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…

<p>Circulating Irisin Levels as a Marker of Osteosarcopenic-Obesity in Cushing’s Disease</p>

Purpose To evaluate circulating irisin levels in patients with active and controlled Cushing's disease (CD). Design Forty-four patients with CD evaluated during the active phase and after 12 months of biochemical remission and 40 controls were recruited. Methods Phenotypic, anthropometric, hormonal and metabolic parameters, including insulin sensitivity estimation by homeostatic model of insulin resistance, Matsuda index and oral disposition index and circulating irisin levels were evaluated. Results Patients with active CD showed lower irisin levels compared to controls (p<0.001) and controlled CD (p<0.001). The independent variables significantly associated with irisin were waist circumf…

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…

Pasireotide versus pituitary surgery: a retrospective analysis of 12 months of treatment in patients with Cushing’s disease

Pituitary surgery represents the first-line treatment for most patients with Cushing’s disease (CD). In the case of surgery failure, additional treatment options are required. Pasireotide has shown favourable results in the first-line treatment of patients with CD, who are not candidates for surgery or in the second-line when surgery has failed. The aim of the current study is to compare the effects of surgery and pasireotide treatment in a cohort of patients with CD, and to evaluate the differences in response rate in terms of hormonal and clinical control, and improvement of metabolic complications

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)

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)

BIOINFORMATICS CERNA ANALYSIS FOR THE STUDY OF STEM FACTOR SOX2 IN ANAPLASTIC THYROID CANCER.

Cancer stem cells (CSC) are believed to play a central role in oncogenesis, but until today their isolation and characterization is still particularly complex. Anaplastic thyroid cancer (ATC) presents several characteristics suggestive of a tumour highly enriched in CSC (high mitotic rate, poor prognosis, high aggressiveness, resistance to treatments, etc). For these reasons ATC represents a good candidate to study CSCs. SOX2 is a key stem transcriptional factor, usually only transiently expressed, that plays a fundamental role in stem cell identity. SOX2 proved to be constitutively expressed in SW1736 cell line, a well established and recognized ATC cell line. The bioinformatics ceRNA anal…

Growth and Osteogenic Differentiation of Discarded Gingiva-Derived Mesenchymal Stem Cells on a Commercial Scaffold

Background In periodontal patients with jawbone resorption, the autologous bone graft is considered a "gold standard" procedure for the placing of dental prosthesis; however, this procedure is a costly intervention and poses the risk of clinical complications. Thanks to the use of adult mesenchymal stem cells, smart biomaterials, and active biomolecules, regenerative medicine and bone tissue engineering represent a valid alternative to the traditional procedures. Aims In the past, mesenchymal stem cells isolated from periodontally compromised gingiva were considered a biological waste and discarded during surgical procedures. This study aims to test the osteoconductive activity of FISIOGRAF…

Circulating Irisin Levels as a Marker of Osteosarcopenic-Obesity in Cushing’s Disease

Purpose: To evaluate circulating irisin levels in patients with active and controlled Cushing’s disease (CD). Design: Forty-four patients with CD evaluated during the active phase and after 12 months of biochemical remission and 40 controls were recruited. Methods: Phenotypic, anthropometric, hormonal and metabolic parameters, including insulin sensitivity estimation by homeostatic model of insulin resistance, Matsuda index and oral disposition index and circulating irisin levels were evaluated. Results: Patients with active CD showed lower irisin levels compared to controls (p&lt;0.001) and controlled CD (p&lt;0.001). The independent variables significantly associated with irisin were wais…

Multiple Pluripotent Stem Cell Markers in Human Anaplastic Thyroid Cancer: The Putative Upstream Role of SOX2

Anaplastic thyroid carcinoma (ATC) is a rare and aggressive endocrine tumor with highly undifferentiated morphology. It has been suggested that cancer stem cells (CSCs) might play a central role in ATC. The objectives of this study were (i) to characterize CSCs from ex vivo ATC specimens by investigating the expression of several pluripotent stem cell markers, and (ii) to evaluate in vitro drug resistance modifications after specific CSC transcription factor switch-off.In ex vivo experiments, eight formalin-fixed, paraffin-embedded ATC specimens were analyzed by reverse-transcription and real-time quantitative PCR and immunohistochemistry. In in vitro experiments using ATC SW1736 cells, the…

ANTITUMOR EFFECTS OF SULPHORAPHANE ON THYROID CARCINOMA CELL LINES

Thyroid cancer is the most frequent endocrine malignancy with a global increasing incidence. Many evidences show that sulphoraphane (SF), a natural isothiocyante found in cruciferous vegetables has a wide range of chemopreventive as well as apoptosis inducing properties. The ability of SF in inducing apoptosis and cell cycle arrest is associated with the regulation of many proteins including Bcl-2 family proteins, caspases, p21, and cyclin dependent kinases. In the present work, we investigated in vitro the activity of SF in three human thyroid cancer cell lines. For this purpose we studied SW1736 (ATC), BC-PAP (PTC) and TT (MTC) cell line by MTT assay, after addition of SF ranging from 0 t…

Myristic acid is associated to low plasma HDL cholesterol levels in a Mediterranean population and increases HDL catabolism by enhancing HDL particles trapping to cell surface proteoglycans in a liver hepatoma cell model

