MSC Pretreatment for Improved Transplantation Viability Results in Improved Ventricular Function in Infarcted Hearts.

Many clinical studies utilizing MSCs (mesenchymal stem cells, mesenchymal stromal cells, or multipotential stromal cells) are underway in multiple clinical settings; however, the ideal approach to prepare these cells in vitro and to deliver them to injury sites in vivo with maximal effectiveness remains a challenge. Here, pretreating MSCs with agents that block the apoptotic pathways were compared with untreated MSCs. The treatment effects were evaluated in the myocardial infarct setting following direct injection, and physiological parameters were examined at 4 weeks post-infarct in a rat permanent ligation model. The prosurvival treated MSCs were detected in the hearts in greater abundance at 1 week and 4 weeks than the untreated MSCs. The untreated MSCs improved ejection fraction in infarcted hearts from 61% to 77% and the prosurvival treated MSCs further improved ejection fraction to 83% of normal. The untreated MSCs improved fractional shortening in the infarcted heart from 52% to 68%, and the prosurvival treated MSCs further improved fractional shortening to 77% of normal. Further improvements in survival of the MSC dose seems possible. Thus, pretreating MSCs for improved in vivo survival has implications for MSC-based cardiac therapies and in other indications where improved cell survival may improve effectiveness.

Concise Review: MSC-Derived Exosomes for Cell-Free Therapy.

Mesenchymal stem cell transplantation is undergoing extensive evaluation as a cellular therapy in human clinical trials. Because MSCs are easily isolated and amenable to culture expansion in vitro there is a natural desire to test MSCs in many diverse clinical indications. This is exemplified by the rapidly expanding literature base that includes many in vivo animal models. More recently, MSC-derived extracellular vesicles (EVs), which include exosomes and microvesicles (MV), are being examined for their role in MSC-based cellular therapy. These vesicles are involved in cell-to-cell communication, cell signaling, and altering cell or tissue metabolism at short or long distances in the body. The exosomes and MVs can influence tissue responses to injury, infection, and disease. MSC-derived exosomes have a content that includes cytokines and growth factors, signaling lipids, mRNAs, and regulatory miRNAs. To the extent that MSC exosomes can be used for cell-free regenerative medicine, much will depend on the quality, reproducibility, and potency of their production, in the same manner that these parameters dictate the development of cell-based MSC therapies. However, the MSC exosome's contents are not static, but rather a product of the MSC tissue origin, its activities and the immediate intercellular neighbors of the MSCs. As such, the exosome content produced by MSCs appears to be altered when MSCs are cultured with tumor cells or in the in vivo tumor microenvironment. Therefore, careful attention to detail in producing MSC exosomes may provide a new therapeutic paradigm for cell-free MSC-based therapies with decreased risk. Stem Cells 2017;35:851-858.

Mesenchymal stem cells preserve neonatal right ventricular function in a porcine model of pressure overload.

Limited therapies exist for patients with congenital heart disease (CHD) who develop right ventricular (RV) dysfunction. Bone marrow-derived mesenchymal stem cells (MSCs) have not been evaluated in a preclinical model of pressure overload, which simulates the pathophysiology relevant to many forms of CHD. A neonatal swine model of RV pressure overload was utilized to test the hypothesis that MSCs preserve RV function and attenuate ventricular remodeling. Immunosuppressed Yorkshire swine underwent pulmonary artery banding to induce RV dysfunction. After 30 min, human MSCs (1 million cells, n = 5) or placebo (n = 5) were injected intramyocardially into the RV free wall. Serial transthoracic echocardiography monitored RV functional indices including 2D myocardial strain analysis. Four weeks postinjection, the MSC-treated myocardium had a smaller increase in RV end-diastolic area, end-systolic area, and tricuspid vena contracta width (P < 0.01), increased RV fractional area of change, and improved myocardial strain mechanics relative to placebo (P < 0.01). The MSC-treated myocardium demonstrated enhanced neovessel formation (P < 0.0001), superior recruitment of endogenous c-kit+ cardiac stem cells to the RV (P < 0.0001) and increased proliferation of cardiomyocytes (P = 0.0009) and endothelial cells (P < 0.0001). Hypertrophic changes in the RV were more pronounced in the placebo group, as evidenced by greater wall thickness by echocardiography (P = 0.008), increased cardiomyocyte cross-sectional area (P = 0.001), and increased expression of hypertrophy-related genes, including brain natriuretic peptide, ß-myosin heavy chain and myosin light chain. Additionally, MSC-treated myocardium demonstrated increased expression of the antihypertrophy secreted factor, growth differentiation factor 15 (GDF15), and its downstream effector, SMAD 2/3, in cultured neonatal rat cardiomyocytes and in the porcine RV myocardium. This is the first report of the use of MSCs as a therapeutic strategy to preserve RV function and attenuate remodeling in the setting of pressure overload. Mechanistically, transplanted MSCs possibly stimulated GDF15 and its downstream SMAD proteins to antagonize the hypertrophy response of pressure overload. These encouraging results have implications in congenital cardiac pressure overload lesions.

