Susanna Benvenuti

Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue

Adipose tissue is a dynamic endocrine organ with a central role in metabolism regulation. Functional differences in adipose tissue seem associated with the regional distribution of fat depots, in particular in subcutaneous and visceral omental pads. Here, we report for the first time the isolation of human adipose‐derived adult stem cells from visceral omental and subcutaneous fat (V‐ASCs and S‐ASCs, respectively) from the same subject. Immunophenotyping shows that plastic culturing selects homogeneous cell populations of V‐ASCs and S‐ASCs from the corresponding stromal vascular fractions (SVFs), sharing typical markers of mesenchymal stem cells. Electron microscopy and electrophysiological and real‐time RT‐PCR analyses confirm the mesenchymal stem nature of both V‐ASCs and S‐ASCs, while no significant differences in a limited pattern of cytokine/chemokine expression can be detected. Similar to S‐ASCs, V‐ASCs can differentiate in vitro toward adipogenic, osteogenic, chondrogenic, muscular, and neuronal lineages, as demonstrated by histochemical, immunofluorescence, real‐time RT‐PCR, and electrophysiological analyses, suggesting the multipotency of such adult stem cells. Our data demonstrate that both visceral and subcutaneous adipose tissues are a source of pluripotent stem cells with multigermline potential. However, the visceral rather than the subcutaneous ASC could represent a more appropriate in vitro cell model for investigating the molecular mechanisms implicated in the pathophysiology of metabolic disorders such as obesity.—Baglioni, S., Francalanci, M., Squecco, R., Lombardi, A., Cantini, G., Angeli, R., Gelmini, S., Guasti, D., Benvenuti, S., Annunziato, F., Bani, D., Liotta, F., Francini, F., Perigli, G., Serio, M., Luconi, M. Characterization of human adult stem‐cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J. 23, 3494–3505 (2009). www.fasebj.org

Adhesion, growth, and matrix production by osteoblasts on collagen substrata

SummaryA number of studies have demonstrated the pivotal role of collagen molecules in modulating cell growth and differentiation. In order to analyze the direct effects of collagen type I on the osteoblastic phenotype, we have devised an in vitro culture system for studying the interactions between bovine collagen type I and Saos-2 cells, a human osteoblastic cell line. Saos-2 cells were cultured both on top of collagen-coated culture dishes as well as inside a three-dimensional collagen network. Plating on dishes treated with collagen induced maximal adhesion of Saos-2 cells after 24-hour incubation. Cells cultured on collagen gel matrix expressed about 2.5-fold more alkaline phosphatase when compared with untreated plastic dishes. On collagen-coated dishes the responsiveness of Saos-2 cells to parathyroid hormone was decreased, whereas no modifications were observed in the effect of vasoactive intestinal peptide on these cells. Using a microfluorimetric measurement of DNA, an increase of proliferation was observed in Saos-2 cells cultured on collagen gel Saos-2 cells were also able to colonize collagen sponges and in this three-dimensional network they were able to synthesize osteocalcin, as assessed both by immunocytochemistry and radioimmunoassay. In this study we have demonstrated that bovine collagen type I exhibits favorable effects on attachment and functional and growth activities of a human osteoblastic cell line, encouraging its use as a bone graft material.

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.

Membrane binding sites and non-genomic effects of estrogen in cultured human preosteoclastic cells

