Reida Menshawe El Oakley

Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury.

Human ESC-derived mesenchymal stem cell (MSC)-conditioned medium (CM) was previously shown to mediate cardioprotection during myocardial ischemia/reperfusion injury through large complexes of 50-100 nm. Here we show that these MSCs secreted 50- to 100-nm particles. These particles could be visualized by electron microscopy and were shown to be phospholipid vesicles consisting of cholesterol, sphingomyelin, and phosphatidylcholine. They contained coimmunoprecipitating exosome-associated proteins, e.g., CD81, CD9, and Alix. These particles were purified as a homogeneous population of particles with a hydrodynamic radius of 55-65 nm by size-exclusion fractionation on a HPLC. Together these observations indicated that these particles are exosomes. These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.

Derivation of clinically compliant MSCs from CD105+, CD24- differentiated human ESCs.

Adult tissue‐derived mesenchymal stem cells (MSCs) have demonstrated therapeutic efficacy in treating diseases or repairing damaged tissues through mechanisms thought to be mediated by either cell replacement or secretion of paracrine factors. Characterized, self‐renewing human ESCs could potentially be an invariable source of consistently uniform MSCs for therapeutic applications. Here we describe a clinically relevant and reproducible manner of generating identical batches of hESC‐derived MSC (hESC‐MSC) cultures that circumvents exposure to virus, mouse cells, or serum. Trypsinization and propagation of HuES9 or H1 hESCs in feeder‐ and serum‐free selection media generated three polyclonal, karyotypically stable, and phenotypically MSC‐like cultures that do not express pluripotency‐associated markers but displayed MSC‐like surface antigens and gene expression profile. They differentiate into adipocytes, osteocytes, and chondrocytes in vitro. Gene expression and fluorescence‐activated cell sorter analysis identified CD105 and CD24 as highly expressed antigens on hESC‐MSCs and hESCs, respectively. CD105+, CD24− monoclonal isolates have a typical MSC gene expression profiles and were identical to each other with a highly correlated gene expression profile (r2 > .90). We have developed a protocol to reproducibly generate clinically compliant and identical hESC‐MSC cultures.

Elucidating the Secretion Proteome of Human Embryonic Stem Cell-derived Mesenchymal Stem Cells

Transplantation of mesenchymal stem cells (MSCs) has been used to treat a wide range of diseases, and the mechanism of action is postulated to be mediated by either differentiation into functional reparative cells that replace injured tissues or secretion of paracrine factors that promote tissue repair. To complement earlier studies that identified some of the paracrine factors, we profiled the paracrine proteome to better assess the relevance of MSC paracrine factors to the wide spectrum of MSC-mediated therapeutic effects. To evaluate the therapeutic potential of the MSC paracrine proteome, a chemically defined serum-free culture medium was conditioned by MSCs derived from human embryonic stem cells using a clinically compliant protocol. The conditioned medium was analyzed by multidimensional protein identification technology and cytokine antibody array analysis and revealed the presence of 201 unique gene products. 86–88% of these gene products had detectable transcript levels by microarray or quantitative RT-PCR assays. Computational analysis predicted that these gene products will significantly drive three major groups of biological processes: metabolism, defense response, and tissue differentiation including vascularization, hematopoiesis, and skeletal development. It also predicted that the 201 gene products activate important signaling pathways in cardiovascular biology, bone development, and hematopoiesis such as Jak-STAT, MAPK, Toll-like receptor, transforming growth factor-β, and mTOR (mammalian target of rapamycin) signaling pathways. This study identified a large number of MSC secretory products that have the potential to act as paracrine modulators of tissue repair and replacement in diseases of the cardiovascular, hematopoietic, and skeletal tissues. Moreover our results suggest that human embryonic stem cell-derived MSC-conditioned medium has the potency to treat a variety of diseases in humans without cell transplantation.

Contractile and Vasorelaxant Effects of Hydrogen Sulfide and Its Biosynthesis in the Human Internal Mammary Artery

This study aimed to test these hypotheses: cystathionine γ-lyase (CSE) is expressed in a human artery, it generates hydrogen sulfide (H2S), and H2S relaxes a human artery. H2S is produced endogenously in rat arteries from cysteine by CSE. Endogenously produced H2S dilates rat resistance arteries. Although CSE is expressed in rat arteries, its presence in human blood vessels has not been described. In this study, we showed that both CSE mRNA, determined by reverse transcription-polymerase chain reaction, and CSE protein, determined by Western blotting, apparently occur in the human internal mammary artery (internal thoracic artery). Artery homogenates converted cysteine to H2S, and the H2S production was inhibited by dl-propargylglycine, an inhibitor of CSE. We also showed that H2S relaxes phenylephrine-precontracted human internal mammary artery at higher concentrations but produces contraction at low concentrations. The latter contractions are stronger in acetylcholine-prerelaxed arteries, suggesting inhibition of nitric oxide action. The relaxation is partially blocked by glibenclamide, an inhibitor of KATP channels. The present results indicate that CSE protein is expressed in human arteries, that human arteries synthesize H2S, and that higher concentrations of H2S relax human arteries, in part by opening KATP channels. Low concentrations of H2S contract the human internal mammary artery, possibly by reacting with nitric oxide to form an inactive nitrosothiol. The possibility that CSE, and the H2S it generates, together play a physiological role in regulating the diameter of arteries in humans, as has been demonstrated in rats, should be considered.

Derivation and characterization of human fetal MSCs: An alternative cell source for large-scale production of cardioprotective microparticles

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10(16-19) cells or 10(7-10) doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r(2)>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50-65 nm. This purified population of microparticles was cardioprotective at approximately 1/10 dosage of the crude secretion.

