Analisi dei meccanismi di controllo dell’apoptosi in una linea cellulare β-pancreatica resistente all’effetto delle citochine.

PFN1 and integrin‐β1/mTOR axis involvement in cornea differentiation of fibroblast limbal stem cells

Abstract Ex vivo limbal stem cell transplantation is the main therapeutic approach to address a complete and functional re‐epithelialization in corneal blindness, the second most common eye disorder. Although important key points were defined, the molecular mechanisms involved in the epithelial phenotype determination are unclear. Our previous studies have demonstrated the pluripotency and immune‐modulatory of fibroblast limbal stem cells (f‐LSCs), isolated from the corneal limbus. We defined a proteomic profile especially enriched in wound healing and cytoskeleton‐remodelling proteins, including Profilin‐1 (PFN1). In this study we postulate that pfn‐1 knock down promotes epithelial lineage…

Identification of Novel Wsf1 Mutations in a Sicilian Child with Wolfram Syndrome

Wolfram Syndrome (WS) is a rare hereditary disease with autosomal recessive inheritance with incomplete penetrance. It is characterized by diabetes mellitus associated with progressive optic atrophy. The diagnosis is essentially clinical and mutation analysis is used to confirm the diagnosis. In the present study we describe the clinical and molecular features of a diabetic child carrying two novel WFS1 mutations. The Sicilian proband and his non-affected family were studied. Ophthalmologic examination included: visual acuity determination and funduscopy, optical coherent tomography, retinal fluorangiography, perimetry and electroretinogram. Molecular methods: automatic sequencing of PCR am…

NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells

The stromal vascular cell fraction (SVF) of visceral and subcutaneous adipose tissue (VAT and SAT) has increasingly come into focus in stem cell research, since these compartments represent a rich source of multipotent adipose-derived stem cells (ASCs). ASCs exhibit a self-renewal potential and differentiation capacity. Our aim was to study the different expression of the embryonic stem cell markers NANOG (homeobox protein NANOG), SOX2 (SRY (sex determining region Y)-box 2) and OCT4 (octamer-binding transcription factor 4) and to evaluate if there exists a hierarchal role in this network in ASCs derived from both SAT and VAT. ASCs were isolated from SAT and VAT biopsies of 72 consenting pat…

Additional file 4: of Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto’s thyroiditis

Jurkat cells as a positive control for apoptosis detection. (A) Untreated Jurkat cells (first row) or treated with activating anti-Fas for 4 or 24 h (second and third row, respectively) after AO/EB staining. The arrows indicate many apoptotic body formations, bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation. Late apoptotic cells were observed with condensed and fragmented nuclei especially after 24 h of anti-Fas treatment. Original magnification: 40×. All pictures are representative of five independent experiments. (PDF 240 kb)

Bioengineering Thymus Organoids to Restore Thymic Function and Induce Donor-Specific Immune Tolerance to Allografts.

One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude …

Additional file 2: Table S1. of Mesenchymal stem cells derived from inflamed dental pulpal and gingival tissue: a potential application for bone formation

The expression values of putative mesenchymal surface stem cells markers. (DOC 31 kb)

Compromised central tolerance of ICA69 induces multiple organ autoimmunity

For reasons not fully understood, patients with an organ-specific autoimmune disease have increased risks of developing autoimmune responses against other organs/tissues. We identified ICA69, a known β-cell autoantigen in Type 1 diabetes, as a potential common target in multi-organ autoimmunity. NOD mice immunized with ICA69 polypeptides exhibited exacerbated inflammation not only in the islets, but also in the salivary glands. To further investigate ICA69 autoimmunity, two genetically modified mouse lines were generated to modulate thymic ICA69 expression: the heterozygous ICA69(del/wt) line and the thymic medullary epithelial cell-specific deletion Aire-ΔICA69 line. Suboptimal central neg…

Identification and enrichment of thyroid stem cells in anaplastic cell lines

Limbal stem cells as a potential source of pancreatic beta cells

BACKGROUND/RATIONAL Beta cell replacement therapy represents an alternative approach to conventional insulin regimen for the management of type 1 diabetes. Stem cells theoretically represent a suitable and renewable source of surrogate beta cells. Recently, it has been demonstrated that corneal epithelium contains stem cells in the basal layer of the limbus. Our aim was to isolate and characterize limbal stem cells (LSCs) from adult corneal-limbal biopsies, and to assess their capability to differentiate into insulin-producing cells. METHODS 2-3 mm2 biopsies were obtained from limbus and central cornea of four patients undergoing surgery for ocular diseases not involving conjuctive or corne…

