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Cell therapy for cardiovascular diseases is regarded as a rapidly growing field within regenerative medicine. Different cellular populations enriched for cardiac progenitor cells (CPCs), or derivate a-cellular products, are currently under preclinical and clinical evaluation. Here, we have reviewed the described mechanisms whereby resident post-natal CPCs, isolated in different ways, act as a therapeutic product on the damaged myocardium. Several biological mechanisms of action have been described which can explain the multiple therapeutic effects of CPC treatment observed on cardiac function and remodelling. These mechanisms span from direct cardiovascular differentiation, through induction of resident progenitor proliferation, to paracrine effects on cardiac and non-cardiac cells mediated by exosomes and non-coding RNAs. All the reported mechanisms of action support an integrated view including cardiomyogenesis, cardioprotection, and anti-fibrotic effects. Moreover, future developments of CPC therapy approaches may support cell-free strategies, exploiting effective pleiotropic cell-derived products, such as exosomes.

Submitted by BIANCA MARIANI GONÇALVES (bia.mariani@yahoo.com.br) on 2018-02-23T04:26:01Z No. of bitstreams: 1 VERSÃO PÓS DEFESA - 22.02.pdf: 2194277 bytes, checksum: 5b59ce8ea0c427305edc63f95e91d00f (MD5) Rejected by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br), reason: Prezada Inserir em seu arquivo a ficha catalográfica - solicite em : fichacatalografica@fca.unesp.br Inserir nos agradecimentos - Agradecimento ao órgão financiador - CNPQ on 2018-02-23T11:14:10Z (GMT) Submitted by BIANCA MARIANI GONÇALVES (bia.mariani@yahoo.com.br) on 2018-02-23T19:37:18Z No. of bitstreams: 1 VERSÃO PÓS DEFESA - 23.02.pdf: 2223850 bytes, checksum: 99d0c3b86baf39d8a2b0bbf2f6a38421 (MD5) Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-02-26T12:22:25Z (GMT) No. of bitstreams: 1 goncalves_bm_me_botfca_par.pdf: 504384 bytes, checksum: e44c795c941c759f9229a26e917a455a (MD5) Made available in DSpace on 2018-02-26T12:22:25Z (GMT). No. of bitstreams: 1 goncalves_bm_me_botfca_par.pdf: 504384 bytes, checksum: e44c795c941c759f9229a26e917a455a (MD5) Previous issue date: 2018-02-16 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) O diabetes mellitus (DM) é uma das doenças que mais tem aumentado sua incidência na última década. As células-tronco mesenquimais derivadas de tecido adiposo (CTMs-TA) possuem características imunomoduladoras e reparadoras, sendo capazes de reverter efeitos sistêmicos da doença. Sendo assim, nosso trabalho teve como objetivo avaliar o impacto do transplante de CTMs-TA pelas vias intravenosa, intrapancreática e intra-capsular renal em ratos diabéticos e comparar a que melhor exerce efeito regenerador. Para o estudo, utilizamos 45 ratos, wistar, fêmeas, divididos em 5 grupos: GCS – controle sadio; GCD – controle diabético; GIV – diabético, via intravenosa; GIP – diabético, via intrapancreática; GIR – diabético, via cápsula renal. Para a indução, utilizamos uma única aplicação intraperitoneal (i.p) de Aloxana (120mg/kg), e após 3 dias, os animais foram submetidos ao transplante com as CTMs-TA (2x106cel/animal). Após 15 dias do transplante, os animais foram eutanasiados. O transplante de CTMs-TA pela cápsula renal foi capaz de reverter a hiperglicemia, além de restaurar o diâmetro das ilhotas e restabelecer a função das massa β-pancreática. Concluímos que a cápsula renal foi a que apresentou melhores resultados na regeneração morfofuncional do pâncreas diabético. As CTMs-TA utilizadas neste estudo foram capazes de exercer um importante efeito parácrino, podendo ser utilizadas como alternativa para a melhoria da qualidade de vida de indivíduos diabéticos. Diabetes mellitus is one of the diseases that has increased your incidence in the last decade. Adipose-Derived Mesenchymal Stem Cells (ATMSCs) have immunomodulatory and regenerate characteristics, thus are capable of reverse the damages of the disease. Therefore, our study aimed to evaluate the impact of ATMSCs transplantation by the intravenous, intrapancreatic and intra-capsular renal routes in diabetic rats and detect the best route to promote regenerative effects. We used 45 female wistar rats, divided into 5 groups: GCS – healthy control; GCD - diabetic control; GIV - diabetic, intravenous; GIP - diabetic, intrapancreatic route; GIR - diabetic, via renal capsule. For induction, we used a single intraperitoneal application (i.p) of Alloxan (120mg / kg) and after 3 days, the animals were transplanted with the ATMSCs (2x106/ animal). After 15 days of transplantation, the animals were euthanized. The transplantation of ATMSCs by the renal capsule were able to reverse hyperglycemia, besides restoring the islet diameter and restoring the function of β-pancreatic mass. We conclude that the renal capsule presented the best results in the morphofunctional regeneration of the diabetic pancreas. The stem cells used in this study were able to exert an important paracrine effect,and could be used as an alternative to improve the quality of life on diabetic patients. CNPQ: 134517/2016-4

