Daniele Guasti

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

Telocytes subtypes in human urinary bladder.

Urinary bladder voiding is a complex mechanism depending upon interplay among detrusor, urothelium, sensory and motor neurons and connective tissue cells. The identity of some of the latter cells is still controversial. We presently attempted to clarify their phenotype(s) in the human urinary bladder by transmission electron microscopy (TEM) and immunohistochemistry. At this latter aim, we used CD34, PDGFRα, αSMA, c‐Kit and calreticulin antibodies. Both, TEM and immunohistochemistry, showed cells that, sharing several telocyte (TC) characteristics, we identified as TC; these cells, however, differed from each other in some ultrastructural features and immunolabelling according to their location. PDGFRα/calret‐positive, CD34/c‐Kit‐negative TC were located in the sub‐urothelium and distinct in two subtypes whether, similarly to myofibroblasts, they were αSMA‐positive and had attachment plaques. The sub‐urothelial TC formed a mixed network with myofibroblasts and were close to numerous nerve endings, many of which nNOS‐positive. A third TC subtype, PDGFRα/αSMA/c‐Kit‐negative, CD34/calret‐positive, ultrastructurally typical, was located in the submucosa and detrusor. Briefly, in the human bladder, we found three TC subtypes. Each subtype likely forms a network building a 3‐D scaffold able to follow the bladder wall distension and relaxation and avoiding anomalous wall deformation. The TC located in the sub‐urothelium, a region considered a sort of sensory system for the micturition reflex, as forming a network with myofibroblasts, possessing specialized junctions with extracellular matrix and being close to nerve endings, might have a role in bladder reflexes. In conclusions, the urinary bladder contains peculiar TC able to adapt their morphology to the organ activity.

Morphofunctional effects of mitotane on mitochondria in human adrenocortical cancer cells

At present, mitotane (MTT) represents the first-line pharmacological approach for the treatment of advanced adrenocortical carcinoma (ACC). Despite clear evidence that the drug can reduce the clinical signs of steroid excess in secreting ACC, the mechanism mediating the possible toxic effect of MTT on tumor cells still remains obscure. This study investigated the intracellular events underlying the toxic effect of MTT by studying qualitative and quantitative alterations in mitochondrial morphology and functions in human adrenocortical cancer cell lines, H295R and SW13. Increasing concentrations of MTT resulted in rapid intracellular accumulation and conversion of the drug. Cytostatic and cytotoxic effects were evident at doses corresponding to the therapeutic window (30-50 μM) through an apoptotic mechanism involving caspase 3/7. Electron microscopic analysis of cell mitochondria displayed MTT-induced dose- and time-dependent alterations in the morphology of the organelle. These alterations were characterized by a marked swelling and a decrease in the number of respiratory cristae, accompanied by a significant depolarization of the mitochondrial membrane potential, finally leading to the disruption of the organelle. A drastic reduction of oxygen consumption was observed due to mitochondrial membrane damage, which was accompanied by a decrease in the levels of VDAC1 integral membrane channel. These findings contribute to better understand the intracellular mechanism of action of MTT in ACC cells, showing that its cytotoxic effect seems to be mainly mediated by an apoptotic process activated by the disruption of mitochondria.

Carbon Nanotube Scaffolds Instruct Human Dendritic Cells: Modulating Immune Responses by Contacts at the Nanoscale

Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immune-modulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile.

Exosomes secreted from human colon cancer cells influence the adhesion of neighboring metastatic cells: Role of microRNA-210

ABSTRACT Cancer-secreted exosomes influence tumor microenvironment and support cancer growth and metastasis. MiR-210 is frequently up-regulated in colorectal cancer tissues and correlates with metastatic disease. We investigated whether exosomes are actively released by HCT-8 colon cancer cells, the role of exosomal miR-210 in the cross-talk between primary cancer cells and neighboring metastatic cells and its contribution in regulating epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). After 7 d of culture, a subpopulation of viable HCT-8 cells detached the monolayer and started to grow in suspension, suggesting anoikis resistance and a metastatic potential. The expression of key proteins of EMT revealed that these cells were E-cadherin negative and vimentin positive further confirming their metastatic phenotype and the acquisition of anoikis resistance. Metastatic cells, in the presence of adherently growing HCT-8, continued to grow in suspension whereas only if seeded in cell-free wells, were able to adhere again and to form E-cadherin positive and vimentin negative new colonies, suggesting the occurrence of MET. The chemosensitivity to 5 fluorouracil and to FOLFOX-like treatment of metastatic cells was significantly diminished compared to adherent HCT-8 cells. Of note, adherent new colonies undergoing MET, were insensitive to both chemotherapeutic strategies. Electron microscopy analysis demonstrated that adherently growing HCT-8, actually secreted exosomes and that exosomes in turn were taken up by metastatic cells. When exosomes secreted by adherently growing HCT-8 were administered to metastatic cells, MET was significantly inhibited. miR-210 was significantly upregulated in exosomes compared to its intracellular levels in adherently growing HCT-8 cells and correlated to anoikis resistance and EMT markers. Exosomes containing miR-210 might be considered as EMT promoting signals that preserve the local cancer-growth permissive milieu and also guide metastatic cells to free, new sites of dissemination.