In Vitro Phenotypic, Genomic and Proteomic Characterization of a Cytokine-Resistant Murine β-TC3 Cell Line

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 resistan…

In Vitro Identification and Characterization of CD133pos Cancer Stem-Like Cells in Anaplastic Thyroid Carcinoma Cell Lines

BackgroundRecent 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 findingsATC 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 …

No association between BRAFV660E and metalloproteinase activity in anaplastic thyroid cell lines

Knockdown of NANOG Reduces Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Human Adipose Stem Cells

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…

Additional file 2: of Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto’s thyroiditis

f-LSCs arrest T-cell division. (A) One-way mixed leukocyte reaction (MLR) was carried out by seeding 2 × 105 of responder healthy PBMCs into 48-well plates (Corning) and adding 1 × 105 mytomycin-C pre-treated stimulator cells for a final volume of 500 μl of lymphocyte medium. Each responder and stimulator cell population was seeded in triplicate. When MLRs were cocultured with f-LSCs the allogenic activation was greatly reduced. Pictures are representative of five independent experiments. (B) Molecular detection of p27Kit and CCND1 mRNA by qRT-PCR. Results are shown as mean ± SD of three independent experiments; *p 

Identification and enrichment of thyroid cancer stem cells in anaplastic cell lines

Caratterizzazione proteomica di una linea beta cellulare resistente all'effetto citochinico

Erratum to: Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells

In regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population.Fibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric…

Heterogeneity of Stem Cells in the Thyroid

Identification of thyroid stem cells in the past few years has made important contributions to our understanding of the cellular and molecular mechanisms that induce tissue regeneration and repair. Embryonic stem (ES) cells and induced-pluripotent stem cells have been used to establish reliable protocols to obtain mature thyrocytes and functional follicles for the treatment of thyroid diseases in mice. In addition, the discovery of resident thyroid progenitor cells, along with other sources of stem cells, has defined in detail the mechanisms responsible for tissue repair upon moderate or severe organ injury.In this chapter, we highlight in detail the current state of research on thyroid ste…

Additional file 5: of Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto’s thyroiditis

Immunomodulation mechanism played from BM-MSCs on healthy activated PBMCs. (A) Acitive-caspase-3 detection by FACS revealed weak induction of the apoptotic cascade in activated healthy PBMCs after 72 h of coculture with BM-MSCs. FACS histogram plots are representative of five experiments of identical design. (B) The expression percentage of the three activation markers (CD28, PD-1, CD69) in activated healthy PBMCs were assessed by flow cytometry and reported in the histogram. Samples were run after 72 h of incubation with or without BM-MSCs. (C) The CD4/CD8 ratio was calculated from data obtained by flow staining in samples of healthy activated PBMCs alone and after coculture with BM-MSCs. …

Mesenchymal stem cells derived from inflamed dental pulpal and gingival tissue: a potential application for bone formation

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, immunophenot…

Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells

AbstractBackgroundIn regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population.MethodsFibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were ev…

Additional file 3: of Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto’s thyroiditis

Inhibitory effect of BM-MSCs on PBMC proliferation. (A) PBMCs from healthy volunteers were stimulated with anti-CD3 for 72 h in the presence or absence of BM-MSCs (1:10 ratio). No activated PBMCs served as negative controls. Original magnification: 40×. Pictures are representative of at least five independent experiments (B). DNA content was assessed using PI staining and analyzed by flow cytometry. Analysis of cell cycle showed the 28 ± 2.7% of PBMCs after 72 h of activation in G2/S phase. The same cells in the presence of BM-MSCs became 15 ± 3.0% confirming the inhibitory action of BM-MSCs on PBMC proliferation. In the transwell system no significant inhibition of PBMC growing was observe…

BrafV600E mutation and hyperexpression of tissue inhibitor of metalloproteinases-1 (TIMP-1) in papillary thyroid carcinoma

Dual-release hydrocortisone vs conventional glucocorticoids in adrenal insufficiency