Pim-1 kinase cooperates with serum signals supporting mesenchymal stem cell propagation.

Mesenchymal stem cells (MSCs) are currently undergoing testing in several clinical settings. The propagation of MSCs from multiple species in culture is an important step in furthering our understanding of these progenitor cells. Pim-1, a proto-oncogenic serine/threonine kinase, regulates cell proliferation, survival, and differentiation. Although it has been shown that Pim-1 participates in signal transduction mediating mitogenic action in MSCs, its roles in the modulation of MSC propagation remain to be defined. Understanding of ovine MSCs transduced with Pim-1 may provide improved ovine models for cellular therapy development. Using genetically modified ovine MSCs that constitutively overexpressed Pim-1 (MSC expressing PIM-1 and ZsGreen protein), we evaluated the impact of elevated Pim-1 activity on the proliferation, survival, and differentiation of MSCs in culture. Our results showed that Pim-1 enhanced the intrinsic molecular signals of growth and survival implicated in the mediation of serum signaling under normal culture conditions (10% serum). We found that Pim-1 promoted MSC proliferation irrespectively of the serum concentration, but with a decreased proliferation rate compared to increased serum concentrations, relative to the control vector-transduced MSC expressing ZsGreen protein. Further, Pim-1 prevented MSC apoptosis induced by hypoxia or serum deprivation as evidenced by enhanced mitochondria integrity and reduced annexin V binding. Interestingly, the phenotype and multilineage differentiation potential of the cells were not influenced by Pim-1. Taken together, these observations demonstrate that Pim-1 kinase cooperates with exogenous serum signals supporting MSC propagation in the ovine model.

Editorial: Our top 10 developments in stem cell biology over the last 30 years.

To celebrate 30 years of peer-reviewed publication of cutting edge stem cell research in Stem Cells, the first journal devoted to this promising field, we pause to review how far we have come in the three-decade lifetime of the Journal. To do this, we will present our views of the 10 most significant developments that have advanced stem cell biology where it is today. With the increasing rate of new data, it is natural that the bulk of these developments would have occurred in recent years, but we must not think that stem cell biology is a young science. The idea of a stem cell has actually been around for quite a long time having appeared in the scientific literature as early as 1868 with Haeckels' concept of a stamzelle as an uncommitted or undifferentiated cell responsible for producing many types of new cells to repair the body [Naturliche Schopfungsgeschichte, 1868; Berlin: Georg Reimer] but it took many years to obtain hard evidence in support of this theory. Not until the work of James Till and Ernest McCulloch in the 1960s did we have proof of the existence of stem cells and until the derivation of embryonal carcinoma cells in the 1960s-1970s and the first embryonic stem cell in 1981, such adult or tissue-specific stem cells were the only known class. The first issue of Stem Cells was published in 1981; no small wonder that most of its papers were devoted to hematopoietic progenitors. More recently, induced pluripotent stem cells (iPSCs) have been developed, and this is proving to be a fertile area of investigation as shown by the volume of publications appearing not only in Stem Cells but also in other journals over the last 5 years. The reader will note that many of the articles in this special issue are concerned with iPSC; however, this reflects the current surge of interest in the topic rather than any deliberate attempt to ignore other areas of stem cell investigation.

Mesenchymal stem cell perspective: cell biology to clinical progress.

