150 research outputs found
Isolation, genomic and proteomic characterization of fibroblastic and epithelial limbal stem cells and evaluation of their multilineage differentiation capability.
ABSTRACT
BACKGROUND: The limbus of the eye, located at the junction of the cornea and conjunctiva of the ocular surface, represents a unique stem cell niche in human body. A critical advantage of limbal cells is that they are easily accessible with a well established and minimally invasive procedure. Several groups have reported the gene expression profile of limbal and corneal epithelial cells that has significantly contributed to the understanding of several cellular pathways and intrinsic factors that underpin the phenotypic difference between the two cell types [1-3]. However the gene expression profile of the human limbal epithelial and limbal stromal cultured cells obtained from the native limbal tissue has not been addressed until now. The lack of specific molecular markers for the identification of the multipotent limbal subpopulation limited the investigation of their differentiation capability. SSEA4 (stage specific embryonic antigen) is recognized marker of pluripotent stem cells, such as embryonic stem cells, and not of multipotent stem cells. Actually, the researcher are agreed that SSEA4 expression is lost from stem cells as they start to differentiate. It is generally agreed that the LESC are characterized by special location in the limbus, clonality, cytokeratin profile, transformation-related protein 63 (p63) delta isomers, and ATP-binding cassette sub-family G member 2 (ABCG2) expression [4-5]. Fibroblast (f)-LSCs represent a multipotent stem cell population characterized as CD105+, CD73+, CD90+, CD34+ and CD45- as well as by their self-renewal and multi-lineage differentiation capability [6-7].
AIM: To identify the better protocol of isolation and expansion for LESC and f-LSCs to obtain two purified and distinct subpopulations for molecular and proteomic characterization to evaluate their multilineage differentiation capability.
MATERIAL AND METHODS: Cytofluorimetric analisys; sphere formation assay; qRT PCR and proteomic assays; multilineage differentiation assays were performed.
RESULTS: Both f-LSC and LESC populations expressed SSEA4 (principal staminal marker) 98.63 ± 3.5%; vs 78.32 ± 2.8%, respectively (at 2PD) and ABCG2 (limbal specific marker).
The f-LSC genomic expression profile in comparison to LESC didn’t show significant differences and both cell populations kept a stem genomic expression profiles since 24 PD. f-LSC kept an high SSEA4 positive expression (98.63 ± 3.5%; 90.36 ± 1.4%; 2PD-14PD) whereas LESC showed a CK15+/ΔNp63+ pattern and a reduced expression of SSEA4 (78.32 ± 2.8% vs 13.90± 4.6 %; 2PD-14PD).
f-LSC, but not LESC, were differentiated into pancreatic β-cell phenotype (insulin-producing beta-like cells). Furthermore, both f-LSC and LESC achieved terminal osteoblastic and adipose differentiation. By contrast, the specific for committed LESC towards definitive epithelial phenotype cytokeratin profile, reduced LESC multilineage differentiation skill.
CONCLUSIONS: The identification of genetic profiles and novel molecular markers characteristic of specific cellular phenotypes is likely to assist in better defining the stages of progression from unspecialized cells to cell types of interest. Yet, translation of these promising basic science discoveries into successful cell replacement therapy in human subjects is likely to require sophisticated methods for assessing genetic and phenotypic stability
Irisin: A Possible Marker of Adipose Tissue Dysfunction in Obesity
Adipose tissue (AT) secretes pro- and anti-inflammatory cytokines involved in AT homeostasis, including tumor necrosis factor-α (TNFα) and irisin. The functionality of AT is based on a regulated equilibrium between adipogenesis and extracellular matrix (ECM) remodeling. We investigated the contributions of adipose progenitors (ASCs) and adipocytes (AMCs) to TNFα-induced ECM remodeling and a possible implication of irisin in AT impairment in obesity. ASCs and AMCs were exposed to TNFα treatment and nuclear factor–kappa (NF-kB) pathway was investigated: Tissue Inhibitor of Metalloproteinase (TIMP-1), Twist Family Transcription Factor 1 (TWIST-1), and peroxisome proliferator-activated receptor-γ (PPARγ) expression levels were analyzed. The proteolytic activity of matrix metalloproteinases (MMPs) -2 and -9 was analyzed by zymography, and the irisin protein content was measured by ELISA. In inflamed AMCs, a TIMP-1/TWIST-1 imbalance leads to a drop in PPARγ. Adipogenesis and lipid storage ability impairment come with local tissue remodeling due to MMP-9 overactivation. In vitro and ex vivo measurements confirm positive correlations among inflammation, adipose secreting irisin levels, and circulating irisin levels in patients with visceral obesity. Our findings identify the NF-kB downstream effectors as molecular initiators of AT dysfunction and suggest irisin as a possible AT damage and obesity predictive factor
A note on preconditioning weighted linear least squares, with consequences for weakly-constrained variational data assimilation
The effect of preconditioning linear weighted least-squares using an
approximation of the model matrix is analyzed, showing the interplay of the
eigenstructures of both the model and weighting matrices. A small example is
given illustrating the resulting potential inefficiency of such
preconditioners. Consequences of these results in the context of the
weakly-constrained 4D-Var data assimilation problem are finally discussed.Comment: 10 pages, 2 figure
(Dipyrido[3,2-a:2',3'-c]phenazine)(glycinato)copper(II) perchlorate: A novel DNA-intercalator with anti-proliferative activity against thyroid cancer cell lines