Background: HDL-C plasma levels are modulated by dietary fatty acid (FA), but studies investigating dietary supplementation in FA gave contrasting results. Saturated FA increased HDL-C levels only in some studies. Mono-unsaturated FA exerted a slight effect while poly-unsaturated FA mostly increased plasma HDL-C. Aims: This study presents two aims: i) to investigate the relationship between HDL-C levels and plasma FA composition in a Sicilian population following a "Mediterranean diet", ii) to investigate if FA that resulted correlated with plasma HDL-C levels in the population study and/or very abundant in the plasma were able to affect HDL catabolism in an "in vitro" model of cultured hep…

In Vitro Generation of Pancreatic Endocrine Cells from Human Adult Fibroblast-Like Limbal Stem Cells

Stem cells might provide unlimited supply of transplantable cells for β-cell replacement therapy in diabetes. The human limbus is a highly specialized region hosting a well-recognized population of epithelial stem cells, which sustain the continuous renewal of the cornea, and the recently identified stromal fibroblast-like stem cells (f-LSCs), with apparent broader plasticity. However, the lack of specific molecular markers for the identification of the multipotent limbal subpopulation has so far limited the investigation of their differentiation potential. In this study we show that the human limbus contains uncommitted cells that could be potentially harnessed for the treatment of diabete…

Anaplastic Thyroid Carcinoma: A ceRNA Analysis Pointed to a Crosstalk between SOX2, TP53, and microRNA Biogenesis.

It has been suggested that cancer stem cells (CSC) may play a central role in oncogenesis, especially in undifferentiated tumours. Anaplastic thyroid carcinoma (ATC) has characteristics suggestive of a tumour enriched in CSC. Previous studies suggested that the stem cell factorSOX2has a preeminent hierarchical role in determining the characteristics of stem cells in SW1736 ATC cell line. In detail, silencing SOX2 in SW1736 is able to suppress the expression of the stem markers analysed, strongly sensitizing the line to treatment with chemotherapeutic agents. Therefore, in order to further investigate the role of SOX2 in ATC, a competing endogenous RNA (ceRNA) analysis was conducted in order…

BRAFV600E mutation, TIMP-1 upregulation, and NF-κB activation: closing the loop on the papillary thyroid cancer trilogy.

BRAFV600E is the most common mutation found in papillary thyroid carcinoma (PTC). Tissue inhibitor of metalloproteinases (TIMP-1) and nuclear factor (NF)-κB have been shown to play an important role in thyroid cancer. In particular, TIMP-1 binds its receptor CD63 on cell surface membrane and activates Akt signaling pathway, which is eventually responsible for its anti-apoptotic activity. The aim of our study was to evaluate whether interplay among these three factors exists and exerts a functional role in PTCs. To this purpose, 56 PTC specimens were analyzed for BRAFV600E mutation, TIMP-1 expression, and NF-κB activation. We found that BRAFV600E mutation occurs selectively in PTC nodules an…

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…

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 

ISOLATION AND CHARACTERIZATION OF VISCERAL- AND SUBCUTANEOUS ADIPOSE-DERIVED MESENCHYMAL STEM CELLS: PUTATIVE ROLE IN OBESITY AND METABOLIC SYNDROME

Espressione dei fattori di trascrizione di staminalità in cellule stromali (HASC) ottenute da tessuto adiposo viscerale e sottocutaneo

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…

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. …

Cut-off points of the visceral adiposity index (VAI) identifying a visceral adipose dysfunction associated with cardiometabolic risk in a Caucasian Sicilian population

Abstract Background The Visceral Adiposity Index (VAI) is a sex-specific mathematical index, based on Waist Circumference (WC), Body Mass Index (BMI), triglycerides (TG) and HDL cholesterol (HDL) levels, indirectly expressing visceral adipose function and insulin sensitivity. Our aim was to find the optimal cut-off points of VAI identifying a visceral adipose dysfunction (VAD) associated with cardiometabolic risk in a Caucasian Sicilian population. Methods Medical check-up data of 1,764 Primary Care patients (PC patients) were retrospectively and cross-sectionally examined using a receiver-operating characteristic (ROC) curve to determine appropriate stratified-for-age cut-off of VAI, for t…

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…

Epigenetic involvement in Hutchinson-Gilford progeria syndrome: a mini-review.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to a severe premature ageing phenotype, caused by mutations in the &lt;i&gt;LMNA&lt;/i&gt; gene. The &lt;i&gt;LMNA&lt;/i&gt; gene codes for lamin-A and lamin-C proteins, which are structural components of the nuclear lamina. HGPS is usually caused by a de novo &lt;i&gt;C1824T&lt;/i&gt; mutation that leads to the accumulation of a dominant negative form of lamin-A called progerin. Progerin also accumulates physiologically in normal ageing cells as a rare splicing form of lamin-A transcripts. From this perspective, HGPS cells seem to be good candidates for the study of the physiological mechanisms of ageing…

Endocrine and pro-inflammatory activity of visceral (VAT) and subcutaneous (SAT) adipose tissue in morbidly obese patients with and without metabolic syndrome.

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.

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 &amp;plusmn; 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…

BRAF mutation influences hypoxia-inducible factor-1α expression levels in papillary thyroid cancer

Hypoxia-inducible factor-1α is found frequently overexpressed in solid tumors cells, exerting an important role in angiogenesis, glucose metabolism, cell proliferation, survival and invasion. In thyroid carcinomas, hypoxia-inducible factor-1α expression was found increased in differentiated, poorly differentiated, medullary and anaplastic variants. Hypoxia represents the principal stimulus responsible for hypoxia-inducible factor-1α induction. Other nonhypoxic stimuli increase hypoxia-inducible factor-1α synthesis through the activation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in a cell-type-specific manner. We have previously shown the role of BRAFV600…

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 &gt; 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