Benedetta Mazzanti

Use of donor bone marrow mesenchymal stem cells for treatment of skin allograft rejection in a preclinical rat model.

Recent studies indicate that mesenchymal stem cells (MSC) exhibit a degree of immune privilege due to their ability to suppress T cell mediated responses causing tissue rejection; however, the impact of allogeneic MSC in the setting of organ transplantation has been poorly investigated so far. The aim of our study was to evaluate the effect of intravenous donor MSC infusion for clinical tolerance induction in allogeneic skin graft transplantations in rats. MSC were isolated from Wistar rats and administered in Sprague-Dawley rats receiving Wistar skin graft with or without cyclosporine A (CsA). Graft biopsies were performed at day 10 post transplantation in all experimental groups for histological and gene expression studies. Intravenous infusion with donor MSC in CsA-treated transplanted rats resulted in prolongation of skin allograft survival compared to control animals. Unexpectedly, donor MSC infusion in immunocompetent rats resulted in a faster rejection as compared to control group. Cytokine expression analysis at the site of skin graft showed that CsA treatment significantly decreased pro-inflammatory cytokines IFN-γ and IL-2 and reduced TNF-α gene expression; however, the level of TNF-α is high in MSC-treated and not immunosuppressed rats. Results of our study in a rat tissue transplantation model demonstrated a possible immunogenic role for donor (allogeneic) MSC, confirming the need of adequate preclinical experimentation before clinical use.

Rosiglitazone stimulates adipogenesis and decreases osteoblastogenesis in human mesenchymal stem cells

Thiazolidinediones (TZD) are widely prescribed for the treatment of Type 2 diabetes. Increased loss of bone mass and a higher incidence of fractures have been associated with the use of this class of drugs in post-menopausal women. In vitro studies performed in rodent cell models indicated that rosiglitazone (RGZ), one of the TZD, inhibited osteoblastogenesis and induced adipogenesis in bone marrow progenitor cells. The objective of the present study was to determine for the first time the RGZ-dependent shift from osteoblastogenesis toward adipogenesis using a human cell model. To this purpose, bone marrow-derived mesenchymal stem cells were characterized and induced to differentiate along osteogenic and adipogenic lineages. We found that the exposure to RGZ potentiated adipogenic differentiation and shifted the differentiation toward an osteogenic phenotype into an adipogenic phenotype, as assessed by the appearance of lipid droplets. Accordingly, RGZ markedly increased the expression of the typical marker of adipogenesis fatty-acid binding protein 4, whereas it reduced the expression of Runx2, a marker of osteoblastogenesis. This is the first demonstration that RGZ counteracts osteoblastogenesis and induces a preferential differentiation into adipocytes in human mesenchymal stem cells.

Inhibition of Immune Synapse by Altered Dendritic Cell Actin Distribution: A New Pathway of Mesenchymal Stem Cell Immune Regulation

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306–318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell–cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC–DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.

Autologous mesenchymal stem cells foster revascularization of ischemic limbs in systemic sclerosis: a case report.

BACKGROUND Mesenchymal stem cells can differentiate into endothelial cells and participate in angiogenesis in adults. In experimental models of acute myocardial infarction, mesenchymal stem cells led to the recovery of cardiac function through the formation of a new vascular network. OBJECTIVE To describe treatment with intravenous infusions of expanded autologous mesenchymal stem cells in 1 patient with critical limb ischemia due to systemic sclerosis. DESIGN Case report. SETTING The rheumatology unit at the University of Florence, Florence, Italy. PATIENT A woman, aged 34 years, with systemic sclerosis who developed acute gangrene of the upper and lower limbs. INTERVENTION 3 intravenous pulses of expanded autologous mesenchymal stem cells. MEASUREMENTS Angiography, skin histopathology, and immunohistochemistry. RESULTS Areas of necrotic skin were reduced after the first mesenchymal stem-cell infusion. After the third infusion, angiography showed revascularization of the patients extremities. Skin section analysis revealed cell clusters with tubelike structures, and angiogenic factors were strongly expressed. LIMITATION Causality cannot be established by a single case. CONCLUSION In patients with systemic sclerosis who have severe peripheral ischemia, intravenous infusion of expanded autologous mesenchymal stem cells may foster the recovery of the vascular network, restore blood flow, and reduce skin necrosis. PRIMARY FUNDING SOURCE Fondazione Cassa di Risparmio di Pistoia e Pescia (partial funding).

