Sveva Bollini

De novo cardiomyocytes from within the activated adult heart after injury

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal—relative to cell transplantation—would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm’s tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.

Cardiac lymphatics are heterogeneous in origin and respond to injury

The lymphatic vasculature is a blind-ended network crucial for tissue-fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. By contrast, here we show that cardiac lymphatic vessels in mice have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple Cre–lox-based lineage tracing revealed a potential contribution from the putative haemogenic endothelium during development, and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2+ and Vav1+ compartments. In the adult heart, myocardial infarction promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C, resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury.

Amniotic fluid stem cells are cardioprotective following acute myocardial infarction.

In recent years, various types of stem cells have been characterized and their potential for cardiac regeneration has been investigated. We have previously described the isolation of broadly multipotent cells from amniotic fluid, defined as amniotic fluid stem (AFS) cells. The aim of this study was to investigate the therapeutic potential of human AFS cells (hAFS) in a model of acute myocardial infarction. Wistar rats underwent 30 min of ischemia by ligation of the left anterior descending coronary artery, followed by administration of hAFS cells and 2 h of reperfusion. Infarct size was assessed by 2,3,5-triphenyltetrazolium chloride staining and planimetry. hAFS cells were also analyzed by enzyme-linked immunosorbent assay to detect secretion of putative paracrine factors, such as the actin monomer-binding protein thymosin β4 (Tβ4). The systemic injection of hAFS cells and their conditioned medium (hAFS-CM) was cardioprotective, improving myocardial cell survival and decreasing the infarct size from 53.9%±2.3% (control animals receiving phosphate-buffered saline injection) to 40.0%±3.0% (hAFS cells) and 39.7%±2.5% (hAFS-CM, P<0.01). In addition, hAFS cells were demonstrated to secrete Tβ4, previously shown to be both cardioprotective and proangiogenic. Our results suggest that AFS cells have therapeutic potential in the setting of acute myocardial infarction, which may be mediated through paracrine effectors such as Tβ4. Therefore, AFS cells might represent a novel source for cell therapy and cell transplantation strategies in repair following ischemic heart disease, with a possible paracrine mechanism of action and a potential molecular candidate for acute cardioprotection.

Amniotic fluid stem cells improve survival and enhance repair of damaged intestine in necrotising enterocolitis via a COX-2 dependent mechanism

Objective Necrotising enterocolitis (NEC) remains one of the primary causes of morbidity and mortality in neonates and alternative strategies are needed. Stem cells have become a therapeutic option for other intestinal diseases, which share some features with NEC. We tested the hypothesis that amniotic fluid stem (AFS) cells exerted a beneficial effect in a neonatal rat model of NEC. Design Rats intraperitoneally injected with AFS cells and their controls (bone marrow mesenchymal stem cells, myoblast) were analysed for survival, behaviour, bowel imaging (MRI scan), histology, bowel absorption and motility, immunofluorescence for AFS cell detection, degree of gut inflammation (myeloperoxidase and malondialdehyde), and enterocyte apoptosis and proliferation. Results AFS cells integrated in the bowel wall and improved rat survival and clinical conditions, decreased NEC incidence and macroscopic gut damage, improved intestinal function, decreased bowel inflammation, increased enterocyte proliferation and reduced apoptosis. The beneficial effect was achieved via modulation of stromal cells expressing cyclooxygenase 2 in the lamina propria, as shown by survival studies using selective and non-selective cyclooxygenase 2 inhibitors. Interestingly, AFS cells differentially expressed genes of the Wnt/β-catenin pathway, which regulate intestinal epithelial stem cell function and cell migration and growth factors known to maintain gut epithelial integrity and reduce mucosal injury. Conclusions We demonstrated here for the first time that AFS cells injected in an established model of NEC improve survival, clinical status, gut structure and function. Understanding the mechanism of this effect may help us to develop new cellular or pharmacological therapies for infants with NEC.