Liver fibrosis is a disease that causes high morbidity and has become a major health problem. Liver fibrosis can lead to the end stage of liver diseases (livercirrhosisand hepatocellularcarcinoma). Currently, liver transplantation is the only effective treatment for end-stage liver disease. However, the shortage of organ donors, high cost of medical surgery, immunological rejection and transplantation complications severely hamper liver transplantation therapy. Mesenchymal stem cells (MSCs) have been regarded as promising cells for clinical applications in stem cell therapy in the treatment of liver diseases due to their unique multipotent differentiation capacity, immunoregulation and paracrine effects. Although liver fibrosis improvements by MSC transplantation in preclinical experiments as well as clinical trials have been reported, the in vivo fate of MSCs after transportation and their therapeutic mechanisms remain unclear. In this present study, we isolated MSCs from the bone marrow of rhesus macaques. The cells exhibited typical MSC markers and could differentiate into chondrocytes, osteocytes, and adipocytes, which were not affected by labeling with enhanced green fluorescent protein (EGFP). The harvested MSCs respond to interferon-γ stimulation and have the ability to inhibit lymphocyte proliferation in vitro. EGFP-labeled MSCs (1 × 106 cells) were transplanted into mice with carbon tetrachloride-induced liver fibrosis via tail vein injection. The ability of the heterogenic MSC infusion to ameliorate liver fibrosis in mice was evaluated by a blood plasma chemistry index, pathological examination and liver fibrosis-associated gene expression. Additionally, a small number of MSCs that homed and engrafted in the mouse liver tissues were evaluated by immunofluorescence analysis. Our results showed that the transplantation of heterogenic MSCs derived from monkey bone marrow can be used to treat liver fibrosis in the mouse model and that the paracrine effects of MSCs may play an important role in the improvement of liver fibrosis.