The terms MSC and MSCs have become the preferred acronym to describe a cell and a cell population of multipotential stem/progenitor cells commonly referred to as mesenchymal stem cells, multipotential stromal cells, mesenchymal stromal cells, and mesenchymal progenitor cells. The MSCs can differentiate to important lineages under defined conditions in vitro and in limited situations after implantation in vivo. MSCs were isolated and described about 30 years ago and now there are over 55,000 publications on MSCs readily available. Here, we have focused on human MSCs whenever possible. The MSCs have broad anti-inflammatory and immune-modulatory properties. At present, these provide the greatest focus of human MSCs in clinical testing; however, the properties of cultured MSCs in vitro suggest they can have broader applications. The medical utility of MSCs continues to be investigated in over 950 clinical trials. There has been much progress in understanding MSCs over the years, and there is a strong foundation for future scientific research and clinical applications, but also some important questions remain to be answered. Developing further methods to understand and unlock MSC potential through intracellular and intercellular signaling, biomedical engineering, delivery methods and patient selection should all provide substantial advancements in the coming years and greater clinical opportunities. The expansive and growing field of MSC research is teaching us basic human cell biology as well as how to use this type of cell for cellular therapy in a variety of clinical settings, and while much promise is evident, careful new work is still needed.

Dual-modality monitoring of targeted intraarterial delivery of mesenchymal stem cells after transient ischemia.

BACKGROUND AND PURPOSE: In animal models of stroke, functional improvement has been obtained after stem cell transplantation. Successful therapy depends largely on achieving a robust and targeted cell engraftment, with intraarterial (IA) injection being a potentially attractive route of administration. We assessed the suitability of laser Doppler flow (LDF) signal measurements and magnetic resonance (MR) imaging for noninvasive dual monitoring of targeted IA cell delivery. METHODS: Transient cerebral ischemia was induced in adult Wistar rats (n=25) followed by IA or intravenous (IV) injection of mesenchymal stem cells (MSCs) labeled with superparamagnetic iron oxide. Cell infusion was monitored in real time with transcranial laser Doppler flowmetry while cellular delivery was assessed with MRI in vivo (4.7 T) and ex vivo (9.4 T). RESULTS: Successful delivery of magnetically labeled MSCs could be readily visualized with MRI after IA but not IV injection. IA stem cell injection during acute stroke resulted in a high variability of cerebral engraftment. The amount of LDF reduction during cell infusion (up to 80%) was found to correlate well with the degree of intracerebral engraftment, with low LDF values being associated with significant morbidity. CONCLUSIONS: High cerebral engraftment rates are associated with impeded cerebral blood flow. Noninvasive dual-modality imaging enables monitoring of targeted cell delivery, and through interactive adjustment may improve the safety and efficacy of stem cell therapy.

Mesenchymal stem cells from adult bone marrow.

Mesenchymal stem cells (MSCs), sometimes referred to as marrow stromal cells or multipotential stromal cells, represent a class of adult progenitor cells capable of differentiation to several mesenchymal lineages. They can be isolated from many tissues although bone marrow has been used most often. The MSCs may prove useful for repair and regeneration of a variety of mesenchymal tissues such as bone, cartilage, muscle, marrow stroma, and the cells produce useful growth factors and cytokines that may help repair additional tissues. There is also evidence for their differentiation to nonmesenchymal lineages, but that work will not be considered here. This chapter will provide the researcher with some background, and then provide details on MSC isolation, expansion and multilineage differentiation. These are the beginning steps toward formulating tissue repair strategies. The methods provided here have been used in many laboratories around the world and the reader can begin by following the methods presented here, and then test other methods if these prove unsatisfactory for your intended purpose.

Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction.

BACKGROUND: Recent results from animal studies suggest that stem cells may be able to home to sites of myocardial injury to assist in tissue regeneration. However, the histological interpretation of postmortem tissue, on which many of these studies are based, has recently been widely debated. METHODS AND RESULTS: With the use of the high sensitivity of a combined single-photon emission CT (SPECT)/CT scanner, the in vivo trafficking of allogeneic mesenchymal stem cells (MSCs) colabeled with a radiotracer and MR contrast agent to acute myocardial infarction was dynamically determined. Redistribution of the labeled MSCs after intravenous injection from initial localization in the lungs to nontarget organs such as the liver, kidney, and spleen was observed within 24 to 48 hours after injection. Focal and diffuse uptake of MSCs in the infarcted myocardium was already visible in SPECT/CT images in the first 24 hours after injection and persisted until 7 days after injection and was validated by tissue counts of radioactivity. In contrast, MRI was unable to demonstrate targeted cardiac localization of MSCs in part because of the lower sensitivity of MRI. CONCLUSIONS: Noninvasive radionuclide imaging is well suited to dynamically track the biodistribution and trafficking of mesenchymal stem cells to both target and nontarget organs.