Toward MSC in Solid Organ Transplantation: 2008 Position Paper of the MISOT Study Group

The following position paper summarizes the recommendations for early clinical trials and ongoing basic research in the field of mesenchymal stem cell-induced solid organ graft acceptance—agreed upon on the first meeting of the Mesenchymal Stem Cells In Solid Organ Transplantation (MISOT) study group in late 2008.

ATP Modulates Cell Proliferation and Elicits Two Different Electrophysiological Responses in Human Mesenchymal Stem Cells

Bone marrow‐derived human mesenchymal stem cells (hMSCs) have the potential to differentiate into several cell lines. Extracellular adenosine 5′‐triphosphate (ATP) acts as a potent signaling molecule mediating cell‐to‐cell communication. Particular interest has been focused in recent years on the role of ATP in stem cell proliferation and differentiation. In the present work, we demonstrate that hMSCs at early stages of culture (P0–P5) spontaneously release ATP, which decreases cell proliferation. Increased hMSC proliferation is induced by the unselective P2 antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS) and by the selective P2Y1 antagonist 2′‐deoxy‐N6‐methyladenosine3′,5′‐bisphosphate (MRS 2179). A functional role of extracellular ATP in modulating ionic conductances with the whole‐cell and/or perforated patch‐clamp techniques was also investigated. Exogenous ATP increased both the voltage‐sensitive outward and inward currents in 47% of cells, whereas, in 31% of cells, only an increase in inward currents was found. Cells responding in this dual manner to ATP presented different resting membrane potentials. Both ATP‐induced effects had varying sensitivity to the P2 antagonists PPADS and MRS 2179. Outward ATP‐sensitive currents are carried by potassium ions, since they are blocked by cesium replacement and are Ca2+‐dependent because they are eliminated in the presence of 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid. On the basis of different electrophysiological and pharmacological characteristics, we conclude that outward ATP‐sensitive currents are due to Ca2+‐dependent K+‐channel activation following stimulation of P2Y receptors, whereas inward ATP‐sensitive currents are mediated by P2X receptor activation. In summary, ATP released in early life stages of hMSCs modulates their proliferation rate and likely acts as one of the early factors determining their cell fate.

Differences in mesenchymal stem cell cytokine profiles between MS patients and healthy donors: implication for assessment of disease activity and treatment.

MSCs have been proposed as possible treatment in MS: In this study MSCs obtained from 10 MS patients and 6 healthy donors (HD) were compared in terms of phenotypical and functional characteristics. We show that MSCs isolated from MS and HD differ significantly for IP10 production. Therefore, although MSCs isolated from MS patients exhibit the same properties of HD MSCs in terms of proliferation, phenotype, in vitro differentiation, TLR expression, immunosuppressive ability, inhibition of DC differentiation and activation, the use of autologous MSCs in cell therapy of autoimmune diseases should be submitted to attentive evaluation and treatment.

Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis exhibit a paracrine machinery and stimulate angiogenesis in vitro

Objective To characterise bone marrow-derived mesenchymal stem cells (MSCs) from patients with systemic sclerosis (SSc) for the expression of factors implicated in MSC recruitment at sites of injury, angiogenesis and fibrosis. The study also analysed whether the production/release of bioactive mediators by MSCs were affected by stimulation with cytokines found upregulated in SSc serum and tissues, and whether MSCs could modulate dermal microvascular endothelial cell (MVEC) angiogenesis. Methods MSCs obtained from five patients with early severe diffuse SSc (SSc-MSCs) and five healthy donors (H-MSCs) were stimulated with vascular endothelial growth factor (VEGF), transforming growth factor β (TGFβ) or stromal cell-derived factor-1 (SDF-1). Transcript and protein levels of SDF-1 and its receptor CXCR4, VEGF, TGFβ1 and receptors TβRI and TβRII were evaluated by quantitative real-time PCR, western blotting and confocal microscopy. VEGF, SDF-1 and TGFβ1 secretion in culture supernatant was measured by ELISA. MVEC capillary morphogenesis was performed on Matrigel with the addition of MSC-conditioned medium. Results In SSc-MSCs the basal expression of proangiogenic SDF-1/CXCR4 and VEGF was significantly increased compared with H-MSCs. SSc-MSCs constitutively released higher levels of SDF-1 and VEGF. SDF-1/CXCR4 were upregulated after VEGF stimulation and CXCR4 redistributed from the cytoplasm to the cell surface. VEGF was increased by SDF-1 challenge. VEGF, TGFβ and SDF-1 stimulation upregulated TGFβ1, TβRI and TβRII in SSc-MSCs. TβRII redistributed from the cytoplasm to focal adhesion contacts. SSc-MSC-conditioned medium showed a greater proangiogenic effect on MVECs than H-MSCs. Experiments with blocking antibodies showed that MSC-derived cytokines were responsible for this potent proangiogenic effect. Conclusion SSc-MSCs constitutively overexpress and release bioactive mediators/proangiogenic factors and potentiate dermal MVEC angiogenesis.

