Triggering Endogenous Cardiac Repair and Regeneration via Extracellular Vesicle-Mediated Communication

A variety of paracrine signals create networks within the myocardium and mediate intercellular communications. Indeed, paracrine stimulation of the endogenous regenerative program of the heart, mainly based on resident cardiac progenitor cell (CPC) activation together with cardiomyocyte proliferation, has become increasingly relevant for future cardiac medicine. In the last years, it has been shown that extracellular vesicles (EV), including exosomes (Ex), are powerful conveyors of relevant biological effects. EV have been proposed not only as promising therapeutic tool for triggering cardiac regeneration and improving repair, but also as means of better understanding the physiological and pathological relationships between specific cardiac components, including cardiomyocytes and fibroblasts. Actually, EV from different kinds of exogenous stem cells have been shown to mediate beneficial effects on the injured myocardium. Moreover, endogenous cells, like CPC can instruct cardiovascular cell types, including cardiomyocytes, while cardiac stromal cells, especially fibroblasts, secrete EV that modulate relevant aspects of cardiomyocyte biology, such as hypertrophy and electrophysiological properties. Finally, cardiomyocytes too may release EV influencing the function of other cardiac cell types. Therefore, EV-based crosstalk is thought to be important in both physiology and pathology, being involved in the responses of the heart to noxious stimuli. In this review we will discuss the role of EV in both regulating cardiac homeostasis and driving heart regeneration. In particular, we will address their role in: (i) providing cardio-protection and enhancing cardiac repair mechanisms; (ii) CPC biology; and (iii) influencing adult cardiomyocyte behavior

Autologous transplantation of amniotic fluid-derived mesenchymal stem cells into sheep fetuses

Long-term engraftment and phenotype correction has been difficult to achieve in humans after in utero stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained during gestation from the amniotic fluid with minimal risk for the fetus and the mother. Using a sheep model, we explored the possibility of using amniotic fluid mesenchymal stem cells (AFMSCs) for autologous in utero stem cell/gene therapy. We collected amniotic fluid (AF) under ultrasound-guided amniocentesis in early gestation pregnant sheep (n = 9, 58 days of gestation, term = 145 days). AFMSCs were isolated and expanded in all sampled fetal sheep. Those cells were transduced using an HIV vector encoding enhanced green fluorescent protein (GFP) with 63.2% (range 38.3-96.2%) transduction efficiency rate. After expansion, transduced AFMSCs were injected into the peritoneal cavity of each donor fetal sheep at 76 days under ultrasound guidance. One ewe miscarried twin fetuses after amniocentesis. Intraperitoneal injection was successful in the remaining 7 fetal sheep giving a 78% survival for the full procedure. Tissues were sampled at postmortem examination 2 weeks later. PCR analysis detected GFP-positive cells in fetal tissues including liver, heart, placenta, membrane, umbilical cord, adrenal gland, and muscle. GFP protein was detected in these tissues by Western blotting and further confirmed by cytofluorimetric and immunofluorescence analyses. This is the first demonstration of autologous stem cell transplantation in the fetus using AFMSCs. Autologous cells derived from AF showed widespread organ migration and could offer an alternative way to ameliorate prenatal congenital diseas

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT(+) hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT(+) hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn(F7/F7) mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT(+) hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355

Guidelines to Analyze Preclinical Studies Using Perinatal Derivatives.

The last 18 years have brought an increasing interest in the therapeutic use of perinatal derivatives (PnD). Preclinical studies used to assess the potential of PnD therapy include a broad range of study designs. The COST SPRINT Action (CA17116) aims to provide systematic and comprehensive reviews of preclinical studies for the understanding of the therapeutic potential and mechanisms of PnD in diseases and injuries that benefit from PnD therapy. Here we describe the publication search and data mining, extraction, and synthesis strategies employed to collect and prepare the published data selected for meta-analyses and reviews of the efficacy of PnD therapies for different diseases and injuries. A coordinated effort was made to prepare the data suitable to make statements for the treatment efficacy of the different types of PnD, routes, time points, and frequencies of administration, and the dosage based on clinically relevant effects resulting in clear increase, recovery or amelioration of the specific tissue or organ function. According to recently proposed guidelines, the harmonization of the nomenclature of PnD types will allow for the assessment of the most efficient treatments in various disease models. Experts within the COST SPRINT Action (CA17116), together with external collaborators, are doing the meta-analyses and reviews using the data prepared with the strategies presented here in the relevant disease or research fields. Our final aim is to provide standards to assess the safety and clinical benefit of PnD and to minimize redundancy in the use of animal models following the 3R principles for animal experimentation