Background Dual-release hydrocortisone (DR-HC) improves metabolism in patients with adrenal insufficiency. The aims of this study were to compare the cardiovascular and metabolic effects of conventional glucocorticoids (GCs) vs. DR-HC and of high vs. low doses of GCs, after 48 months of observation. Methods We selected 27 patients on hydrocortisone (mean dose 17.5 ± 4.2 mg/day) and 20 patients on cortisone acetate (mean dose 37.5 ± 12.1 mg/day) who maintained this treatment (group A) and 53 patients switched to DR-HC (mean dose 22 ± 4.8 mg/day) (group B). At baseline and after 48 months, clinical and metabolic parameters and Framingham Risk Score (FRS) were obtained. Results After 48 month…

Differential protein expression in cytokine-sensitive and -resistant pancreatic beta cell lines

Erratum: Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells (Stem Cell Research and Therapy (2016) 7 (8))

Following publication of the original article in Stem Cell Research and Therapy [1], it was brought to our attention that panel 5E in Fig. 5 is a duplicate of panel 5F. Please find below the figure with the correct panel E. We apologize for the inconvenience this may have caused.

Production of a Double-Layer Scaffold for the “On-Demand” Release of Fibroblast-like Limbal Stem Cells

The production and characterization of a double layer scaffold, to be used as a system for the “on demand” release of corneal limbal stem cells are here reported. The devices used in the clinics and proposed so far in the scientific literature, for the release of corneal stem cells in the treatment of limbal stem cell deficiency, cannot control the in vivo space-time release of cells since the biomaterial of which they are composed is devoid of stimuli responsiveness features. Our approach was to produce a scaffold composed of two different polymeric layers that give the device the appropriate mechanical properties to be placed on the ocular surface and the possibility of releasing the stem…

Anti-Inflammatory Action of Heterogeneous Nuclear Ribonucleoprotein A2/B1 in Patients with Autoimmune Endocrine Disorders

Our previous studies documented that human fibroblast-limbal stem cells (f-LSCs) possess immunosuppressive capabilities, playing a role in regulating T-cell activity. This study highlights the molecular activities by which human f-LSCs can attenuate the inflammatory responses of self-reactive peripheral blood mononuclear cells (PBMCs) collected from patients with autoimmune endocrine diseases (AEDs). Anti-CD3 activated PBMCs from twenty healthy donors and fifty-two patients with AEDs were cocultured on f-LSC monolayer. 2D-DIGE proteomic experiments, mass spectrometry sequencing and functional in vitro assays were assessed in cocultured PBMCs. We identified the downmodulation of several huma…

NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells (ASCs)

The stromal vascular cell fraction (SVF) of visceral and subcutaneous adipose tissue (VAT and SAT) has increasingly come into focus in stem cell research, since these compartments represent a rich source of multipotent adipose-derived stem cells (ASCs). ASCs exhibit a self- renewal potential and differentiation capacity. Our aim was to study the different expression of embryonic stem cell markers NANOG, SOX2 and OCT3/4 and to evaluate if there exists a hierarchal role in this network in ASCs derived from both SAT and VAT. ASCs were isolated from SAT and VAT biopsies of 72 consenting patients (23 men, 47 women; age 45 ± 10; BMI between 25 and 30 range) undergoing elective open-abd…

Additional file 1: of Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto’s thyroiditis

Immunophenotyping of BM-MSCs. (A) The immunological profile of BM-MSCs was assessed by flow cytometry before (upper panel) and after 48 h of cytokine-treatment (IL-1β, IL-6, and IFNγ, 500 U/ml) (lower panel) using the same Abs for f-LSC immune characterization. All histogram plots include the percentage of expression of each protein as representative value of five independent experiments. (PDF 572 kb)

Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto’s thyroiditis

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 pr…

Additional file 1: Figure S1. of Mesenchymal stem cells derived from inflamed dental pulpal and gingival tissue: a potential application for bone formation

A) Bar graph represents the MTT absorbance mean values ± SD of P-DPSCs and P-GMSCs vs. their healthy control. The differences were not significant. B) The histograms represent the comparison of DT. Mean values ± SD are based on three independent experiments. n.s = not significant (p value > 0.05). C) Each field shows a cell cycle analysis representative of each sample investigated. (TIF 11728 kb)

Additional file 3: Figure S2. of Mesenchymal stem cells derived from inflamed dental pulpal and gingival tissue: a potential application for bone formation

A, B) Basal gene expression of IL-1β and TNF-α receptors in P-DPSCs, H-DPSCs, P-GMSCs, and H-GMSCs. C, D) Gene expression of IL-1β and TNF-α receptors under cytokine stimulation at 24, 48, and 72 h (cytokine treatment: 20 ng/ml IL-1β + 40 ng/ml TNF-α). *p value