Mesenchymal stromal cells affect cardiomyocyte growth through juxtacrine Notch-1/Jagged-1 signaling and paracrine mechanisms: clues for cardiac regeneration.

The possibility to induce myocardial regeneration by the activation of resident cardiac stem cells (CSCs) has raised great interest. However, to propose endogenous CSCs as therapeutic options, a better understanding of the complex mechanisms controlling heart morphogenesis is needed, including the cellular and molecular interactions that cardiomyocyte precursors establish with cells of the stromal compartment. In the present study, we co-cultured immature cardiomyocytes from neonatal mouse hearts with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte growth in vitro. We found that cardiomyocyte proliferation was enhanced by direct co-culture with MSCs compared with the single cultures. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch-1 receptor by its ligand Jagged-1 expressed by the adjacent MSCs. In fact, the cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notch-intracellular domain (Notch-ICD) as compared with those cultured alone and this response was significantly attenuated when MSCs were silenced for Jagged-1. The presence of various cardiotropic cytokines and growth factors in the conditioned medium of MSCs underscored the contribution of paracrine mechanisms to Notch-1 up-regulation by the cardiomyocytes. In conclusions these findings unveil a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation through Notch-1/Jagged-1 pathway and suggest that stromal-myocardial cell juxtacrine and paracrine interactions may contribute to the development of new and more efficient cell-based myocardial repair strategies.

Testosterone treatment improves metabolic syndrome-induced adipose tissue derangements

We recently demonstrated that testosterone dosing ameliorated the metabolic profile and reduced visceral adipose tissue (VAT) in a high-fat diet (HFD)-induced rabbit model of metabolic syndrome (MetS). We studied the effects of HFD and in vivo testosterone dosing on VAT function and the adipogenic capacity of rabbit preadipocytes isolated from VAT of regular diet (RD), HFD, and testosterone-treated HFD rabbits. VAT was studied by immunohistochemistry, western blot, and RT-PCR. Isolated rPADs were exposed to adipocyte differentiating mixture (DIM) to evaluate adipogenic potential. Adipocyte size was significantly increased in HFD VAT compared with RD, indicating adipocyte dysfunction, which was normalized by testosterone dosing. Accordingly, perilipin, an anti-lipolytic protein, was significantly increased in HFD VAT, when compared with other groups. HFD VAT was hypoxic, while testosterone dosing normalized VAT oxygenation. In VAT, androgen receptor expression was positively associated with mRNA expression of GLUT4 (SLC2A4) (insulin-regulated glucose transporter) and STAMP2 (STEAP4) (androgen-dependent gene required for insulin signaling). In testosterone-treated HFD VAT, STAMP2 mRNA was significantly increased when compared with the other groups. Moreover, GLUT4 membrane translocation was significantly reduced in HFD VAT, compared with RD, and increased by testosterone. In DIM-exposed preadipocytes from HFD, triglyceride accumulation, adipocyte-specific genes, insulin-stimulated triglyceride synthesis, glucose uptake, and GLUT4 membrane translocation were reduced compared with preadipocytes from RD and normalized by in vivo testosterone dosing. In conclusion, testosterone dosing in a MetS animal model positively affects VAT functions. This could reflect the ability of testosterone in restoring insulin sensitivity in VAT, thus counteracting metabolic alterations.