Over the past decade, human term placenta has become more than a vital organ during pregnancy, but also a precious reservoir of cells. An increasing number of studies have shown that these cells harbor beneficial properties. In fact, we and others have reported the therapeutic effects of placental cells in preclinical models of lung and liver fibrosis, sepsis, inflammatory bowel disease, autoimmune encephalomyelitis, cardiac ischemia and hind limb ischemia. Remarkably, the diseases which were most attenuated by placental cell treatment were those with underlying altered immune reactions. We have significantly contributed to the understanding of the immune-modulatory properties of cells from the amniotic membrane in vitro, showing that they can reduce the proliferation of T cell subsets, down regulate Th1 and Th17 subsets, and increase T lymphocytes with regulatory functions (Treg). Contributing to their regenerative potential, placenta-derived cells have been reported to secrete a variety of growth factors that could act on progenitor and/or resident cells to favor tissue regeneration. For example, placental cells can release pro-angiogenic factors, such as hepatocyte growth factor, and mediators in extracellular matrix degradation, such as matrix metalloproteinases (MMPs). Moreover, the release of growth factors could stimulate resident stem cells to proliferate, altogether contributing to tissue regeneration. Interestingly, to further substantiate the observation that secreted factors are the main players in the therapeutic properties of placental cells, an increasing number of studies have shown that these beneficial effects are evident when conditioned medium obtained from cell culture is used or when cells are cultured in transwell systems.In conclusion, the placenta is a rich resource of therapeutic derivatives, such as cells, their secreted factors, and also others such as amniotic membrane patches, the latter of which have been successfully used in medicine for over a century. More recently, placental cells and their derivatives are being testing in clinical trials in patients with immune-dysregulated diseases.This work was supported by Fondazione Poliambulanza, Fondazione Cariplo, Ministero dell’Istruzione, dell’Università e della Ricerca, and Ministero della Salute., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

WOS:000346650300017; International audience; Objective: The anti-inflammatory and anti-catabolic effects of neonatal Mesenchymal Stromal Cell (MSC) were investigated in a xenogeneic model of mild osteoarthritis (OA). The paracrine properties of MSC on synoviocytes were further investigated in vitro. Study design: OA was induced by medial meniscal release (MMR) in 30 rabbit knees. A single early (day 3) or delayed (day 15) intra-articular (IA) injection of MSC isolated from equine Umbilical Cord Wharton's jelly (UC-MSC) was performed. Rabbits were euthanized on days 15 or 56. OA grading was performed and gene expression of inflammatory cytokines and metalloproteinases was measured in synovial tissue. Paracrine effects of UC-MSC were investigated using UC-conditioned vs control medium on rabbit primary synoviocytes stimulated with interleukin 1 beta in vitro. Results: No adverse local or systemic responses were observed clinically after xenogeneic UC-MSC injection. At study end point, cartilage fibrillation was lower in early treatment than in delayed treatment group. Cellular infiltrate was observed in the synovium of both UC-MSC groups. OA synovium exhibited a reduced expression of metalloproteinases-1, -3, -13 in the early cell-treated group at d56. In vitro, UC-conditioned medium exerted anti-inflammatory and anti-catabolic effects on synoviocytes exposed to pro-inflammatory stimulus. Conclusions: Early IA injection of equine UC-MSC was effective in preventing OA signs in rabbit knees following MMR. UC-MSC target the synovium and modulate the gene expression pattern of synoviocytes to promote an anti-catabolic environment. This confirms the synovium is a major target and mediator of MSC therapy, modulating the expression of matrix-degrading enzymes. (C) 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Background Our aim was to examine the roles of mesenchymal stem cell (MSC) transplantation in the repair of large uterine defects. Methods Uterine defects were created in both uterine horns of female rats by a punch instrument, and bone marrow-derived MSCs, MSC-conditioned medium (MSC-CM) or vehicle were injected into the myometrium around the defect. The rate of uterine defect repair was monitored on day 2 and 4 after operation. Cytokine array of MSC-CM was performed, followed by neutralizing antibody experiments to clarify the exact cytokine participating in the MSC-CM-enhanced wound repair. Results Transplantation of MSCs, but not myometrial cells, significantly enhanced uterine defect repair. The transplanted MSCs were detected in the uterine horn with no signs of rejection on day 4 after transplantation, when the MSC-transplanted uterine wound was nearly healed. Moreover, uterine defect repair was also accelerated by injection of MSC-CM, indicating the paracrine effects of MSCs on uterine wound healing. Cytokine array analysis further revealed that MSC-CM contained abundant cytokines and chemokines, among which high levels of interleukin-6 (IL-6) were found. Additionally, antibodies against IL-6 were shown to block MSC-CM-enhanced uterine defect repair. Conclusion This study demonstrated that transplantation of MSCs could enhance uterine defect repair by paracrine effects involving IL-6, which are findings that may be applied to facilitate uterine wound healing in the removal of huge intramural masses.