Mesenchymal stem cells and their potential as cardiac therapeutics.

Mesenchymal stem cells (MSCs) represent a stem cell population present in adult tissues that can be isolated, expanded in culture, and characterized in vitro and in vivo. MSCs differentiate readily into chondrocytes, adipocytes, osteocytes, and they can support hematopoietic stem cells or embryonic stem cells in culture. Evidence suggests MSCs can also express phenotypic characteristics of endothelial, neural, smooth muscle, skeletal myoblasts, and cardiac myocyte cells. When introduced into the infarcted heart, MSCs prevent deleterious remodeling and improve recovery, although further understanding of MSC differentiation in the cardiac scar tissue is still needed. MSCs have been injected directly into the infarct, or they have been administered intravenously and seen to home to the site of injury. Examination of the interaction of allogeneic MSCs with cells of the immune system indicates little rejection by T cells. Persistence of allogeneic MSCs in vivo suggests their potential "off the shelf" therapeutic use for multiple recipients. Clinical use of cultured human MSCs (hMSCs) has begun for cancer patients, and recipients have received autologous or allogeneic MSCs. Research continues to support the desirable traits of MSCs for development of cellular therapeutics for many tissues, including the cardiovascular system. In summary, hMSCs isolated from adult bone marrow provide an excellent model for development of stem cell therapeutics, and their potential use in the cardiovascular system is currently under investigation in the laboratory and clinical settings.

Unraveling the Mesenchymal Stromal Cells' Paracrine Immunomodulatory Effects.

In the last 10 years, the role of mesenchymal stromal cells (MSCs) in modulating inflammatory and immune responses has been characterized using both in vitro studies and in vivo models of immune disorders. Mesenchymal stromal cell immunomodulatory properties have been linked to various paracrine factors which expression varies depending on the pathologic condition to which the MSCs are exposed. These factors may directly impact key cells of the adaptive immune system, such as T cells. Indeed, coculturing MSCs with T cells in a mixed lymphocyte reaction assay inhibits T-cell proliferation through the secretion of immunomodulatory cytokines. However, in a context of inflammation, MSCs may secrete paracrine factors that influence other immune cell subpopulations such as dendritic cells and macrophages and polarize them toward a tolerogenic phenotype. In vivo, these same immunomodulatory factors are shown to be increased in the serum of animal models presenting with inflammatory diseases treated with MSC administration. In light of the results from these landmark studies, we review the main MSC secreted factors identified to play a role in modulating inflammatory immune responses either in vitro or in vivo, and we assess the impact of these factors on the therapeutic applications of MSC-based cell therapies in immune diseases.

Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.

BACKGROUND: Cellular cardiomyoplasty has been proposed as an alternative strategy for augmenting the function of diseased myocardium. We investigated the potential of human mesenchymal stem cells (hMSCs) from adult bone marrow to undergo myogenic differentiation once transplanted into the adult murine myocardium. METHODS AND RESULTS: A small bone marrow aspirate was taken from the iliac crest of healthy human volunteers, and hMSCs were isolated as previously described. The stem cells, labeled with lacZ, were injected into the left ventricle of CB17 SCID/beige adult mice. At 4 days after injection, none of the engrafted hMSCs expressed myogenic markers. A limited number of cells survived past 1 week and over time morphologically resembled the surrounding host cardiomyocytes. Immunohistochemistry revealed de novo expression of desmin, beta-myosin heavy chain, alpha-actinin, cardiac troponin T, and phospholamban at levels comparable to those of the host cardiomyocytes; sarcomeric organization of the contractile proteins was observed. In comparison, neither cardiac troponin T nor phospholamban was detected in the myotubes formed in vitro by MyoD-transduced hMSCs. CONCLUSIONS: The purified hMSCs from adult bone marrow engrafted in the myocardium appeared to differentiate into cardiomyocytes. The persistence of the engrafted hMSCs and their in situ differentiation in the heart may represent the basis for using these adult stem cells for cellular cardiomyoplasty.

In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction.

