| 1. |
- Pivoriunas, Augustas, et al.
(författare)
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Proteomic Analysis of Stromal Cells Derived from the Dental Pulp of Human Exfoliated Deciduous Teeth
- 2010
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Ingår i: STEM CELLS AND DEVELOPMENT. - : Mary Ann Leibert. - 1547-3287 .- 1557-8534. ; 19:7, s. 1081-1093
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Tidskriftsartikel (refereegranskat)abstract
- Human dental pulp derived from exfoliated deciduous teeth has been described as a promising alternative source of multipotent stem cells. While these cells share certain similarities with mesenchymal stem-like cells (MSC) isolated from other tissues, basically they are still poorly characterized. In this study, for the first time, a proteomic map of abundantly expressed proteins in stromal cells derived from the dental pulp of human exfoliated deciduous teeth (SHED) was established. We also analyzed proteomic signatures of 2 clonal strains derived from SHEDs by single-cell cloning. The SHEDs were established from enzyme-disaggregated deciduous dental pulp from 6-year-old children. They had typical fibroblastoid morphology and high colony-forming efficiency index (16.4%). Cloning was performed at the second passage using limiting dilution in a 96-well plate (0.3 cell/well). Differentiation assessment revealed strong osteogenic but no adipogenic potential of the SHEDs in either clonal strain. The cells expressed characteristic antigens of MSC-like cells, including CD73, CD90, CD105, CD146, and did not express hematopoietic markers CD14, CD34, and CD45, as assessed with FACS analysis. For proteomic studies, cytosolic and nuclear proteins were analyzed with 2-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). All proteins were identified with high level of confidence (the lowest sequence coverage was 27%). Identification of highly expressed proteins in SHEDs revealed proteomic profiles very similar to that of MSC-like cells derived from other tissues. We also found a high degree of similarity between proteomic signatures of primary SHEDs and clonal cell strains. Thus, our data confirm a close resemblance between SHEDs and MSC-like cells from other tissues and may serve as starting point for creating-comprehensive proteomic maps.
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| 2. |
- Tunaitis, Virginijus, et al.
(författare)
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Effects of different sera on adipose tissue-derived mesenchymal stromal cells
- 2011
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Ingår i: JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE. - : John Wiley and Sons. - 1932-6254. ; 5:9, s. 733-746
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Tidskriftsartikel (refereegranskat)abstract
- Current cell therapy protocols require considerable numbers of mesenchymal stromal cells (MSCs), which can be obtained only by in vitro expansion. The most important issue is a choice of optimal growth supplements for cell culture. Ideally, these should be of known composition, free of animal components and allow production of large homogenic populations of MSCs in a considerably short period of time. Since this standard has not been achieved to date, we aimed to assess the molecular responses of MSCs to different growth supplements commonly in use. MSCs were isolated from breast or abdominal adipose tissue and plated into DMEM supplemented with one of four different sera: fetal calf serum (FCS), pretested fetal calf serum (FCS-Sp), human allogeneic serum (HS) or artificial serum substitute (AS). MSCs cultivated with different serum supplements demonstrated distinct morphologies, high adipogenic and osteogenic differentiation potential and expressed characteristic antigens. Using real-time PCR, we found a large increase in PPAR gamma and Msx2 gene expression in both lines of proliferating MSCs cultivated with AS. We found that MSCs cultivated in the presence of different sera had similar global proteomic expression patterns, but comparisons of identified proteins revealed most differences in the MSCs cultivated with AS. Our results indicate that MSCs cultivated in the presence of FCS and HS display similar growth, differentiation, immunophenotypic and proteomic properties, while AS induces more profound changes in the physiology of MSCs, suggesting that further fundamental studies should be done before its introduction into clinical practice.