Background Cardiac regenerative medicine is a rapidly evolving field, with promising future developments for effective personalized treatments. Several stem/progenitor cells are candidates for cardiac cell therapy, and emerging evidence suggests how multiple metabolic and biochemical pathways strictly regulate their fate and renewal. Scope of review In this review, we will explore a selection of areas of common interest for biology and biochemistry concerning stem/progenitor cells, and in particular cardiac progenitor cells. Numerous regulatory mechanisms have been identified that link stem cell signaling and functions to the modulation of metabolic pathways, and vice versa. Pharmacological treatments and culture requirements may be exploited to modulate stem cell pluripotency and self-renewal, possibly boosting their regenerative potential for cell therapy. Major conclusions Mitochondria and their many related metabolites and messengers, such as oxygen, ROS, calcium and glucose, have a crucial role in regulating stem cell fate and the balance of their functions, together with many metabolic enzymes. Furthermore, protein biochemistry and proteomics can provide precious clues on the definition of different progenitor cell populations, their physiology and their autocrine/paracrine regulatory/signaling networks. General significance Interdisciplinary approaches between biology and biochemistry can provide productive insights on stem/progenitor cells, allowing the development of novel strategies and protocols for effective cardiac cell therapy clinical translation. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

In recent years, exosomes have attracted increasing scientific interest and are no longer considered just as containers for cell waste, but as important mediators of intercellular communication. Among many biomedical research topics, a possible direct role of exosomes in the regenerative medicine field has been underlined in recent studies, including those regarding the so called "paracrine hypothesis". In this perspective, a therapeutic role and/or use of exosomes for tissue regeneration seems to be plausible. However, the majority of the cells isolated and cultured in vitro are exposed to an exogenous exosomes source because of the wide use of foetal bovine serum as cell culture supplement. Bovine serum has been gradually considered as a major biological stimulus, but with still unknown outcome. In this review, we present the state of the art about the role of exosomes in regenerative medicine, particularly for the cardiovascular system. We also analyse the most commonly used exosome isolation techniques that, since their discovery, have undergone continuous development to reach the highest degree of scalability for future clinical translation.

Objectives The goal of this study was to conduct a direct head-to-head comparison of different stem cell types in vitro for various assays of potency and in vivo for functional myocardial repair in the same mouse model of myocardial infarction. Background Adult stem cells of diverse origins (e.g., bone marrow, fat, heart) and antigenic identity have been studied for repair of the damaged heart, but the relative utility of the various cell types remains unclear. Methods Human cardiosphere-derived cells (CDCs), bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and bone marrow mononuclear cells were compared. Results CDCs revealed a distinctive phenotype with uniform expression of CD105, partial expression of c-kit and CD90, and negligible expression of hematopoietic markers. In vitro, CDCs showed the greatest myogenic differentiation potency, highest angiogenic potential, and relatively high production of various angiogenic and antiapoptotic-secreted factors. In vivo, injection of CDCs into the infarcted mouse hearts resulted in superior improvement of cardiac function, the highest cell engraftment and myogenic differentiation rates, and the least-abnormal heart morphology 3 weeks after treatment. CDC-treated hearts also exhibited the lowest number of apoptotic cells. The c-kit(+) subpopulation purified from CDCs produced lower levels of paracrine factors and inferior functional benefit when compared with unsorted CDCs. To validate the comparison of cells from various human donors, selected results were confirmed in cells of different types derived from individual rats. Conclusions CDCs exhibited a balanced profile of paracrine factor production and, among various comparator cell types/subpopulations, provided the greatest functional benefit in experimental myocardial infarction.

