Gianluca Carnevale

Human Serum Promotes Osteogenic Differentiation of Human Dental Pulp Stem Cells In Vitro and In Vivo

Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

Human Dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations

BackgroundHuman dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency.ResultsThe STRO-1+/c-Kit+/CD34+ hDPSCs showed a slower proliferation, gradual loss of stemness, early cell senescence and apoptosis, compared to STRO-1+/c-Kit+/CD34− hDPSCs. Both the subpopulations demonstrated similar abilities to differentiate towards mesoderm lineages, whereas a significant difference was observed after the neurogenic induction, with a greater commitment of STRO-1+/c-Kit+/CD34+ hDPSCs. Moreover, undifferentiated STRO-1+/c-Kit+/CD34− hDPSCs did not show any expression of CD271 and nestin, typical neural markers, while STRO-1+/c-Kit+/CD34+ hDPSCs expressed both.ConclusionsThese results suggest that STRO-1+/c-Kit+/CD34− hDPSCs and STRO-1+/c-Kit+/CD34+ hDPSCs might represent two distinct stem cell populations, with different properties. These results trigger further analyses to deeply investigate the hypothesis that more than a single stem cell population resides within the dental pulp, to better define the flexibility of application of hDPSCs in regenerative medicine.

Human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization

IntroductionThe main aim of this study is to evaluate potential human stem cells, such as dental pulp stem cells and amniotic fluid stem cells, combined with collagen scaffold to reconstruct critical-size cranial bone defects in an animal model.MethodsWe performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. After 4 and 8 weeks, cranial tissue samples were taken for histological and immunofluorescence analysis.ResultsWe observed a new bone formation in all of the samples but the most relevant differences in defect correction were shown by stem cell–collagen samples 4 weeks after implant, suggesting a faster regeneration ability of the combined constructs. The presence of human cells in the newly formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Furthermore, human cells were found to be an essential part of new vessel formation in the scaffold.ConclusionThese data confirmed the strong potential of bioengineered constructs of stem cell–collagen scaffold for correcting large cranial defects in an animal model and highlighting the role of stem cells in neovascularization during skeletal defect reconstruction.

Influence of ferutinin on bone metabolism in ovariectomized rats. II: Role in recovering osteoporosis

The aim of the present investigation, which represents an extension of a previous study, was to investigate the effect of ferutinin in recovering severe osteoporosis due to estrogen deficiency after rat ovariectomy and to compare phytoestrogen effects with those of estrogens commonly used in hormone replacement therapy (HRT) by women with postmenopausal osteoporosis. The animal model used was the Sprague–Dawley ovariectomized rat. Ferutinin was orally administered (2 mg kg−1 per day) for 30 or 60 days starting from 2 months after ovariectomy (i.e. when osteoporosis was clearly evident) and its effects were compared with those of estradiol benzoate (1.5 μg per rat twice a week, subcutaneously injected) vs. vehicle‐treated ovariectomized (OVX) and sham‐operated (SHAM) rats. Histomorphometric analyses were performed on trabecular bone of lumbar vertebrae (4th and 5th) and distal femoral epiphysis, as well as on cortical bone of femoral diaphysis. Bone histomorphometric analyses showed that ferutinin seems to display the same effects on bone mass recorded with estradiol benzoate, thus suggesting that it could enhance the recovery of bone loss due to severe estrogen deficiency in OVX rats. On this basis, the authors propose listing ferutinin among the substances representing a potential alternative for the treatment of postmenopausal osteoporosis, which occurs as a result of estrogen deficiency.

In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp

AIM To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic β-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration.

The protease inhibitor atazanavir triggers autophagy and mitophagy in human preadipocytes.

Background:The association between HAART and lipodystrophy is well established, but lipodystrophy pathogenesis is still poorly understood. Drugs, and in particular protease inhibitors, accumulate in adipose tissue affecting adipocyte physiology and gene expression by several mechanisms. Recent studies have identified autophagy as another process affected by these classes of drugs, but no studies have been performed in adipose cells. Methods:SW872 preadipocytic human cell line was used to evaluate changes induced by amprenavir (APV), ritonavir (RTV), or atazanavir (ATV), all used at 10–200 &mgr;mol/l. A subline was stably transfected with murine stem cell virus (pMSCV)-enhanced green fluorescent protein (EGFP)-LC3 plasmid (to obtain a fluorescent LC3 protein) and treated with ATV at different doses. The distribution of LC3 and the colocalization of mitochondria, lysosome, and autophagosome were assessed by confocal microscopy. Transmission electron microscopy of ATV-treated cells was also performed. The cellular content of lysosomes was assessed using Lysotracker Green; apoptosis was evaluated by annexin V/propidium iodide staining, and mitochondrial superoxide anion (mtO2-) was analyzed by mitoSOX red. Lysosomes, apoptosis, and mtO2- were studied by flow cytometry and multispectral imaging flow cytometry. Results:In SW872 cells, RTV caused massive apoptosis, more than autophagy, whereas APV was almost ineffective. ATV induced both apoptosis (high doses) and autophagy (low doses). ATV-treated cells displayed LC3-specific punctae, suggesting the formation of autophagosomes that enclosed mitochondria, as revealed by electron microscopy. At low doses, ATV promoted mitochondrial superoxide generation, whereas at high doses, it induced mitochondrial membrane depolarization. Conclusion:Autophagy/mitophagy can be considered a mechanism triggered by ATV in SW872 preadipocytes.

Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells

Lon is a nuclear‐encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up‐regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon‐silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to β‐oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5‐fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2‐deoxy‐D‐glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3‐ and 1.3‐fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2‐cyano‐3,12‐dioxooleana‐1,9,‐dien‐28‐oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.—Gibellini, L., Pinti, M., Boraldi, F., Giorgio, V., Bernardi, P., Bartolomeo, R., Nasi, M., De Biasi, S., Missiroli, S., Carnevale, G., Losi, L., Tesei, A., Pinton, P., Quaglino, D., Cossarizza, A., Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells. FASEB J. 28, 5122–5135 (2014). www.fasebj.org

Inhibition of Lon protease by triterpenoids alters mitochondria and is associated to cell death in human cancer cells

Mitochondrial Lon protease (Lon) regulates several mitochondrial functions, and is inhibited by the anticancer molecule triterpenoid 2-cyano-3, 12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), or by its C-28 methyl ester derivative (CDDO-Me). To analyze the mechanism of action of triterpenoids, we investigated intramitochondrial reactive oxygen species (ROS), mitochondrial membrane potential, mitochondrial mass, mitochondrial dynamics and morphology, and Lon proteolytic activity in RKO human colon cancer cells, in HepG2 hepatocarcinoma cells and in MCF7 breast carcinoma cells. We found that CDDO and CDDO-Me are potent stressors for mitochondria in cancer cells, rather than normal non-transformed cells. In particular, they: i) cause depolarization; ii) increase mitochondrial ROS, iii) alter mitochondrial morphology and proteins involved in mitochondrial dynamics; iv) affect the levels of Lon and those of aconitase and human transcription factor A, which are targets of Lon activity; v) increase level of protein carbonyls in mitochondria; vi) lead to intrinsic apoptosis. The overexpression of Lon can rescue cells from cell death, providing an additional evidence on the role of Lon in conditions of excessive stress load.

Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells

AIMS The phytoestrogen Ferutinin plays an important role in prevention of osteoporosis caused by ovariectomy-induced estrogen deficiency in rats, but there is no evidence of its effect on osteoblastic differentiation in vitro. In this study we investigated the effect of Ferutinin on proliferation and osteoblastic differentiation of two different human stem cells populations, one derived from the amniotic fluid (AFSCs) and the other from the dental pulp (DPSCs). MAIN METHODS AFSCs and DPSCs were cultured in a differentiation medium for 14 or 21days with or without the addition of Ferutinin at a concentration ranging from 10(-11) to 10(-4)M. 17β-Estradiol was used as a positive drug at 10(-8)M. Cell proliferation and expression of specific osteoblast phenotype markers were analyzed. KEY FINDINGS MTT assay revealed that Ferutinin, at concentrations of 10(-8) and 10(-9)M, enhanced proliferation of both AFSCs and DPSCs after 72h of exposure. Moreover, in both stem cell populations, Ferutinin treatment induced greater expression of the osteoblast phenotype markers osteocalcin (OCN), osteopontin (OPN), collagen I, RUNX-2 and osterix (OSX), increased calcium deposition and osteocalcin secretion in the culture medium compared to controls. These effects were more pronounced after 14days of culture in both populations. SIGNIFICANCE The enhancing capabilities on proliferation and osteoblastic differentiation displayed by the phytoestrogen Ferutinin make this compound an interesting candidate to promote bone formation in vivo.

Sirtuin 3 interacts with Lon protease and regulates its acetylation status

Lon is a mitochondrial protease that degrades oxidized damaged proteins, assists protein folding and participates in maintaining mitochondrial DNA levels. Changes in Lon mRNA levels, protein levels and activity are not always directly correlated, suggesting that Lon could be regulated at post translational level. We found that Lon and SIRT3, the most important mitochondrial sirtuin, colocalize and coimmunoprecipitate in breast cancer cells, and silencing or inhibition of Lon did not alter SIRT3 levels. Silencing of SIRT3 increased the levels of Lon protein and of its acetylation, suggesting that Lon is a target of SIRT3, likely at K917.