Mesenchymal Stem Cells Secrete Multiple Cytokines That Promote Angiogenesis and Have Contrasting Effects on Chemotaxis and Apoptosis

We have previously shown that mesenchymal stem cells (MSC) improve function upon integration in ischemic myocardium. We examined whether specific cytokines and growth factors produced by MSCs are able to affect angiogenesis, cellular migration and apoptosis. Conditioned media (CM) was prepared by culturing MSC for 48 hours. CM displayed significantly elevated levels of VEGF, Monocyte Chemoattractant Protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monokine induced by IFN-γ (MIG) compared to control media. MSC contained RNA for these factors as detected by RT-PCR. CM was able to induce angiogenesis in canine vascular endothelial cells. MCP-1 and MIP-1α increased cell migration of MSC while VEGF reduced it. H9c2 cells treated with CM under hypoxic conditions for 24 hours displayed a 16% reduction in caspase-3 activity compared to controls. PI 3-kinase γ inhibitor had no effect on controls but reversed the effect of CM on caspase-3 activity. MCP-1 alone mimicked the protective effect of CM while the PI 3-Kγ inhibitor did not reverse the effect of MCP-1. CM reduced phospho-BAD (Ser112) and phospho-Akt (Ser473) while increasing phospho-Akt (Thr308). MCP-1 reduced the level of phospho-Akt (Ser473) while having no effect on the other two; the PI 3-Kγ inhibitor did not alter the MCP-1 effect. ERK 1/2 phosphorylation was reduced in CM treated H9c2 cells, and inhibition of ERK 1/2 reduced the phosphorylation of Akt (Ser473), Akt (Thr308) and Bad (Ser112). In conclusion, MSC synthesize and secrete multiple paracrine factors that are able to affect MSC migration, promote angiogenesis and reduce apoptosis. While both MCP-1 and PI3-kinase are involved in the protective effect, they are independent of each other. It is likely that multiple pro-survival factors in addition to MCP-1 are secreted by MSC which act on divergent intracellular signaling pathways

WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics

Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear Β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector Β-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.Peer reviewe

Growth factor stimulation of cardiomyocytes induces changes in the transcriptional contents of secreted exosomes

Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes.Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1) with or without growth factor treatment (TGF-β2 and PDGF-BB), with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS), electron microscopy (EM) and Western blot and analyzed with Illumina whole genome microarray gene expression.The exosomes were rounded in shape and had an average size of 50–90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-β2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217) were found in all 3 groups.We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system. To access the supplementary material to this article, please see Supplementary files under Article Tools online

Intramyocardial Transplantation of Undifferentiated Rat Induced Pluripotent Stem Cells Causes Tumorigenesis in the Heart

BACKGROUND: Induced pluripotent stem cells (iPSCs) are a novel candidate for use in cardiac stem cell therapy. However, their intrinsic tumorigenicity requires further investigation prior to use in a clinical setting. In this study we investigated whether undifferentiated iPSCs are tumorigenic after intramyocardial transplantation into immunocompetent allogeneic recipients. METHODOLOGY/PRINCIPAL FINDINGS: We transplanted 2 × 10(4), 2 × 10(5), or 2 × 10(6) cells from the established rat iPSC line M13 intramyocardially into intact or infarcted hearts of immunocompetent allogeneic rats. Transplant duration was 2, 4, or 6 weeks. Histological examination with hematoxylin-eosin staining confirmed that undifferentiated rat iPSCs could generate heterogeneous tumors in both intracardiac and extracardiac sites. Furthermore, tumor incidence was independent of cell dose, transplant duration, and the presence or absence of myocardial infarction. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that allogeneic iPSC transplantation in the heart will likely result in in situ tumorigenesis, and that cells leaked from the beating heart are a potential source of tumor spread, underscoring the importance of evaluating the safety of future iPSC therapy for cardiac disease

Direct targets of Klf5 transcription factor contribute to the maintenance of mouse embryonic stem cell undifferentiated state

<p>Abstract</p> <p>Background</p> <p>A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed.</p> <p>Results</p> <p>By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes.</p> <p>Conclusions</p> <p>Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype.</p> <p>See: <url>http://www.biomedcental.com/1741-7007/8/125</url></p

Effects of histocompatibility and host immune responses on the tumorigenicity of pluripotent stem cells

Pluripotent stem cells hold great promises for regenerative medicine. They might become useful as a universal source for a battery of new cell replacement therapies. Among the major concerns for the clinical application of stem cell-derived grafts are the risks of immune rejection and tumor formation. Pluripotency and tumorigenicity are closely linked features of pluripotent stem cells. However, the capacity to form teratomas or other tumors is not sufficiently described by inherited features of a stem cell line or a stem cell-derived graft. The tumorigenicity always depends on the inability of the recipient to reject the tumorigenic cells. This review summarizes recent data on the tumorigenicity of pluripotent stem cells in immunodeficient, syngeneic, allogeneic, and xenogeneic hosts. The effects of immunosuppressive treatment and cell differentiation are discussed. Different immune effector mechanisms appear to be involved in the rejection of undifferentiated and differentiated cell populations. Elements of the innate immune system, such as natural killer cells and the complement system, which are active also in syngeneic recipients, appear to preferentially reject undifferentiated cells. This effect could reduce the risk of tumor formation in immunocompetent recipients. Cell differentiation apparently increases susceptibility to rejection by the adaptive immune system in allogeneic hosts. The current data suggest that the immune system of the recipient has a major impact on the outcome of pluripotent stem cell transplantation, whether it is rejection, engraftment, or tumor development. This has to be considered when the results of experimental transplantation models are interpreted and even more when translation into clinics is planned

IL-4 as a Repurposed Biological Drug for Myocardial Infarction through Augmentation of Reparative Cardiac Macrophages: Proof-of-Concept Data in Mice.