Purpose of review For heart failure patients, the urgent need for heart transplantation exceeds the availability of donor hearts. Therefore, cell transplantation has emerged as an interesting and potential solution. This review will focus on the capability of different types of stem cells to regenerate the heart. Moreover, the mechanism for success will be addressed, focusing on the specific (and indispensable?) role of the cells. Recent findings In recent years, many types of stem cells have been described as a possible source for cell transplantation in failing hearts, with mixed outcomes. Cell transplantation is hampered by suboptimal delivery techniques, limited survival of cells, and reduced proliferation and differentiation rates in vivo. Interestingly, the number of injected cells that engrafted the heart successfully cannot explain the observed beneficial effects and, therefore, paracrine effects are suggested for the success in cell therapy. Summary This review summarizes the current types of stem or progenitor cells used in cardiac cell therapy and beneficial effects on heart function and morphology in preclinical studies. Currently, the observed effects suggest that paracrine effects might be responsible, thereby triggering mobilization and activation of resident (stem) cells, which challenges the classical concept and true regenerative capacity of cell therapy at this point.

In the last year, the promising features of mesenchymal stem cells (MSCs), including their regenerative properties and ability to differentiate into diverse cell lineages, have generated great interest among researchers whose work has offered intriguing perspectives on cell-based therapies for various diseases. Currently the most commonly used adult stem cells in regenerative medicine, MSCs can be isolated from several tissues, exhibit a strong capacity for replication in vitro, and can differentiate into osteoblasts, chondrocytes, and adipocytes. However, heterogeneous procedures for isolating and cultivating MSCs among laboratories have prompted the International Society for Cellular Therapy (ISCT) to issue criteria for identifying unique populations of these cells. Consequently, the isolation of MSCs according to ISCT criteria has produced heterogeneous, non-clonal cultures of stromal cells containing stem cells with different multipotential properties, committed progenitors, and differentiated cells. Though the nature and functions of MSCs remain unclear, non-clonal stromal cultures obtained from bone marrow and other tissues currently serve as sources of putative MSCs for therapeutic purposes, and several findings underscore their effectiveness in treating different diseases. To date, 493 MSC-based clinical trials, either complete or ongoing, appear in the database of the US National Institute of Health. In the present paper, we provide a comprehensive review of MSC-based clinical trials conducted worldwide that scrutinizes MSCs' biological properties, elucidates recent clinical findings and clinical trial phases of investigation, highlights MSCs' therapeutic effects, and identifies principal criticisms of the use of these cells. In particular, we analyze clinical trials using MSCs for representative diseases, including hematological disease, graft-versus-host disease, organ transplantation, diabetes, inflammatory diseases, and diseases in the liver, kidney, and lung, as well as cardiovascular, bone and cartilage, neurological, and autoimmune diseases.