PARP Inhibition Delays Progression of Mitochondrial Encephalopathy in Mice

Mitochondrial disorders are deadly childhood diseases for which therapeutic remedies are an unmet need. Given that genetic suppression of the nuclear enzyme poly (adenine diphosphate-ribose) polymerase(PARP)-1 improves mitochondrial functioning, we investigated whether pharmacological inhibition of the enzyme affords protection in a mouse model of a mitochondrial disorder. We used mice lacking the Ndufs4 subunit of the respiratory complex I (Ndufs4 knockout [ KO] mice); these mice undergo progressive encephalopathy and die around postnatal day 50. Mice were treated daily with the potent PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acetamide hydrochloride (PJ34); neurological parameters, PARP activity, and mitochondrial homeostasis were evaluated. We found that mice receiving N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acetamide hydrochloride from postnatal day 30 to postnatal day 50 show reduced neurological impairment, and increased exploratory activity and motor skills compared with vehicle-treated animals. However, drug treatment did not delay or reduce death. We found no evidence of increased PARP activity within the brain of KO mice compared with heterozygous, healthy controls. Conversely, a 10-day treatment with the PARP inhibitor significantly reduced basal poly(ADP-ribosyl)ation in different organs of the KO mice, including brain, skeletal muscle, liver, pancreas, and spleen. In keeping with the epigenetic role of PARP-1, its inhibition correlated with increased expression of mitochondrial respiratory complex subunits and organelle number. Remarkably, pharmacological targeting of PARP reduced astrogliosis in olfactory bulb and motor cortex, but did not affect neuronal loss of KO mice. In light of the advanced clinical development of PARP inhibitors, these data emphasize their relevance to treatment of mitochondrial respiratory defects.

Searching for Classical Brown Fat in Humans: Development of a Novel Human Fetal Brown Stem Cell Model

The potential therapeutic applications of targeting brown adipose tissue open new clinical avenues in fighting against metabolic pathologies. However, due to the limited extension in adult humans of brown depots, which are dramatically reduced after birth, solid cell models to study human brown adipogenesis and its regulatory factors in pathophysiology are urgently needed. Here, we generated a novel human model of brown adipose stem cells, hfB‐ASC, derived for the first time from fetal interscapular brown fat depots. Besides the characterization of their stem and classical brown adipose properties, we demonstrated that these cells retain a specific intrinsic differentiation program to functional brown adipocytes, even spontaneously generating organoid structures with brown features. Moreover, for the first time, we investigated the thermogenic and electrophysiological activity of the in vitro‐derived fetal brown adipocytes compared to their undifferentiated precursors hfB‐ASC, in basal and norepinephrine‐induced conditions. In conclusion, from interscapular brown fat of the human fetus we developed and functionally characterized a novel physiological brown adipose stem cell model early programmed to brown differentiation, which may represent a unique opportunity for further studies on brown adipogenesis processes in humans as well as the most suitable target to study novel therapeutic approaches for stimulating brown activity in metabolic pathologies. Stem Cells 2016;34:1679–1691

Tadalafil reduces visceral adipose tissue accumulation by promoting preadipocytes differentiation towards a metabolically healthy phenotype: Studies in rabbits