A novel copper(II) heteroleptic complex of dipyrido[3,2-a:2′,3′-c]phenazine (dppz) and glycinato (gly) as chelating ancillary ligand, [Cu(dppz)(gly)]ClO4 (1), was synthesized and characterized. X-ray crystallography revealed that the coordination geometry of the cationic [Cu(dppz)(gly)]+ unit is hexacoordinated and shows a distorted octahedral coordination geometry in the solid state, with the N,N and N,O chelating atoms of dppz and glycinato, respectively, in the square plane and in which the planar units are connected in a monodimensional polymeric array by the apical copper coordination of the second carboxylic oxygen atom. Biological assays showed that 1 exhibits a remarkable anti-proliferative activity against the two human anaplastic thyroid cancer cell lines 8505c (BrafV600E/V600E) and SW1736 (BrafWT/V600E), in a dose- and time-dependent manner. In details, the IC50 after 48 h of drug exposure was 2.86±0.54 μM for SW1736 and 1.05±0.48 μM for 8505c. On the other hand, the IC50 shown by cis-diamminedichloroplatinum(II) (cisplatin) against the same cell lines was 2.50±0.40 μM and 6.03±0.78 μM, respectively. Optical microscopy observations, after 48 h of treatment, showed morphological cell changes typical of apoptosis, confirmed by DNA ladder assays. DNA interaction studies, performed by UV absorption spectrophotometry, circular dichroism and viscosimetry, clearly showed that [Cu(dppz)(gly)]ClO4 is a DNA-intercalator, with a DNA-binding constant, Kb, of 2.1×106 M−1, suggesting that the mechanism of the cytotoxic activity can be related to its DNA-binding
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 amplified WFS1 gene fragments and qRT-PCR analysis of WFS1 transcripts. 3 WSF1 mutations have been identified in the proband. One allele carries 2 paternally inherited mutations (c.1332 C>G and c.1631C>G) in exon-8, never annotated before, in heterozygosis with one “de novo” classic mutation (c.505 G>A) in exon-5. In addition, we report an unexpected molecular feature: higher WFS1 mRNA levels in the proband compared to the father
Anaplastic Thyroid Carcinoma: A ceRNA Analysis Pointed to a Crosstalk between SOX2
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 factor SOX2 has 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 to isolate new functional partners of SOX2. Among the interactors,
of particular interest are genes involved in the biogenesis of miRNAs (DICER1, RNASEN, and EIF2C2), in the control cell cycle
(TP53, CCND1), and in mitochondrial activity (COX8A). The data suggest that stemness, microRNA biogenesis and functions, p53
regulatory network, cyclin D1, and cell cycle control, together with mitochondrial activity, might be coregulated
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.
Conclusion: Matriderm represents a biocompatible scaffold able to support the in vitro cell growth and osteodifferentiation ability of gingival mesenchymal stem cells isolated from waste gingiva, and could be employed to develop low-cost and painless strategy of autologous bone tissue regeneration. This study aims to test the osteoconductive activity of FISIOGRAFT Bone Granular and Matriderm collagen scaffolds on mesenchymal stem cells isolated from periodontally compromised gingiva as a low-cost and painless strategy of autologous bone tissue regeneration.
Materials and Methods: We isolated human mesenchymal stem cells from 22 healthy and 26 periodontally compromised gingival biopsy tissues and confirmed the stem cell phenotype by doubling time assay, colony-forming unit assay, and expression of surface
and nuclear mesenchymal stem cell markers, respectively by cytofluorimetry and realtime quantitative PCR. Healthy and periodontally compromised gingival mesenchymal stem cells were seeded on FISIOGRAFT Bone GranularR and MatridermR scaffolds, and in vitro cell viability and bone differentiation were then evaluated.
Results: Even though preliminary, the results demonstrate that FISIOGRAFT Bone GranularR is not suitable for in vitro growth and osteogenic differentiation of healthy and periodontally compromised mesenchymal stem cells, which, instead, are able to grow, homogeneously distribute, and bone differentiate in the MatridermR collagen scaffold
The impact of reproductive life on breast cancer risk in women with family history or BRCA mutation
Reproductive history and exogenous hormonal exposures are acknowledged risk factors for breast cancer in the general population. In women at increased breast cancer risk for genetic predisposition or positive family history, data regarding these risk factors are limited or con icting, and recommendations for these categories are unclear. We evaluated the characteristics of reproductive life in 2522 women at increased genetic or familial breast cancer risk attending our Family Cancer Center. Breast cancers in BRCA mutation carriers were more likely to be hormone receptor negative, diagnosed at 35 years or before and multiple during the lifetime than tumors in women at increased familial risk, while the distribution of invasive cancers and HER2 positive tumors was similar in the different risk groups. At least one full- term pregnancy (HR 0.27; 95% CI 0.12\u20130.58; p = 0.001), breastfeeding either less (HR 0.24; 95% CI 0.09\u20130.66; p = 0.005) or more (HR 0.25; 95% IC 0.08\u20130.82; p = 0.022) than one year and late age at menopause (HR 0.10; 95% CI 0.01\u20130.82; p = 0.033) showed to be protective factors in BRCA mutation carriers, while in women at increased familial risk early age at rst full-term pregnancy (HR 0.62; 95% IC 0.38\u20130.99; p = 0.048) and late menarche (HR 0.61; 95% CI 0.42\u20130.85; p = 0.004) showed to be the main protective factors. Finally, for the entire population, combined hormonal contraceptives demonstrated to do not increase breast cancer risk. The results of our study suggest that women at high familial risk and mutation carries develop tumors with different clinical-pathological characteristics and, consequently, are in uenced by different protective and risk factors
Rabbit slaughter hygiene: evaluation of process hygiene criteria for the supposed food category carcasses of rabbit
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