Myocardial infarction is underoxygenation-driven limited necrosis of heart tissues which results in elimination of ca. 0.5 to 1 billion spontaneously contracting cardiomyocytes (CM). Since the ability of human heart to regenerate is limited, efforts have been undertaken to increase the number of cardiomyocytes in post-infarction myocardium. Theoretically, such proposals might involve transplantation of 1) skeletal myoblasts and cardiomyocytes, or 2) progenitor/stem cells, theoretically capable of differentiating into cardiomyocytes, or 3) pluripotent cells such as embryonal stem cells (ESC) and induced pluripotent stem cells (iPSC) differentiating into cardiomyocytes. The efforts to increase CM could also involve 4) in situ reprogramming of fibroblasts into active cardiomyocyte-like cells, or 5) stimulating in situ proliferation of cardiomyocytes using pharmacological agents. Only three proposals merit closer scrutiny (2, 4 and 5). However, preclinical and clinical data have demonstrated weak ability of progenitor cells to differentiate (proposal 2). Nevertheless, transplanted cell-induced paracrine effects accompanying such therapy do improve functioning of the damaged heart muscle. The proposals that would permit the number of CM to be increased include in situ reprogramming of fibroblasts into active cardiomyocytes (proposal 4), as well as in situ stimulation of quiescent cardiomyocytes' proliferation (proposal 5). It appears that an optimized therapeutic solution (increasing left ventricular ejection fraction and decreasing the post-infarct scar) might combine agents stimulating paracrine effects and reprogramming of fibroblasts.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., Many studies in different biological systems have revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) modulates signaling pathways triggered at the plasma membrane by agents such as Wnt, transforming growth factor (TGF)-β, epidermal growth factor (EGF), and others. In addition, 1α,25(OH)2D3 may affect gene expression by paracrine mechanisms that involve the regulation of cytokine or growth factor secretion by neighboring cells. Moreover, post-transcriptional and post-translational effects of 1α,25(OH)2D3 add to or overlap with its classical modulation of gene transcription rate. Together, these findings show that vitamin D receptor (VDR) cannot be considered only as a nuclear-acting, ligand-modulated transcription factor that binds to and controls the transcription of target genes. Instead, available data support the view that much of the complex biological activity of 1α,25(OH)2D3 resides in its capacity to interact with membrane-based signaling pathways and to modulate the expression and secretion of paracrine factors. Therefore, we propose that future research in the vitamin D field should focus on the interplay between 1α,25(OH)2D3 and agents that act at the plasma membrane, and on the analysis of intercellular communication. Global analyses such as RNA-Seq, transcriptomic arrays, and genome-wide ChIP are expected to dissect the interactions at the gene and molecular levels., The work in the authors’ laboratory is supported by Ministerio de Economía y Competitividad of Spain (SAF2010-18302,BFU2010-19659), Fondo Europeo de Desarrollo Regional-Instituto de Salud Carlos III(RD12/0036/0021,RD12/0036/0041), and Comunidad de Madrid (S2010/BMD-2344, Colomics2).

Several investigators have cultivated marrow stromal cells and have identified a population of mesenchymal stem cells (MSCs). These cells expand extensively in vitro and exhibit multilineage differentiation potential. The lack of MSC-specific markers impedes identification of MSC functions. Further in vivo studies of these cells may elucidate the nature of MSCs. Although the nature of MSCs remains unclear, nonclonal stromal cultures are used as a source of putative MSCs for therapeutic purposes. Preclinical studies and clinical trials assumed that transplanted MSCs exert their effects through their differentiation properties or through the release of molecules that restore tissue functions and modulate immune cells. These studies reported contradictory results and failed to meet expectations. Thus, it is important to note that current protocols for MSC therapy are primarily based on the use of in vitro expanded nonclonal MSCs. Clearly defining the physiological features of in situ MSCs and the in vitro and in vivo properties of nonclonal cultures of stromal cells, which are often misidentified as pure stem cell cultures, may explain the reported failures of MSC therapy. This review will address these issues. (C) 2014 Wiley Periodicals, Inc.

Patients suffering from heart failure as a result of myocardial infarction are in need of heart transplantation. Unfortunately the number of donor hearts is very low and therefore new therapies are subject of investigation. Cell transplantation therapy upon myocardial infarction is a very promising strategy to replace the dead myocardium with viable cardiomyocytes, smooth muscle cells and endothelial cells, thereby reducing scarring and improving cardiac performance. Despite promising results, resulting in reduced infarct size and improved cardiac function on short term, only a few cells survive the ischemic milieu and are retained in the heart, thereby minimizing long-term effects. Although new capillaries and cardiomyocytes are formed around the infarcted area, only a small percentage of the transplanted cells can be detected months after myocardial infarction. This suggests the stimulation of an endogenous regenerative capacity of the heart upon cell transplantation, resulting from release of growth factor, cytokine and other paracrine molecules by the progenitor cells – the so-called paracrine hypothesis. Here, we focus on a relative new component of paracrine signalling, i.e. exosomes. We are interested in the release and function of exosomes derived from cardiac progenitor cells and studied their effects on the migratory capacity of endothelial cells.