased the expression of IL-10 in peri-infarct myocardium.
MSC transplantation modulated the immuno-inflammatory response in MI. The immuno-inflammatory regulatory effect of MSC transplantation might partly account for the cardiac protection in myocardial infarction.
Induced pluripotent stem (iPS) cells are a novel stem cell population induced from mouse and human adult somatic cells through reprogramming by transduction of defined transcription factors. However, detailed differentiation properties and the directional differentiation system of iPS cells have not been demonstrated.
Previously, we established a novel mouse embryonic stem (ES) cell differentiation system that can reproduce the early differentiation processes of cardiovascular cells. We applied our ES cell system to iPS cells and examined directional differentiation of mouse iPS cells to cardiovascular cells. Flk1 (also designated as vascular endothelial growth factor receptor-2)-expressing mesoderm cells were induced from iPS cells after approximately 4-day culture for differentiation. Purified Flk1(+) cells gave rise to endothelial cells and mural cells by addition of vascular endothelial growth factor and serum. Arterial, venous, and lymphatic endothelial cells were also successfully induced. Self-beating cardiomyocytes could be induced from Flk1(+) cells by culture on OP9 stroma cells. Time course and efficiency of the differentiation were comparable to those of mouse ES cells. Occasionally, reexpression of transgene mRNAs, including c-myc, was observed in long-term differentiation cultures.
Various cardiovascular cells can be systematically induced from iPS cells. The differentiation properties of iPS cells are almost completely identical to those of ES cells. This system would greatly contribute to a novel understanding of iPS cell biology and the development of novel cardiovascular regenerative medicine.
The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease- and human patient-specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line.
With the use of a standard embryoid body-based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell-derived cardiomyocytes show typical features of ES cell-derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), alpha-myosin heavy chain (alpha-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric alpha-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca(2+) fluctuations with amplitudes of Ca(2+) transients comparable to ES cell cardiomyocytes. Simultaneous Ca(2+) release within clusters of iPS cell-derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the beta-adrenergic and muscarinic signaling cascade in these cells.
iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.
Direct intramyocardial but not intracoronary injection of bone marrow cells induces ventricular arrhythmias in a rat chronic ischemic heart failure model
- Fukushima
Reprogramming of human somatic cells to pluripotency with defined factors
- Park