In Vitro and In Vivo Cardiomyogenic Differentiation of Amniotic Fluid Stem Cells

Cell therapy has developed as a complementary treatment for myocardial regeneration. While both autologous and allogeneic uses have been advocated, the ideal candidate has not been identified yet. Amniotic fluid-derived stem (AFS) cells are potentially a promising resource for cell therapy and tissue engineering of myocardial injuries. However, no information is available regarding their use in an allogeneic context. c-kit-sorted, GFP-positive rat AFS (GFP-rAFS) cells and neonatal rat cardiomyocytes (rCMs) were characterized by cytocentrifugation and flow cytometry for the expression of mesenchymal, embryonic and cell lineage-specific antigens. The activation of the myocardial gene program in GFP-rAFS cells was induced by co-culture with rCMs. The stem cell differentiation was evaluated using immunofluorescence, RT-PCR and single cell electrophysiology. The in vivo potential of Endorem-labeled GFP-rAFS cells for myocardial repair was studied by transplantation in the heart of animals with ischemia/reperfusion injury (I/R), monitored by magnetic resonance imaging (MRI). Three weeks after injection a small number of GFP-rAFS cells acquired an endothelial or smooth muscle phenotype and to a lesser extent CMs. Despite the low GFP-rAFS cells count in the heart, there was still an improvement of ejection fraction as measured by MRI. rAFS cells have the in vitro propensity to acquire a cardiomyogenic phenotype and to preserve cardiac function, even if their potential may be limited by poor survival in an allogeneic setting.

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 disease.

Different cardiovascular potential of adult- and fetal-type mesenchymal stem cells in a rat model of heart cryoinjury

Efficacy of adult (bone marrow, BM) versus fetal (amniotic fluid, AF) mesenchymal stem cells (MSCs) to replenish damaged rat heart tissues with new cardiovascular cells has not yet been established. We investigated on the differentiation potential of these two rat MSC populations in vitro and in a model of acute necrotizing injury (ANI) induced by cryoinjury. Isolated BM-MSCs and AF-MSCs were characterized by flow cytometry and cytocentrifugation and their potential for osteogenic, adipogenic, and cardiovascular differentiation assayed in vitro using specific induction media. The left anterior ventricular wall of syngeneic Fisher 344 (n = 48) and athymic nude (rNu) rats (n = 6) was subjected to a limited, nontransmural epicardial ANI in the approximately one third of wall thickness without significant hemodynamic effects. The time window for in situ stem cell transplantation was established at day 7 postinjury. Fluorochrome (CMTMR)-labeled BM-MSCs (2 × 106) or AF-MSCs (2 × 106) were injected in syngeneic animals (n = 26) around the myocardial lesion via echocardiographic guidance. Reliability of CMTMR cell tracking in this context was ascertained by transplanting genetically labeled BM-MSCs or AF-MSCs, expressing the green fluorescent protein (GFP), in rNu rats with ANI. Comparison between the two methods of cell tracking 30 days after cell transplantation gave slightly different values (1420,58 ± 129,65 cells/mm2 for CMTMR labeling and 1613.18 ± 643.84 cells/mm2 for genetic labeling; p = NS). One day after transplantation about one half CMTMR-labeled AF-MSCs engrafted to the injured heart (778.61 ± 156.28 cells/mm2) in comparison with BM-MSCs (1434.50± 173.80 cells/mm2, p < 0.01). Conversely, 30 days after cell transplantation survived MSCs were similar: 1275.26 ± 74.51/mm2 (AF-MSCs) versus 1420.58 ± 129.65/mm2 for BM-MSCs (p = NS). Apparent survival gain of AF-MSCs between the two time periods was motivated by the cell proliferation rate calculated at day 30, which was lower for BM-MSCs (6.79 ± 0.48) than AF-MSCs (10.83 ± 3.50; p < 0.01), in the face of a similar apoptotic index (4.68 ± 0.20 for BM-MSCs and 4.16 ± 0.58 for AF-MSCs; p = NS). These cells were also studied for their expression of markers specific for endothelial cells (ECs), smooth muscle cells (SMCs), and cardiomyocytes (CMs) using von Willebrand factor (vWf), smooth muscle (SM) α-actin, and cardiac troponin T, respectively. Grafted BM-MSCs or AF-MSCs were found as single cell/small cell clusters or incorporated in the wall of microvessels. A larger number of ECs (227.27 ± 18.91 vs. 150.36 ± 24.08 cells/mm2, p < 0.01) and CMs (417.91 ± 100.95 vs. 237.43 ± 79.99 cells/mm2, p < 0.01) originated from AF-MSCs than from BM-MSCs. Almost no SMCs were seen with AF-MSCs, in comparison to BM-MSCs (98.03 ± 40.84 cells/mm2), in concordance with lacking of arterioles, which, instead, were well expressed with BM-MSCs (71.30 ± 55.66 blood vessels/mm2). The number of structurally organized capillaries was slightly different with the two MSCs (122.49± 17.37/mm2 for AF-MSCs vs. 148.69 ± 54.41/mm2 for BM-MSCs; p = NS). Collectively, these results suggest that, in the presence of the same postinjury microenvironment, the two MSC populations from different sources are able to activate distinct differentiation programs that potentially can bring about a myocardial-capillary or myocardial-capillary-arteriole re-constitution.

