First steps to define murine amniotic fluid stem cell microenvironment

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells

Decellularized diaphragmatic muscle drives a constructive angiogenic response in vivo

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1\u3b1. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31+, \u3b1SMA+, and vWF+ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF+ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration

N-Palmitoyl-D-Glucosamine Inhibits TLR-4/NLRP3 and Improves DNBS-Induced Colon Inflammation through a PPAR-α-Dependent Mechanism

Similar to canine inflammatory enteropathy, inflammatory bowel disease (IBD) is a chronic idiopathic condition characterized by remission periods and recurrent flares in which diarrhea, visceral pain, rectal bleeding/bloody stools, and weight loss are the main clinical symptoms. Intestinal barrier function alterations often persist in the remission phase of the disease without ongoing inflammatory processes. However, current therapies include mainly anti-inflammatory compounds that fail to promote functional symptoms-free disease remission, urging new drug discoveries to handle patients during this step of the disease. ALIAmides (ALIA, autacoid local injury antagonism) are bioactive fatty acid amides that recently gained attention because of their involvement in the control of inflammatory response, prompting the use of these molecules as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. N-palmitoyl-D-glucosamine (PGA), an under-researched ALIAmide, resulted in being safe and effective in preclinical models of inflammation and pain, suggesting its potential engagement in the treatment of IBD. In our study, we demonstrated that micronized PGA significantly and dose-dependently reduces colitis severity, improves intestinal mucosa integrity by increasing the tight junction proteins expression, and downregulates the TLR-4/NLRP3/iNOS pathway via PPAR-α receptors signaling in DNBS-treated mice. The possibility of clinically exploiting micronized PGA as support for the treatment and prevention of inflammation-related changes in IBD patients would represent an innovative, effective, and safe strategy

Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia

open12siopenSchiavo, Andrea Alex; Franzin, Chiara; Albiero, Mattia; Piccoli, Martina; Spiro, Giovanna; Bertin, Enrica; Urbani, Luca; Visentin, Silvia; Cosmi, Erich; Fadini, Gian Paolo; De Coppi, Paolo; Pozzobon, MichelaSchiavo, ANDREA ALEX; Franzin, Chiara; Albiero, Mattia; Piccoli, Martina; Spiro, Giovanna; Bertin, Enrica; Urbani, Luca; Visentin, Silvia; Cosmi, Erich; Fadini, GIAN PAOLO; DE COPPI, Paolo; Pozzobon, Michel

Allogenic tissue-specific decellularized scaffolds promote long-term muscle innervation and functional recovery in a surgical diaphragmatic hernia model

Congenital diaphragmatic hernia (CDH) is a neonatal defect in which the diaphragm muscle does not develop properly, thereby raising abdominal organs into the thoracic cavity and impeding lung development and function. Large diaphragmatic defects require correction with prosthetic patches to close the malformation. This treatment leads to a consequent generation of unwelcomed mechanical stress in the repaired diaphragm and hernia recurrences, thereby resulting in high morbidity and significant mortality rates. We proposed a specific diaphragm-derived extracellular matrix (ECM) as a scaffold for the treatment of CDH. To address this strategy, we developed a new surgical CDH mouse model to test the ability of our tissue-specific patch to regenerate damaged diaphragms. Implantation of decellularized diaphragmatic ECM-derived patches demonstrated absence of rejection or hernia recurrence, in contrast to the performance of a commercially available synthetic material. Diaphragm-derived ECM was able to promote the generation of new blood vessels, boost long-term muscle regeneration, and recover host diaphragmatic function. In addition, using a GFP\u202f+\u202fSchwann cell mouse model, we identified re-innervation of implanted patches. These results demonstrated for the first time that implantation of a tissue-specific biologic scaffold is able to promote a regenerating diaphragm muscle and overcome issues commonly related to the standard use of prosthetic materials

Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides

Endothelial dysfunction is a hallmark of LPS-induced acute kidney injury (AKI). Endothelial cells (ECs) acquired a fibroblast-like phenotype and contributed to myofibroblast generation through the endothelial-to-mesenchymal transition (EndMT) process. Of note, human adult renal stem/progenitor cells (ARPCs) enhance the tubular regenerative mechanism during AKI but little is known about their effects on ECs. Following LPS exposure, ECs proliferated, decreased EC markers CD31 and vascular endothelial cadherin, and up-regulated myofibroblast markers, collagen I, and vimentin. The coculture with ARPCs normalized the EC proliferation rate and abrogated the LPS-induced EndMT. The gene expression analysis showed that most of the genes modulated in LPS-stimulated ARPCs belong to cell activation and defense response pathways. We showed that the ARPC-specific antifibrotic effect is exerted by the secretion of CXCL6, SAA4, and BPIFA2 produced after the anaphylatoxin stimulation. Next, we investigated the molecular signaling that underlies the ARPC protective mechanism and found that renal progenitors diverge from differentiated tubular cells and ECs in myeloid differentiation primary response 88-independent pathway activation. Finally, in a swine model of LPS-induced AKI, we observed that activated ARPCs secreted CXCL6, SAA4, and BPIFA2 as a defense response. These data open new perspectives on the treatment of both sepsis- and endotoxemia-induced AKI, suggesting an underestimated role of ARPCs in preventing endothelial dysfunction and novel strategies to protect the endothelial compartment and promote kidney repair.-Sallustio, F., Stasi, A., Curci, C., Divella, C., Picerno, A., Franzin, R., De Palma, G., Rutigliano, M., Lucarelli, G., Battaglia, M., Staffieri, F., Crovace, A., Pertosa, G. B., Castellano, G., Gallone, A., Gesualdo, L. Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides

Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1β, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.Fil: Tolomeo, Anna Maria. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Università di Padova; Italia. Consorzio per la Ricerca Sanitaria; ItaliaFil: Castagliuolo, Ignazio. Università di Padova; ItaliaFil: Piccoli, Martina. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Grassi, Michele. Università di Padova; ItaliaFil: Magarotto, Fabio. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Università di Padova; ItaliaFil: De Lazzari, Giada. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; Italia. Consorzio per la Ricerca Sanitaria; Italia. Università di Padova; ItaliaFil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; Argentina. Consorzio per la Ricerca Sanitaria; Italia. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Caicci, Federico. Università di Padova; ItaliaFil: Franzin, Chiara. Fondazione Istituto di Ricerca Pediatrica Città della Speranza; ItaliaFil: Scarpa, Melania. Veneto Institute of Oncology; ItaliaFil: Macchi, Veronica. Università di Padova; ItaliaFil: De Caro, Raffaele. Università di Padova; Italia. Consorzio Per la Ricerca Sanitaria; ItaliaFil: Angriman, Imerio. Università di Padova; ItaliaFil: Viola, Antonella. Università di Padova; ItaliaFil: Porzionato, Andrea. Consorzio Per la Ricerca Sanitaria; Italia. Università di Padova; ItaliaFil: Pozzobon, Michela. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Italia. Università di Padova; ItaliaFil: Muraca, Maurizio. Università di Padova; Italia. Consorzio Per la Ricerca Sanitaria; Italia. Fondazione Istituto Di Ricerca Pediatrica Città Della Speranza; Itali

Complement Activation During lschemia/Reperfusion Injury Induces Pericyte-to-Myofibroblast Transdifferentiation Regulating Peritubular Capillary Lumen Reduction Through pERK Signaling

Nephrolog

Clonal Characterization of Rat Muscle Satellite Cells: Proliferation, Metabolism and Differentiation Define an Intrinsic Heterogeneity

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described

Double Labeling of PDGFR-β and α-SMA in Swine Models of Acute Kidney Injury to Detect Pericyte-to-Myofibroblast Transdifferentation as Early Marker of Fibrosis

Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to transdifferentiate toward alpha-SMA(+) myofibroblasts leading to interstitial fibrosis. Even if precise pericytes identification requires transmission electron microscopy and the co-immunostaining of several markers (i. e., Gli, NG2 chondroitin sulphate proteoglycan, CD146, desmin or CD73) and emerging new markers (CD248 or TEM1, endosialin), previous studies suggested that PDGFR-beta could be used as marker for renal pericytes characterization. Recently, double immunofluorescence staining of PDGFR-beta and alpha-SMA was performed to identify the damage activated pericytes(PDGFR-beta(+)/alpha-SMA(+) cells) in the early phase of fibrosis development. Our data highlighted the crucial role of renal pericytes in the physiopathology of sepsis and I/R associated AKI. In this protocol, we describe the procedure for double immunofluorescence staining of PDGFR-beta and alpha-SMA in swine Formalin-Fixed Paraffin-Embedded (FFPE) kidney biopsies and the method for image analysis and quantification