General consensus on multimodal functions and validation analysis of perinatal derivatives for regenerative medicine applications.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration

Investigating the Paracrine Role of Perinatal Derivatives: Human Amniotic Fluid Stem Cell-Extracellular Vesicles Show Promising Transient Potential for Cardiomyocyte Renewal

Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid-derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of R26pFUCCI2 mice with cell cycle fluorescent tagging by mutually exclusive nuclear signal. mNVCM were stimulated by fetal versus perinatal hAFSC-CM and hAFSC-EVs to identify the most promising formulation for in vivo assessment in a R26pFUCCI2 neonatal mouse model of myocardial infarction (MI) via intraperitoneal delivery. While the perinatal hAFSC secretome did not provide any significant cardiogenic effect, fetal hAFSC-EVs significantly sustained mNVCM transition from S to M phase by 2-fold, while triggering cytokinesis by 4.5-fold over vehicle-treated cells. Treated mNVCM showed disorganized expression of cardiac alpha-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with a 40% significant downregulation of Cofilin-2 and a positive trend of polymerized F-Actin. Fetal hAFSC-EVs increased cardiomyocyte cell cycle progression by 1.8-fold in the 4-day-old neonatal left ventricle myocardium short term after MI; however, such effect was lost at the later stage. Fetal hAFSC-EVs were enriched with a short isoform of Agrin, a mediator of neonatal heart regeneration acting by YAP-related signaling; yet in vitro application of YAP inhibitor verteporfin partially affected EV paracrine stimulation on mNVCM. EVs secreted by developmentally juvenile fetal hAFSC can support cardiomyocyte renewal to some extension, via intercellular conveyance of candidates possibly involving Agrin in combination with other factors. These perinatal derivative promising cardiogenic effects need further investigation to define their specific mechanism of action and enhance their potential translation into therapeutic opportunity

BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease

Epicardium-derived cells (EPDCs) contribute cardiovascular cell types during development and in adulthood respond to Thymosin \u3b24 (T\u3b24) and myocardial infarction (MI) by reactivating a fetal gene programme to promote neovascularization and cardiomyogenesis. The mechanism for epicardial gene (re-)activation remains elusive. Here we reveal that BRG1, the essential ATPase subunit of the SWI/SNF chromatin-remodelling complex, is required for expression of Wilms' tumour 1 (Wt1), fetal EPDC activation and subsequent differentiation into coronary smooth muscle, and restores Wt1 activity upon MI. BRG1 physically interacts with T\u3b24 and is recruited by CCAAT/enhancer-binding protein \u3b2 (C/EBP\u3b2) to discrete regulatory elements in the Wt1 locus. BRG1-T\u3b24 co-operative binding promotes optimal transcription of Wt1 as the master regulator of embryonic EPDCs. Moreover, chromatin immunoprecipitation-sequencing reveals BRG1 binding at further key loci suggesting SWI/SNF activity across the fetal epicardial gene programme. These findings reveal essential functions for chromatin-remodelling in the activation of EPDCs during cardiovascular development and repair

Cardiomyogenic Potential of Amniotic Fluid Stem Cells As A New Tool For Cell Based Cardiac Tissue Engineering