Myocardial infarction in the C57BL/6J mouse: a quantifiable and highly reproducible experimental model.

INTRODUCTION The laboratory mouse is a powerful tool in cardiovascular research. In this report, we describe a method for a reproducible mouse myocardial infarction model that would allow subsequent comparative and quantitative studies on molecular and pathophysiological variables. METHODS (A) The distribution of the major coronary arteries including the septal artery in the left ventricle of the C57BL/6J mice (n=20) was mapped by perfusion of latex dye or fluorescent beads through the aorta. (B) The territory of myocardial infarction after the ligation of the most proximal aspect of the left anterior descending (LAD) coronary artery was quantified

Myocyte Transplantation for Myocardial Repair: A Few Good Cells Can Mend a Broken Heart

Cell transplantation is a potential therapeutic approach for patients with chronic myocardial failure. Experimental transplantation of neonatal and fetal cardiac myocytes showed that the grafted cells can functionally integrate with and augment the function of the recipient heart. Clinical application of this approach will be limited by shortage of donors, chronic rejection, and because it is ethically contentious. By contrast skeletal myoblasts (satellite cells) are abundant and can be grafted successfully into the animals own heart even after genetic manipulation in vitro. Functional integration of myoblasts, however, is hampered by the lack of intercellular gap junction communication and the difference in excitation-contraction coupling between skeletal and cardiac myocytes. In experimental studies several other cell types have been used to augment cardiac function. In this review we discuss the published results of myocyte transplantation with emphasis on potential sources of cells, the ethics of using donor embryonic and fetal cardiomyocytes, genetic transformation of skeletal myoblasts for myocardial repair, and the functional benefits of cell transplantation to the failing heart.

Choice of Prosthetic Heart Valve in Today's Practice

In this update, current guidelines addressing prosthesis selection published by the American College of Cardiology/American Heart Association (ACC/AHA) and the European Society of Cardiology (ESC) are discussed, along with additional data that affect choices in valve prostheses. The case of a 50-year-old man undergoing aortic valve replacement is used to address anticipated operative mortality, risk of reoperation, and valve-related morbidity and mortality. The apparent advantages associated with the use of a bioprosthesis even in a relatively young patient help to explain current clinical trends toward the increasing use of tissue valves. We also provide a simplified algorithm that may be used to facilitate the choice of valve procedure in patients with heart valve disease. The outcomes after surgery for valvular heart disease in terms of survival, functional status, and quality of life are determined primarily by patient-related factors such as age, ventricular function, and other comorbidities.1 However, outcomes also are influenced by surgical factors; the best clinical outcomes often are associated with valve repair, although mitral repair is not always possible and aortic valve repair in adults remains the exception rather than the rule. For patients who require valve replacement, the valve prosthesis can significantly influence outcome. The ideal prosthetic valve that combines excellent hemodynamic performance and long-term durability without increased thromboembolic risk or the need for long-term anticoagulation does not exist. Hence, patients and their physicians need to choose between a mechanical and a tissue (bioprosthetic) valve. In general, the advantageous durability of mechanical valves is offset by the risk of thromboembolism and the need for long-term anticoagulation and its associated risk of bleeding. In contrast, bioprosthetic valves do not require long-term anticoagulation yet carry the risk of structural failure and reoperation.2,3 Two historic randomized clinical trials compared outcomes after valve replacement with a first-generation porcine heterograft …

Embryonic Cell Lines with Endothelial Potential: An In Vitro System for Studying Endothelial Differentiation

Objective—Endothelial differentiation is a fundamental process in angiogenesis and vasculogenesis with implications in development, normal physiology, and pathology. To better understand this process, an in vitro cellular system that recapitulates endothelial differentiation and is amenable to experimental manipulations is required. Methods and Results—Embryonic cell lines that differentiate exclusively into endothelial cells were derived from early mouse embryos using empirical but reproducible culture techniques without viral or chemical transformation. The cells were not pluripotent and expressed reduced levels of Oct 4 and Rex-1. They were non-tumorigenic with a population doubling time of ≈15 hours. When plated on matrigel, they readily differentiated to form patent tubular structures with diameters of 30 to 150 &mgr;m. The differentiated cells endocytosed acetylated low-density lipoprotein (LDL) and began to express endothelial-specific markers such as CD34, CD31, Flk-1, TIE2, P-selectin, Sca-1, and thy-1. They also expressed genes essential for differentiation and maintenance of endothelial lineages, eg, Flk-1, vascular endothelial growth factor (VEGF), and angiopoietin-1. When transplanted into animal models, these cells incorporated into host vasculature. Conclusions—These cell lines can undergo in vitro and in vivo endothelial differentiation that recapitulated known endothelial differentiation pathways. Therefore, they are ideal for establishing an in vitro cellular system to study endothelial differentiation.

Hepatic expression of PPARα, a molecular target of fibrates, is regulated during inflammation in a gender‐specific manner

Dyslipidemia, inflammation and gender are major risk factors in cardiovascular disease. Here we show that hepatic expression of Peroxisome proliferator‐activated receptor α (PPARα), a nuclear receptor that regulates lipid metabolism and inflammation, is regulated in a gender‐specific manner during lipopolysaccharide (LPS)‐induced systemic inflammation. Immediately following LPS‐induced systemic inflammation, hepatic PPARα mRNA level decreased dramatically in mice. It was restored to baseline within 24 h in females but remained below baseline for >72 h in male mice. In gonadectomized mice of both sexes, PPARα mRNA level was restored to baseline within 48 h after the initial decrease.