Development of metabolically healthy adipocytes within dysfunctional adipose tissue may represent an attractive way to counteract metabolic syndrome (MetS). In an experimental animal model of high fat diet (HFD)-induced MetS, in vivo, long- and short-term tadalafil treatments were able to reduce visceral adipose tissue (VAT) accumulation and hypertriglyceridemia, and to induce the expression in VAT of the brown fat-specific marker, uncoupling protein 1 (UCP1). VAT preadipocytes (PAD), isolated from the tadalafil-treated HFD rabbits, showed: i) a multilocular morphology; ii) an increased expression of brown fat-specific genes (such as UCP1 and CIDEA); iii) improved mitochondrial structure and dynamic and reduced superoxide production; iv) improved insulin sensitivity. Similar effects were obtained after in vitro tadalafil treatment in HFD rPAD. In conclusion, tadalafil counteracted HFD-associated VAT alterations, by restoring insulin-sensitivity and prompting preadipocytes differentiation towards a metabolically healthy phenotype.

Dissecting the Origin of Inducible Brown Fat in Adult Humans Through a Novel Adipose Stem Cell Model from Adipose Tissue Surrounding Pheochromocytoma

CONTEXT The recent discovery of inducible brown adipose tissue (BAT) in adult humans, in which it is involved in controlling adiposity, is pivotal in the development of treatment strategies for obesity. However, the origin of these adipocytes in white adipose tissue (WAT) is still unclear, and no human brown adipose cell models are currently available. OBJECTIVE The objective of the study was to define the origin of inducible BAT (iBAT) by isolating brown adipose stem cells (B-ASCs) from periadrenal fat in adult patients with catecholamine-secreting pheochromocytoma. DESIGN This was a 1-year study to enroll adrenal tumor patients undergoing surgery. SETTING The study was conducted at a university hospital. PATIENTS AND INTERVENTION Eight patients operated for pheochromocytoma and three for adrenocortical adenoma participated in the study. MAIN OUTCOME MEASURES Isolation of inducible B-ASCs from fat surrounding the pheochromocytoma was measured. RESULTS We demonstrated the presence of iBAT islets dispersed in periadrenal WAT in patients operated for pheochromocytoma. From this fat depot, which expresses brite/classical BAT markers and high levels of uncoupling protein-1, we isolated B-ASCs and compared their properties with precursors from sc WAT (S-ASC) of the same patients. B-ASCs showed mesenchymal, stem, and multipotency features and expression of brite/classical BAT markers. When differentiated toward white phenotype, B-ASCs accumulated lipid droplets smaller than S-ASCs and expressed adiponectin. Upon induction of brown differentiation, brown commitment was found only in B-ASCs and not in S-ASCs, with no mature brown adipocytes. CONCLUSIONS Our findings demonstrate that iBAT developed in periadrenal WAT derives from adult stem cells, unlike WAT precursors, confirming an independent origin of the two fat depots. These stem cells represent a unique in vitro stem cell model to study brown adipogenesis and develop novel antiobesity therapies targeting WAT-BAT conversion.

Immunohistochemical analysis of dendritic cells in skin lesions: correlations with survival time

The response to wounds until healing requires the activity of many cell types coordinate in space and time, so that the types of cells in a wound and their localization may be of help to date lesions with respect to death, which would be useful in forensic pathology. Cells reacting to injury include dendritic cells; the early reaction of these cells to skin wounding has not yet been investigated in humans, which was the aim of this study. Samples of wounded and control skin were taken at autopsy and analyzed by affinity histochemistry. Both epidermal and dermal MHC-II+ cells increased transiently in number within the first hour after wounding, then decreased. In the epidermis the increase affected also CD1a+ cells, i.e. well differentiated Langherhans cells, which however increased less, earlier and for a shorter time period than MHC-II+ cells. Dermal MHC-II+ cells became part of a perivascular mononuclear cell infiltrate visible in the subpapillary dermis by 60 min after wounding, which contained also mast cells. The immediately perivascular MHC-II+ cells were DC-SIGN- and CD11c-, while MHC-II+, DC-SIGN+, CD11c+ dendritic cells were predominantly located at the periphery of infiltrates and some were near the epidermis. Mast cells underwent degranulation, besides increase in number, in the first hours after wounding. The results suggest that skin dendritic cells, including Langerhans cells, participate to the early response to wounding in concert with mast cells, and that subpapillary blood vessels are primary sites of cell infiltration during that response in humans. The results show that the ratio between CD1a positive and MHC-II positive cells in the epidermis, the degranulation index of mast cells and the relative volume of MHC-II positive cells in the dermis can be added to the tools useful to distinguish vital from post mortem lesions and, the first two of them, to estimate the interval between a lesion and death.