Besides functional estrogen receptors, the presence of signalling cell surface binding sites for 17beta-estradiol (17betaE2) has been reported in osteoblast- and osteoclast-like cells, suggesting that 17betaE2 may influence bone remodelling by a dual mechanism of action: to affect gene expression mediated by the nuclear activity of the steroid-receptor complex, and to initiate rapid responses triggered by a signal-generating receptor on the cell surface. Recently, we demonstrated that the human pre-osteoclastic cell line FLG 29.1 bears functional estrogen receptors. In this study we examined FLG 29.1 cells for the presence of cell surface binding sites for 17betaE2, and whether 17betaE2 could elicit cell signalling. Using a cell-impermeant and fluorescent estrogen conjugate, 17beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, we demonstrated the presence of specific plasma membrane binding sites for 17betaE2. Stimulation of FLG 29.1 cells with low (1 nM) and high (1 microM) doses of 17betaE2 induced a prompt and significant (P < 0.05) increase of cellular pH, as measured in single cells using an image analysis system. In addition, both cAMP and cGMP were significantly increased by 17betaE2 with a dose-dependent response. Finally, a rapid increase of intracellular calcium ion concentration [Ca2+] was also induced by 1 nM 17betaE2, as measured in single cells using an image analysis system. Our findings strongly suggest a non-genomic action of 17betaE2 on osteoclast precursors.

Prosurvival Effect of DHCR24/Seladin-1 in Acute and Chronic Responses to Oxidative Stress

ABSTRACT DHCR24/seladin-1, a crucial enzyme in sterol synthesis, is of lower abundance in brain areas affected by Alzheimers disease. While high levels of DHCR24/seladin-1 exert antiapoptotic function by conferring resistance against oxidative stress, the molecular mechanism for this protective effect is not fully understood. Here we show that DHCR24/seladin-1 expression is up-regulated in an acute response and down-regulated in a chronic response to oxidative stress. High levels of DHCR24/seladin-1 were associated with elevated cholesterol concentrations and a general increase in cholesterol biosynthesis upon oxidative stress exposure in neuroblastoma SH-SY5Y cells. DHCR24/seladin-1 overexpression conferred resistance to oxidative stress in a cholesterol-dependent manner. Mutating the reductase activity within DHCR24/seladin-1 abolished this protective effect. Conversely, DHCR24/seladin-1 levels diminished upon chronic exposure to oxidative stress. Low levels of DHCR24/seladin-1 were associated with reduced p53 levels, independent of DHCR24 activity and cholesterol concentrations. Additionally, ablation of DHCR24/seladin-1 prevented apoptosis of primary neurons in a p53-dependent manner and reduced the response of critical p53 targets due to deficient stabilization of p53 and therefore elevated p53 ubiquitination and degradation. Our findings reveal a dual capacity of DHCR24/seladin-1, which appears to be involved in two mechanistically independent prosurvival effects, exerting an acute response and a chronic response to oxidative stress.

Seladin-1 Is a Fundamental Mediator of the Neuroprotective Effects of Estrogen in Human Neuroblast Long-Term Cell Cultures

Estrogen exerts neuroprotective effects and reduces beta-amyloid accumulation in models of Alzheimers disease (AD). A few years ago, a new neuroprotective gene, i.e. seladin-1 (for selective AD indicator-1), was identified and found to be down-regulated in AD vulnerable brain regions. Seladin-1 inhibits the activation of caspase-3, a key modulator of apoptosis. In addition, it has been demonstrated that the seladin-1 gene encodes 3beta-hydroxysterol Delta24-reductase, which catalyzes the synthesis of cholesterol from desmosterol. We have demonstrated previously that in fetal neuroepithelial cells, 17beta-estradiol (17betaE2), raloxifene, and tamoxifen exert neuroprotective effects and increase the expression of seladin-1. The aim of the present study was to elucidate whether seladin-1 is directly involved in estrogen-mediated neuroprotection. Using the small interfering RNA methodology, significantly reduced levels of seladin-1 mRNA and protein were obtained in fetal neuroepithelial cells. Seladin-1 silencing determined the loss of the protective effect of 17betaE2 against beta-amyloid and oxidative stress toxicity and caspase-3 activation. A computer-assisted analysis revealed the presence of half-palindromic estrogen responsive elements upstream from the coding region of the seladin-1 gene. A 1490-bp region was cloned in a luciferase reporter vector, which was transiently cotransfected with the estrogen receptor alpha in Chinese hamster ovarian cells. The exposure to 17betaE2, raloxifene, tamoxifen, and the soy isoflavones genistein and zearalenone increased luciferase activity, thus suggesting a functional role for the half-estrogen responsive elements of the seladin-1 gene. Our data provide for the first time a direct demonstration that seladin-1 may be considered a fundamental mediator of the neuroprotective effects of estrogen.

Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells

Abstract.Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as α-smooth muscle actin (αSMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated αSMA. More importantly, S1P challenge was responsible for the functional appearance of Ca2+ currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P2 turned out to be critical for the pro-differentiating effect of S1P, while S1P3 appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration.

Characterization and function of the receptor for IGF-I in human preosteoclastic cells

Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cells migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.

Fluid Shear Stress Increases Interleukin‐11 Expression in Human Osteoblast‐like Cells: Its Role in Osteoclast Induction

It is unclear how mechanical stress influences bone cells. Mechanical stress causes fluid shear stress (FSS) in the bone. Osteoblast lineage cells are thought to sense FSS and regulate bone remodeling. We therefore investigated the effects of FSS on human osteoblast‐like osteosarcoma cells: SaOS‐2 cells in vitro. The conditioned medium of the SaOS‐2 cells after 24 h of FSS (24 h‐FSS CM) showed such osteoclastic phenotype inductions as significantly increasing the number of tartrate‐resistant acid phosphatase (TRAP) positive multinuclear cells in rat bone marrow cells and TRAP‐positive cells in human preosteoclastic cells: FLG 29.1 cells. An enzyme‐linked immunosorbent assay showed interleukin‐11 (IL‐11) protein to increase 7‐fold in the 24 h‐FSS CM. A Northern analysis showed that IL‐11 mRNA increased 4‐fold in the SaOS‐2 cells after 6 h‐FSS; however, no IL‐6 mRNA expression was detected. Furthermore, the anti‐human IL‐11 antibody significantly neutralized the osteoclastic phenotype induction of the 24 h‐FSS CM. The IL‐11 mRNA up‐regulation in SaOS‐2 cells by the 6 h‐FSS was not inhibited by the anti‐human transforming growth factor‐β1 antibody, but it was significantly inhibited by indomethacin. An enzymeimmunoassay showed prostaglandin E2 to increase 7‐fold in the 1 h‐FSS CM. These findings thus suggest that FSS induces osteoblasts to produce IL‐11 (mediated by prostaglandins) and thus stimulates bone remodeling.

Dermal matrix scaffold engineered with adult mesenchymal stem cells and platelet-rich plasma as a potential tool for tissue repair and regeneration.

The purpose of this study was to investigate the efficacy of Integra®, an artificial dermal matrix used as a dermal template for skin regeneration, to form a multifunctional scaffold with human bone marrow‐derived mesenchymal stem cells (hMSCs) and platelet‐rich plasma (PRP) for tissue engineering and regenerative technology. First, we showed that PRP, used as a supplement for growth medium, represented an optimal substitute for animal serum as well as a source of multiple growth factors, was able to satisfactorily support cell viability and cell proliferation and influence stemness gene expression in hMSCs. Moreover, Integra appeared to be a suitable substrate for hMSCs colonization, as judged by two‐photon microscopy combined with fluorescence lifetime imaging (FLIM) and confocal analysis. The cells were then seeded on Integra + PRP for 24 and 48 h. Notably, in these conditions, the seeded cells exhibited a greater aptitude to colonize the scaffold, showed improved cell adhesion and spreading as compared with those cultured on Integra alone, and acquired a fibroblast‐like phenotype, indicating that the bioengineered scaffold provided an appropriate environment for cellular growth and differentiation. In conclusion, these results, although preliminary, provide clues for the design of new therapeutic strategies for skin regeneration, consisting in the combination of mesenchymal stem cells with engineered biomaterials. Copyright