BACKGROUND: We investigated the potential of magnetic resonance imaging (MRI) to track magnetically labeled mesenchymal stem cells (MR-MSCs) in a swine myocardial infarction (MI) model. METHODS AND RESULTS: Adult farm pigs (n=5) were subjected to closed-chest experimental MI. MR-MSCs (2.8 to 16x107 cells) were injected intramyocardially under x-ray fluoroscopy. MRIs were obtained on a 1.5T MR scanner to demonstrate the location of the MR-MSCs and were correlated with histology. Contrast-enhanced MRI demonstrated successful injection in the infarct and serial MSC tracking was demonstrated in two animals. CONCLUSIONS: MRI tracking of MSCs is feasible and represents a preferred method for studying the engraftment of MSCs in MI.

Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects.

BACKGROUND: A novel therapeutic option for the treatment of acute myocardial infarction involves the use of mesenchymal stem cells (MSCs). The purpose of this study was to investigate whether implantation of autologous MSCs results in sustained engraftment, myogenic differentiation, and improved cardiac function in a swine myocardial infarct model. METHODS: MSCs were isolated and expanded from bone marrow aspirates of 14 domestic swine. A 60-minute left anterior descending artery occlusion was used to produce anterior wall infarction. Piezoelectric crystals were placed within the ischemic region for measurement of regional wall thickness and contractile function. Two weeks later animals autologous, Di-I-labeled MSCs (6 x 10(7)) were implanted into the infarct by direct injection. Hemodynamic and functional measurements were obtained weekly until the time of sacrifice. Immunohistochemistry was used to assess MSC engraftment and myogenic differentiation. RESULTS: Microscopic analysis showed robust engraftment of MSCs in all treated animals. Expression of muscle-specific proteins was seen as early as 2 weeks and could be identified in all animals at sacrifice. The degree of contractile dysfunction was significantly attenuated at 4 weeks in animals implanted with MSCs (5.4% +/- 2.2% versus -3.37% +/- 2.7% in control). In addition, the extent of wall thinning after myocardial infarction was markedly reduced in treated animals. CONCLUSIONS: Mesenchymal stem cells are capable of engraftment in host myocardium, demonstrate expression of muscle specific proteins, and may attenuate contractile dysfunction and pathologic thinning in this model of left ventricular wall infarction. MSC cardiomyoplasty may have significant clinical potential in attenuating the pathology associated with myocardial infarction.

Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction.

Progressive cardiac remodeling, including the myopathic process in the adjacent zone following myocardial infarction (MI), contributes greatly to the development of cardiac failure. Cardiomyoplasty using bone marrow-derived mesenchymal stem cells (MSCs) has been demonstrated to protect cardiomyocytes and/or repair damaged myocardium, leading to improved cardiac performance, but the therapeutic effects on cardiac remodeling are still under investigation. Here, we tested the hypothesis that MSCs could improve the pathological remodeling of the adjacent myocardium abutting the infarct. Allogeneic ovine MSCs were transplanted into the adjacent zone by intracardiac injection 4 hours after infarction. Results showed that remodeling and contractile strain alteration were reduced in the adjacent zone of the MSC-treated group. Cardiomyocyte hypertrophy was significantly attenuated with the normalization of the hypertrophy-related signaling proteins phosphatidylinositol 3-kinase α (PI3Kα), PI3Kγ, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (p-ERK) in the adjacent zone of the MSC-treated group versus the MI-alone group. Moreover, the imbalance of the calcium-handling proteins sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA2a), phospholamban (PLB), and sodium/calcium exchanger type 1 (NCX-1) induced by MI was prevented by MSC transplantation, and more strikingly, the activity of SERCA2a and uptake of calcium were improved. In addition, the upregulation of the proapoptotic protein Bcl-xL/Bcl-2-associated death promoter (BAD) was normalized, as was phospho-Akt expression; there was less fibrosis, as revealed by staining for collagen; and the apoptosis of cardiomyocytes was significantly inhibited in the adjacent zone by MSC transplantation. Collectively, these data demonstrate that MSC implantation improved the remodeling in the region adjacent to the infarct after cardiac infarction in the ovine infarction model.

Sleuthing the source of regeneration by MSCs.

Mesenchymal stem cells (MSCs) can differentiate into useful cell types and also have the ability to modulate inflammation. In this issue of Cell Stem Cell, Lee et al. (2009) investigate the production of the soluble inflammation inhibitor TSG-6 by MSCs.