Recent research has shown that reparative (alternatively activated or M2) macrophages play a role in repair of damaged tissues, including the infarcted hearts. Administration of IL-4 is known to augment M2 macrophages. This translational study thus aimed to investigate whether IL-4 administration is useful for the treatment of myocardial infarction. Long-acting IL-4 complex (IL-4c; recombinant IL-4 mixed with anti-IL-4 monoclonal antibody as a stabilizer) was administered after coronary artery ligation in mice. It was observed that IL-4c administration increased accumulation of CD206+F4/80+ M2-like macrophages predominantly in the injured myocardium, compared to the control. Sorted cardiac M2-like macrophages highly expressed wide-ranging tissue repair-related genes. Indeed, IL-4c administration enhanced cardiac function in association with reduced infarct size and enhanced tissue repair (strengthened connective tissue formation, improved microvascular formation and attenuated cardiomyocyte hypertrophy). Experiments using Trib1 -/- mice that had a depleted ability to develop M2 macrophages and other in-vitro studies supported that these IL-4-mediated effects were induced via M2-like macrophages. On the other hand, when administered at Day 28 post-MI, the effects of IL-4c were diminished, suggesting a time-frame for IL-4 treatment to be effective. These data represent proof-of-concept of efficacy of IL-4 treatment for acute myocardial infarction, encouraging its further development.This project was funded by the Heart Research UK Translational Research Grant (RG2653/15/16), British Heart Foundation Programme Grant (RG/15/31236), and Queen Mary Innovation Proof of Concept Grant (2015). The National Institute for Health Researchfunded Barts Cardiovascular Biomedical Research Unit also supported this project.This project was funded by the Heart Research UK Translational Research Grant (RG2653/15/16), British Heart Foundation Programme Grant (RG/15/31236), and Queen Mary Innovation Proof of Concept Grant (2015). The National Institute for Health Research-funded Barts Cardiovascular Biomedical Research Unit also supported this project

A systematic review of cerebral microdialysis and outcomes in TBI: relationships to patient functional outcome, neurophysiologic measures, and tissue outcome

OBJECTIVE: To perform a systematic review on commonly measured cerebral microdialysis (CMD) analytes and their association to: (A) patient functional outcome, (B) neurophysiologic measures, and (C) tissue outcome; after moderate/severe TBI. The aim was to provide a foundation for next-generation CMD studies and build on existing pragmatic expert guidelines for CMD. METHODS: We searched MEDLINE, BIOSIS, EMBASE, Global Health, Scopus, Cochrane Library (inception to October 2016). Strength of evidence was adjudicated using GRADE. RESULTS: (A) Functional Outcome: 55 articles were included, assessing outcome as mortality or Glasgow Outcome Scale (GOS) at 3-6 months post-injury. Overall, there is GRADE C evidence to support an association between CMD glucose, glutamate, glycerol, lactate, and LPR to patient outcome at 3-6 months. (B) Neurophysiologic Measures: 59 articles were included. Overall, there currently exists GRADE C level of evidence supporting an association between elevated CMD measured mean LPR, glutamate and glycerol with elevated ICP and/or decreased CPP. In addition, there currently exists GRADE C evidence to support an association between elevated mean lactate:pyruvate ratio (LPR) and low PbtO2. Remaining CMD measures and physiologic outcomes displayed GRADE D or no evidence to support a relationship. (C) Tissue Outcome: four studies were included. Given the conflicting literature, the only conclusion that can be drawn is acute/subacute phase elevation of CMD measured LPR is associated with frontal lobe atrophy at 6 months. CONCLUSIONS: This systematic review replicates previously documented relationships between CMD and various outcome, which have driven clinical application of the technique. Evidence assessments do not address the application of CMD for exploring pathophysiology or titrating therapy in individual patients, and do not account for the modulatory effect of therapy on outcome, triggered at different CMD thresholds in individual centers. Our findings support clinical application of CMD and refinement of existing guidelines

Mesenchymal stem cells: from experiment to clinic

There is currently much interest in adult mesenchymal stem cells (MSCs) and their ability to differentiate into other cell types, and to partake in the anatomy and physiology of remote organs. It is now clear these cells may be purified from several organs in the body besides bone marrow. MSCs take part in wound healing by contributing to myofibroblast and possibly fibroblast populations, and may be involved in epithelial tissue regeneration in certain organs, although this remains more controversial. In this review, we examine the ability of MSCs to modulate liver, kidney, heart and intestinal repair, and we update their opposing qualities of being less immunogenic and therefore tolerated in a transplant situation, yet being able to contribute to xenograft models of human tumour formation in other contexts. However, such observations have not been replicated in the clinic. Recent studies showing the clinical safety of MSC in several pathologies are discussed. The possible opposing powers of MSC need careful understanding and control if their clinical potential is to be realised with long-term safety for patients