Marco Ghionzoli

A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration

Management of intestinal failure remains a clinical challenge and total parenteral nutrition, intestinal elongation and/or transplantation are partial solutions. In this study, using a detergent-enzymatic treatment (DET), we optimize in rats a new protocol that creates a natural intestinal scaffold, as a base for developing functional intestinal tissue. After 1 cycle of DET, histological examination and SEM and TEM analyses showed removal of cellular elements with preservation of the native architecture and connective tissue components. Maintenance of biomechanical, adhesion and angiogenic properties were also demonstrated strengthen the idea that matrices obtained using DET may represent a valid support for intestinal regeneration.

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.

ES, iPS, MSC, and AFS cells. Stem cells exploitation for Pediatric Surgery: current research and perspective

Despite the advancements that have been made in treating infants with congenital malformations, these still represent a major cause of disease and death during the first years of life and childhood. Regeneration of natural tissue from living cells to restore damaged tissues and organs is the main purpose of regenerative medicine. This relatively new field has emerged by the combination of tissue engineering and stem cell transplantation as a possible strategy for the replacement of damaged organs or tissues. This review would like to offer an insight on the latest evolution of stem cells with a glance at their possible application for regenerative medicine, particularly in the Paediatric Surgery field.

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.

Amniotic Fluid Stem Cells Rescue Both In Vitro and In Vivo Growth, Innervation, and Motility in Nitrofen-Exposed Hypoplastic Rat Lungs Through Paracrine Effects

Lung hypoplasia can be prevented in vitro by retinoic acid (RA). Recent evidence suggests that amniotic fluid stem (AFS) cells may integrate injured lungs and influence their recovery. We tested the hypothesis that AFS cells might improve lung growth and motility by paracrine mechanisms. Pregnant rats received either nitrofen or vehicle on E9.5. In vitro E13 embryonic lungs were cultured in the presence of culture medium alone or with RA, basophils, or AFS cells. In vivo green fluorescent protein-expressing (GFP+) rat AFS cells were transplanted in nitrofen-exposed rats on E10.5. E13 lung explants were cultured before analysis. The surface, the number of terminal buds, and the frequency of bronchial contractions were assessed. Protein gene product 9.5 (PGP 9.5) and α-actin protein levels were measured. The lung explants transplanted with AFS cells were stained for α-actin, PGP 9.5, and TTF-1. The levels of FGF-10, VEGFα, and TGF-β1 secreted by the AFS cells in the culture medium were measured. Comparison between groups was made by ANOVA. In vitro, the surface, the number of terminal buds, and the bronchial peristalsis were increased in nitrofen + AFS cell explants in comparison with nitrofen-exposed lungs. While nitrofen + RA lungs were similar to nitrofen + AFS ones, basophils did not normalize these measurements. PGP 9.5 protein was decreased in nitrofen lungs, but after adding AFS cells, the value was similar to controls. No differences were found in the expression of α-actin. In vivo, the surface, number of terminal buds, and peristalsis were similar to control after injection of AFS cells in nitrofenexposed rats. Colocalization with TTF-1-positive cells was found. The levels of FGF-10 and VEGFα were increased in nitrofen + AFS cell explants, while the levels of TGF-β1 were similar to controls. Lung growth, bronchial motility, and innervation were decreased in nitrofen explants and rescued by AFS cells both in vitro and in vivo, similarly to that observed before with RA. The AFS cell beneficial effect was probably related to paracrine action of growth factor secretion.