Re-Activated Adult Epicardial Progenitor Cells Are a Heterogeneous Population Molecularly Distinct from Their Embryonic Counterparts

Cardiovascular disease remains the major cause of mortality, and cardiac cell therapy has recently emerged as a paradigm for heart repair. The epicardium is a layer of mesothelial cells covering the heart that during development contributes to different cardiovascular lineages, including cardiomyocytes, but which becomes quiescent after birth. We previously revealed that the peptide thymosin beta 4 (Tβ4) can reactivate adult epicardium-derived cells (EPDCs) after myocardial infarction (MI), to proliferate, and differentiate into cardiovascular derivatives. The aim of this study was to provide a lineage characterization of the adult EPDCs relative to the embryonic epicardial lineage and to determine prospective cell fate biases within the activated adult population during cardiovascular repair. Wt1(GFPCre/+) mice were primed with Tβ4 and MI induced by ligation of the left anterior descending coronary artery. Adult WT1(+) GFP(+) EPDCs were fluorescence-activated cell sorted (FACS) at 2, 4, and 7 days after MI. Embryonic WT1(+) GFP(+) EPDCs were isolated from embryonic hearts (E12.5) by FACS, and sorted cells were characterized by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunostaining. Adult WT1(+) GFP(+) EPDCs were highly heterogeneous, expressing cardiac progenitor and mesenchymal stem markers. Based on the expression of stem cell antigen-1 (Sca-1), CD44, and CD90, we identified different subpopulations of EPDCs of varying cardiovascular potential, according to marker gene profiles, with a molecular phenotype distinct from the source embryonic epicardial cells at E12.5. Thus, adult WT1(+) GFP(+) cells are a heterogeneous population that when activated can restore an embryonic gene programme, but do not revert entirely to adopt an embryonic phenotype. Potential biases in cardiovascular cell fate suggest that discrete subpopulations of EPDCs might be clinically relevant for regenerative therapy.

Thymosin β4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac wound healing

The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide (H2O2) released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that Thymosin β4-Sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, lies downstream of H2O2 in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T cell/CD14+ monocyte co-cultures, Tβ4-SO inhibits IFN-γ and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, Tβ4-SO is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.

Human Bone Marrow-Derived CD133+ Cells Delivered to a Collagen Patch on Cryoinjured Rat Heart Promote Angiogenesis and Arteriogenesis:

Transplanting hematopoietic and peripheral blood-derived stem/progenitor cells can have beneficial effects in slowing the effects of heart failure. We investigated whether human bone marrow CD133+-derived cells (BM-CD133+ cells) might be used for cell therapy of heart injury in combination with tissue engineering. We examined these cells for: 1) their in vitro capacity to be converted into cardiomyocytes (CMs), and 2) their potential for in vivo differentiation when delivered to a tissue-engineered type I collagen patch placed on injured hearts (group II). To ensure a microvascular network ready for use by the transplanted cells, cardiac injury and patching were scheduled 2 weeks before cell injection. The cardiovascular potential of the BM-CD133+ cells was compared with that of a direct injection (group I) of the same cells in heart tissue damaged according to the same schedule as for group II. While a small fraction (2 ± 0.5%) of BM-CD133+cells cocultured with rat CMs switched in vitro to a CM-like cell phenotype, in vivo—and in both groups of nude rats transplanted with BM-CD133+—there was no evidence of any CM differentiation (as detected by cardiac troponin I expression), but there were signs instead of new capillaries and small arterioles. While capillaries prevailed over arterioles in group II, the opposite occurred in group I. The transplanted cells further contributed to the formation of new microvessels induced by the patch (group II) but the number of vessels did not appear superior to the one developed after directly injecting the BM-CD133+cells into the injured heart. Although chimeric human–rat microvessels were consistently found in the hearts of both groups I and II, they represented a minority (1.5–2.3%) compared with those of rat origin. Smooth muscle myosin isoform expression suggested that the arterioles achieved complete differentiation irrespective of the presence or absence of the collagen patch. These findings suggest that: 1) BM-CD133+ cells display a limited propensity for in vitro conversion to CMs; 2) the preliminarily vascularized bioscaffold did not confer a selective homing and differentiation advantage for the phenotypic conversion of BM-CD133+ cells into CMs; and 3) combined patching and cell transplantation is suitable for angiogenesis and arteriogenesis, but it does not produce better results, in terms of endothelial and smooth muscle cell differentiation, than the “traditional” method of cell injection into the myocardium.