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  1. Article ; Online: Knockdown of NANOG Reduces Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Human Adipose Stem Cells

    Maria Pitrone / Giuseppe Pizzolanti / Antonina Coppola / Laura Tomasello / Stefania Martorana / Gianni Pantuso / Carla Giordano

    International Journal of Molecular Sciences, Vol 20, Iss 10, p

    2019  Volume 2580

    Abstract: The core components of regenerative medicine are stem cells with high self-renewal and tissue regeneration potentials. Adult stem cells can be obtained from many organs and tissues. NANOG , SOX2 and OCT4 represent the core regulatory network that ... ...

    Abstract The core components of regenerative medicine are stem cells with high self-renewal and tissue regeneration potentials. Adult stem cells can be obtained from many organs and tissues. NANOG , SOX2 and OCT4 represent the core regulatory network that suppresses differentiation-associated genes, maintaining the pluripotency of mesenchymal stem cells. The roles of NANOG in maintaining self-renewal and undifferentiated status of adult stem cells are still not perfectly established. In this study we define the effects of downregulation of NANOG in maintaining self-renewal and undifferentiated state in mesenchymal stem cells (MSCs) derived from subcutaneous adipose tissue (hASCs). hASCs were expanded and transfected in vitro with short hairpin Lentivirus targeting NANOG . Gene suppressions were achieved at both transcript and proteome levels. The effect of NANOG knockdown on proliferation after 10 passages and on the cell cycle was evaluated by proliferation assay, colony forming unit (CFU), qRT-PCR and cell cycle analysis by flow-cytometry. Moreover, NANOG involvement in differentiation ability was evaluated. We report that downregulation of NANOG revealed a decrease in the proliferation and differentiation rate, inducing cell cycle arrest by increasing p27 / CDKN1B (Cyclin-dependent kinase inhibitor 1B) and p21 / CDKN1A (Cyclin-dependent kinase inhibitor 1A) through p53 and regulate DLK1 / PREF1 . Furthermore, NANOG induced downregulation of DNMT1 , a major DNA methyltransferase responsible for maintaining methylation status during DNA replication probably involved in cell cycle regulation. Our study confirms that NANOG regulates the complex transcription network of plasticity of the cells, inducing cell cycle arrest and reducing differentiation potential.
    Keywords human adipose stem cell ; NANOG ; cell cycle regulation ; DNMT1 ; lentiviral transduction ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 571
    Language English
    Publishing date 2019-05-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto’s thyroiditis

    Antonina Coppola / Laura Tomasello / Maria Pitrone / Salvatore Cillino / Pierina Richiusa / Giuseppe Pizzolanti / Carla Giordano

    Stem Cell Research & Therapy, Vol 8, Iss 1, Pp 1-

    2017  Volume 14

    Abstract: Abstract Background Due to their “natural immune privilege” and immunoregulatory properties human fibroblast-like limbal stem cells (f-LSCs) have acquired great interest as a potential tool for achieving immunotolerance. Hashimoto’s thyroiditis (HT) is ... ...