Is early delivery beneficial in gastroschisis

PURPOSE Gastroschisis neonates have delayed time to full enteral feeds (ENT), possibly due to bowel exposure to amniotic fluid. We investigated whether delivery at <37weeks improves neonatal outcomes of gastroschisis and impact of intra/extra-abdominal bowel dilatation (IABD/EABD). METHODS A retrospective review of gastroschisis (1992-2012) linked fetal/neonatal data at 2 tertiary referral centers was performed. Primary outcomes were ENT and length of hospital stay (LOS). Data (median [range]) were analyzed using parametric/non-parametric tests, positive/negative predictive values, and regression analysis. RESULTS Two hundred forty-six patients were included. Thirty-two were complex (atresia/necrosis/perforation/stenosis). ENT (p<0.0001) and LOS (p<0.0001) were reduced with increasing gestational age. IABD persisted to last scan in 92 patients, 68 (74%) simple (intact/uncompromised bowel), 24 (26%) complex. IABD or EABD diameter in complex patients was not significantly greater than simple gastroschisis. Combined IABD/EABD was present in 22 patients (14 simple, 8 complex). When present at <30weeks, the positive predictive value for complex gastroschisis was 75%. Two patients with necrosis and one atresia had IABD and collapsed extra-abdominal bowel from <30weeks. CONCLUSION Early delivery is associated with prolonged ENT/LOS, suggesting elective delivery at <37weeks is not beneficial. Combined IABD/EABD or IABD/collapsed extra-abdominal bowel is suggestive of complex gastroschisis.

Gastroschisis with intestinal atresia—predictive value of antenatal diagnosis and outcome of postnatal treatment

PURPOSE The purpose of this study is to evaluate (1) the predictive value of fetal bowel dilatation (FBD) for intestinal atresia in gastroschisis and (2) the postnatal management and outcome of this condition. METHODS A retrospective review of all gastroschisis cases diagnosed in our fetal medicine unit between 1992 and 2010 and treated postnatally in our center was performed. RESULTS One hundred thirty cases had full postnatal data available. Intestinal atresia was found at surgery in 14 neonates (jejunum, n = 6; ileum, n = 3; ascending colon, n = 3; multiple, n = 2). Polyhydramnios and FBD were more likely in the atresia group compared with infants with no atresia (P = .0003 and P = .005, respectively). Fetal bowel dilatation had 99% negative predictive value (95% confidence interval, 0.9-0.99) and 17% positive predictive value (95% confidence interval, 0.1-0.3) for atresia. Treatment of intestinal atresia included primary anastomosis (n = 5), delayed anastomosis (n = 2), and stoma formation followed by anastomosis (n = 7). Infants with atresia had longer duration of parenteral nutrition, higher incidence of sepsis, and cholestasis compared with infants with no atresia (P = .0003). However, the presence of atresia did not increase mortality. CONCLUSIONS Polyhydramnios and FBD are associated with atresia. Absence of FBD in gastroschisis excludes intestinal atresia. In our experience, atresia is associated with a longer duration of parenteral nutrition but does not influence mortality. These findings may be relevant for antenatal counseling.

Human amniotic fluid stem cell differentiation along smooth muscle lineage

Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c‐kit+ hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF‐BB and TGF‐β1. Molecular assays revealed higher level of α smooth muscle actin (α‐SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the α‐SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell‐like electrophysiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.—Ghionzoli, M., Repele, A., Sartiani, L., Costanzi, G., Parenti, A., Spinelli, V., David, A. L., Garriboli, M., Totonelli, G., Tian, J., Andreadis, S. T., Cerbai, E., Mugelli, A., Messineo, A., Pierro, A., Eaton, S., De Coppi, P., Human amniotic fluid stem cell differentiation along smooth muscle lineage. FASEB J. 27, 4853–4865 (2013). www.fasebj.org

Pectus excavatum and heritable disorders of the connective tissue

Pectus excavatum, the most frequent congenital chest wall deformity, may be rarely observed as a sole deformity or as a sign of an underlying connective tissue disorder. To date, only few studies have described correlations between this deformity and heritable connective tissue disorders such as Marfan, Ehlers-Danlos, Poland, MASS (Mitral valve prolapse, not progressive Aortic enlargement, Skeletal and Skin alterations) phenotype among others. When concurring with connective tissue disorder, cardiopulmonary and vascular involvement may be associated to the thoracic defect. Ruling out the concomitance of pectus excavatum and connective tissue disorders, therefore, may have a direct implication both on surgical outcome and long term prognosis. In this review we focused on biological bases of connective tissue disorders which may be relevant to the pathogenesis of pectus excavatum, portraying surgical and clinical implication of their concurrence.