Background. In the last years tissue engineering for cardiac pathologies has been broadly developed with the aim to restore or improve the diseased or damaged heart. Novel cardiac tissue engineering approaches combine the use of biocompatible scaffolds with stem cells to conjugate material science, surgery and cell therapy techniques. So far, different kinds of stem cells have been described and their potential for cardiac regeneration broadly investigated. We have previously described that it is possible to derive lines of broadly multipotent cells from the amniotic fluid (Amniotic Fluid Stem cells; AFS cells). The aim of this study was to characterize more in detail the AFS cells cardiomyogenic potential both in vitro and in vivo. Methods. Neonatal rat cardiomyocyte (rCM) cells were obtained by enzymatic digestion of 2-3-days old rat hearts. GFP-positive rat AFS (gfp+rAFS) cells were obtained from amniotic fluid samples from GFP-positive transgenic pregnant rats. Human AFS (hAFS) cells were obtained from healthy amniotic fluid back up samples from prenatal diagnosis, following informed consent. AFS cells were isolated by immunosorting for the stem marker c-kit. Before applying a tissue engineering approach, using biocompatible scaffolds, to the AFS and rCM cells coculture, the AFS cells “cardiomyocyte-like” phenotype, acquired in cocolture, had been functionally evaluated by patch-clamp analysis. In this work two different kinds of bidimensional micropatterned scaffolds were used: hydrogel films and PDMS (silicon) membranes. The scaffolds were obtained by microcontact printing technique and using a mold scratched with the desidered micropattern and their viability was tested using, at first, the rat neonatal primary culture. AFS and rCM cells were seeded together on the micropatterned PDMS membranes and analyzed for the expression of troponin T by immunostaining after 6 and 10 days of culture. For the in vivo study, immunodeficient nude male rats underwent a cryoinjury on the heart left ventricle with a 3D collagen scaffold implantation and 5x10e6 hAFS cells/animal local or systemic injection after 15 days. hAFS cells were previously labelled with the red intracellular fluorescent dye CMTMR. Animals were sacrificed at 24 hours, 15 and 30 days after cells injection and hearts stained for cardiac and inflammatory markers. For the acute myocardial infarct model, male Wistar rats underwent an ischemic injury by left anterior descendent coronary artery ligation for 30 minutes and then they were reperfused injecting via the external jugular vein 10e7 or 10e6 gfp+rAFS and 10e7 or 5x10e6 hAFS cells/animal for 2 hours; rats were sacrificed afterwards and hearts analyzed for infarct size measurement by Evans blue staining, by 2,3,5-triphenolltetrazolium chloride (TTC) staining and planimetry with the software Image J. Heart, lungs, spleen and liver were analyzed as well by immunostaining for evaluating hAFS cells content. hAFS cells were also analyzed for the presence of a subpopulation of cardiac progenitors, by RT-PCR analysis, for the expression of early cardiac commitment genes as Isl1 and Kdr. The cells were then studied by ELISA essay to speculate if they can secrete in the culture medium the protein thymosin beta 4, paracrine and cardioprotector factor. Results and Conclusions. Regarding the in vitro results, AFS cells were demonstrated to express a “pace maker cell-like” action potential, when cocultured with rat neonatal cardiomyocyte cells. Moreover, when cultured on the bidimensional scaffold, AFS cells showed to follow the longitudinal orientation of the microstruttured membrane, expressing beating activity and the cardiac protein troponin T. Our in vivo data revealed that hAFS cells, injected into the cryoinjured rat heart, survived in the host up to 30 days, moved from the injection site to the lesioned area in the heart and gave rise to new chimeric capillaries in the patch and cryoinjury area. In the acute myocardial infarct model the results obtained suggested that hAFS cells could exert a paracrine effect in vivo, decreasing the infarct size (measured as the ratio between the infarct area and the ischemic area at risk of necrosis) from a 53,9 ± 2,3% (obtained in control animals receiving PBS injection) to 40,0 ± 3,0% of the ischemic area. Furthermore, hAFS cells were also demonstrated to have a subpopulation of cardiac progenitors, positive for the expression of the early cardiac commitment genes Isl1 and Kdr and to to secrete in the culture medium thymosin beta 4, a paracrine factor previously shown to act as cardioprotector and angiogenic agent. In conclusions, our results are very encouraging and challenging, suggesting that AFS cells can show cardiomyogenic potential and cardioprotective therapeutic application in cell based therapy tissue engineering