    Abstract Abstract Background Due to their “natural immune privilege” and immunoregulatory properties human fibroblast-like limbal stem cells (f-LSCs) have acquired great interest as a potential tool for achieving immunotolerance. Hashimoto’s thyroiditis (HT) is the most common thyroid autoimmune disease and cause of hypothyroidism. To date, conventional hormone replacement therapy and unspecific immunosuppressive regimens cannot provide a definitive cure for HT subjects. We explored the immunosuppressant potential of human f-LSCs on circulating lymphomonocytes (PBMCs) collected from healthy donors and female HT patients. Methods We assessed the immunophenotyping of f-LSCs, both untreated and after 48 h of proinflammatory cytokine exposure, by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and flow cytometry. The immunosuppressant effects of f-LSCs on healthy activated PBMCs were investigated in cell-cell contact and transwell settings through cell cycle assay, acridine orange staining, and caspase-3 detection. We also studied T-cell responses and possible Treg conversion by means of flow cytometry. Functional assays were conducted in activated HT lymphocytes cocultured with f-LSCs after carboxyfluorescein succinimidyl ester labeling and intracellular detection of pro- and anti-inflammatory cytokines. Results The hypo-immunogenicity of the f-LSC population depended on both cell contact and soluble factors produced, as well as the undetectable expression of all those molecules required to fully activate T lymphocytes. Following exposure to Th1 cytokines, f-LSCs augmented expression of programmed death-ligand 1 and 2 (PDL-1 and -2), indoleamine-pyrrole-2,3-dioxygenase (IDO), interleukin (IL)-6, and monocyte chemotactic protein 1 (MCP-1) while maintaining their negative phenotype for major histocompatibility (MHC) class II and costimulatory molecules. During coculture, f-LSCs suppressed up to 40% of proliferation in healthy activated PBMCs, arrested them in the G0/G1 cell cycle phase without ...
    Keywords Human limbal stem cells ; Hashimoto’s thyroiditis ; Immunoregulation ; Tolerance induction ; Inflammatory diseases ; Medicine (General) ; R5-920 ; Biochemistry ; QD415-436
    Subject code 610
    Language English
    Publishing date 2017-07-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Mesenchymal stem cells derived from inflamed dental pulpal and gingival tissue

    Laura Tomasello / Rodolfo Mauceri / Antonina Coppola / Maria Pitrone / Giuseppe Pizzo / Giuseppina Campisi / Giuseppe Pizzolanti / Carla Giordano

    Stem Cell Research & Therapy, Vol 8, Iss 1, Pp 1-

    a potential application for bone formation

    2017  Volume 15

    Abstract: Abstract Background Chronic periodontal disease is an infectious disease consisting of prolonged inflammation of the supporting tooth tissue and resulting in bone loss. Guided bone regeneration procedures have become common and safe treatments in ... ...

    Abstract Abstract Background Chronic periodontal disease is an infectious disease consisting of prolonged inflammation of the supporting tooth tissue and resulting in bone loss. Guided bone regeneration procedures have become common and safe treatments in dentistry, and in this context dental stem cells would represent the ideal solution as autologous cells. In this study, we verified the ability of dental pulp mesenchymal stem cells (DPSCs) and gingival mesenchymal stem cells (GMSCs) harvested from periodontally affected teeth to produce new mineralized bone tissue in vitro, and compared this to cells from healthy teeth. Methods To characterize DPSCs and GMSCs, we assessed colony-forming assay, immunophenotyping, mesenchymal/stem cell phenotyping, stem gene profiling by means of flow cytometry, and quantitative polymerase chain reaction (qPCR). The effects of proinflammatory cytokines on mesenchymal stem cell (MSC) proliferation and differentiation potential were investigated. We also observed participation of several heat shock proteins (HSPs) and actin-depolymerizing factors (ADFs) during osteogenic differentiation. Results DPSCs and GMSCs were successfully isolated both from periodontally affected dental tissue and controls. Periodontally affected dental MSCs proliferated faster, and the inflamed environment did not affect MSC marker expressions. The calcium deposition was higher in periodontally affected MSCs than in the control group. Proinflammatory cytokines activate a cytoskeleton remodeling, interacting with HSPs including HSP90 and HSPA9, thioredoxin-1, and ADFs such as as profilin-1, cofilin-1, and vinculin that probably mediate the increased acquisition in the inflamed environment. Conclusions Our findings provide evidence that periodontally affected dental tissue (both pulp and gingiva) can be used as a source of MSCs with intact stem cell properties. Moreover, we demonstrated that the osteogenic capability of DPSCs and GMSCs in the test group was not only preserved but increased by the overexpression of ...
    Keywords Inflammation ; Dental disease ; Pulpal and gingival mesenchymal stem cells ; Bone formation ; Heat shock protein ; ADFs ; Medicine (General) ; R5-920 ; Biochemistry ; QD415-436
    Subject code 571
    Language English
    Publishing date 2017-08-01T00:00:00Z
    Publisher BMC
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: In vitro phenotypic, genomic and proteomic characterization of a cytokine-resistant murine β-TC3 cell line.

    Antonina Coppola / Laura Tomasello / Giuseppe Pizzolanti / Ida Pucci-Minafra / Nadia Albanese / Gianluca Di Cara / Patrizia Cancemi / Maria Pitrone / Alessandra Bommarito / Elvira Carissimi / Giovanni Zito / Angela Criscimanna / Aldo Galluzzo / Carla Giordano

    PLoS ONE, Vol 7, Iss 2, p e

    2012  Volume 32109

    Abstract: Type 1 diabetes mellitus (T1DM) is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a ... ...

    Abstract Type 1 diabetes mellitus (T1DM) is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in β-cell survival.The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced β-cell death. To this purpose, we created a cytokine-resistant β-cell line (β-TC3R) by chronically treating the β-TC3 murine insulinoma cell line with IL-1β + IFN-γ. β-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of β-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that β-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that β-TC3R cells were characterized by downregulation of IL-1β and IFN-γ receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in β-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling.In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant β-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to cytokine-mediated β-cell death. This knowledge may be of potential benefit for ...
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2012-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: In vitro identification and characterization of CD133(pos) cancer stem-like cells in anaplastic thyroid carcinoma cell lines.

    Giovanni Zito / Pierina Richiusa / Alessandra Bommarito / Elvira Carissimi / Leonardo Russo / Antonina Coppola / Monica Zerilli / Vito Rodolico / Angela Criscimanna / Marco Amato / Giuseppe Pizzolanti / Aldo Galluzzo / Carla Giordano

    PLoS ONE, Vol 3, Iss 10, p e

    2008  Volume 3544

    Abstract: Background Recent publications suggest that neoplastic initiation and growth are dependent on a small subset of cells, termed cancer stem cells (CSCs). Anaplastic Thyroid Carcinoma (ATC) is a very aggressive solid tumor with poor prognosis, characterized ...

    Abstract Background Recent publications suggest that neoplastic initiation and growth are dependent on a small subset of cells, termed cancer stem cells (CSCs). Anaplastic Thyroid Carcinoma (ATC) is a very aggressive solid tumor with poor prognosis, characterized by high dedifferentiation. The existence of CSCs might account for the heterogeneity of ATC lesions. CD133 has been identified as a stem cell marker for normal and cancerous tissues, although its biological function remains unknown. Methodology/principal findings ATC cell lines ARO, KAT-4, KAT-18 and FRO were analyzed for CD133 expression. Flow cytometry showed CD133(pos) cells only in ARO and KAT-4 (64+/-9% and 57+/-12%, respectively). These data were confirmed by qRT-PCR and immunocytochemistry. ARO and KAT-4 were also positive for fetal marker oncofetal fibronectin and negative for thyrocyte-specific differentiating markers thyroglobulin, thyroperoxidase and sodium/iodide symporter. Sorted ARO/CD133(pos) cells exhibited higher proliferation, self-renewal, colony-forming ability in comparison with ARO/CD133(neg). Furthermore, ARO/CD133(pos) showed levels of thyroid transcription factor TTF-1 similar to the fetal thyroid cell line TAD-2, while the expression in ARO/CD133(neg) was negligible. The expression of the stem cell marker OCT-4 detected by RT-PCR and flow cytometry was markedly higher in ARO/CD133(pos) in comparison to ARO/CD133(neg) cells. The stem cell markers c-KIT and THY-1 were negative. Sensitivity to chemotherapy agents was investigated, showing remarkable resistance to chemotherapy-induced apoptosis in ARO/CD133(pos) when compared with ARO/CD133(neg) cells. Conclusions/significance We describe CD133(pos) cells in ATC cell lines. ARO/CD133(pos) cells exhibit stem cell-like features--such as high proliferation, self-renewal ability, expression of OCT-4--and are characterized by higher resistance to chemotherapy. The simultaneous positivity for thyroid specific factor TTF-1 and onfFN suggest they might represent putative thyroid cancer stem-like cells. Our in vitro findings might provide new insights for novel therapeutic approaches.
    Keywords Medicine ; R ; Science ; Q
    Subject code 610
    Language English
    Publishing date 2008-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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    Inter-library loan at ZB MED

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