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| 3. |
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| 4. |
- Aldonyte, Ruta
(författare)
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Chronic Obstructive Pulmonary Disease: Role of Alpha-1-antitrypsin
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The only proven genetic risk factor for Chronic Obstructive Pulmonary Disease (COPD) is an inherited Z (Glu342¡úLys) deficiency of alpha-1-antitrypsin (AAT), a major inhibitor of neutrophil elastase. In vivo, AAT is found in both native (inhibitory) and modified (non-inhibitory) forms. There is now increasing evidence that the different forms of AAT may exhibit biological activities independent of protease inhibition. The aim of my studies was to validate polymerized (non inhibitory) form of AAT as a potential COPD biomarker and to investigate its biological effects in vitro. We have discovered that a mouse monoclonal antibody, ATZ11, raised against the mutant Z AAT, does not detect the mutation per se, but recognizes a conformation-dependent epitope in polymerized and elastase-complexed AAT. By using this antibody we show that in Z deficiency subjects a predominant fraction of plasma AAT is in a polymerized form. In addition, a positive staining of endothelial cells with ATZ11 is detected in both wild-type M and deficiency Z individuals. The levels of total and polymerized serum AAT and inflammatory biomarkers in M and Z COPD patients and controls were correlated. Hypothetically, by using factor analysis, we were able to segregate the variables measured into two independent components: the first containing MMP9, MCP-1, IL-8 and VEGF and the second ¨C total and polymerized AAT, sE-selectin and ICAM-1. We demonstrate that 95% of originally grouped individuals can be correctly classified on the basis of the measured variables. This suggests that the combinations of biomarkers may provide useful diagnostic tools. We next investigated the release of pro-inflammatory molecules by monocytes isolated from blood of COPD patients and controls with and without Z AAT deficiency under basal conditions and after stimulation with endotoxin. Dependent on disease state and AAT genotype the different profiles of pro-inflammatory molecules are released by monocytes. The development of COPD may also be ascribed to acquired AAT deficiency, which results from the post-translational modifications of the protein. We have tested whether native and modified (polymerized) forms of AAT differ in their effects on primary human monocytes in vitro. Both native and polymerized AAT exhibit similar pro-inflammatory effects at lower, but not at physiological, concentrations. These properties of AAT appear to be dependent on protein concentration, but not on molecular form. Our studies support a central role of AAT in inflammation and serve as a basis for the future research in identifying new biological role(s) of AAT in vivo.
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| 5. |
- Aldonyte, Ruta, et al.
(författare)
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Circulating monocytes from healthy individuals and COPD patients.
- 2003
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Ingår i: Respiratory Research. - : Springer Science and Business Media LLC. - 1465-9921 .- 1465-993X. ; 4:1, s. 11-11
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Tidskriftsartikel (refereegranskat)abstract
- Background: Chronic obstructive pulmonary disease (COPD) is characterized by incompletely reversible airflow obstruction associated with inflammation in which monocytes/macrophages are the predominant inflammatory cells. The only known genetic factor related to COPD is inherited PiZZ deficiency of alpha1-antitrypsin (AAT), an inhibitor of serine proteases. Methods: We investigated the basal and LPS-stimulated release of pro-inflammatory molecules from blood monocytes isolated from age and gender matched healthy (n = 30) and COPD (n = 20) individuals with and without AAT deficiency. Results: After 18 h of cell culture the basal release of MMP-9 was 2.5-fold, p < 0.02 greater, whereas IL-8 was 1.8-fold (p < 0.01) lower from COPD patient monocytes than from controls. LPS-stimulated release of IL-6 and MCP-1 was greater from COPD patient's monocytes relative to controls, while activation of control cells resulted in enhanced secretion of ICAM-1 and MMP-9 compared to COPD patients. Independent of disease status, monocytes from PiZZ AAT carriers released less TNFalpha (by 2.3-fold, p < 0.03). Conclusions: The basal and LPS-stimulated secretion of specific pro-inflammatory molecules from circulating monocytes differs between healthy and COPD subjects. These findings may be valuable for further studies on the mechanisms involved in recruitment and activation of inflammatory cells in COPD.
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| 6. |
- Aldonyte, Ruta, et al.
(författare)
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Concentration-dependent effects of native and polymerised alpha 1-antitrypsin on primary human monocytes, in vitro
- 2004
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Ingår i: BMC Cell Biology. - : Springer Science and Business Media LLC. - 1471-2121. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Background: alpha1-antitrypsin (AAT) is one of the major serine proteinase inhibitors controlling proteinases in many biological pathways. There is increasing evidence that AAT is able to exert other than antiproteolytic effects. To further examine this question we compared how various doses of the native (inhibitory) and the polymerised (non- inhibitory) molecular form of AAT affect pro-inflammatory responses in human monocytes, in vitro. Human monocytes isolated from different donors were exposed to the native or polymerised form of AAT at concentrations of 0.01, 0.02, 0.05, 0.1, 0.5 and 1 mg/ml for 18 h, and analysed to determine the release of cytokines and to detect the activity of NF-kappaB. Results: We found that native and polymerised AAT at lower concentrations, such as 0.1 mg/ml, enhance expression of TNFalpha (10.9- and 4.8-fold, p < 0.001), IL-6 (22.8- and 23.4-fold, p < 0.001), IL-8 (2.4- and 5.5-fold, p < 0.001) and MCP-1 (8.3- and 7.7-fold, p < 0.001), respectively, compared to buffer exposed cells or cells treated with higher doses of AAT ( 0.5 and 1 mg/ml). In parallel to increased cytokine levels, low concentrations of either conformation of AAT (0.02-0.1 mg/ml) induced NF-kappaB p50 activation, while 1 mg/ml of either conformation of AAT suppressed the activity of NF-kappaB, compared to controls. Conclusions: The observations reported here provide further support for a central role of AAT in inflammation, both as a regulator of proteinase activity, and as a signalling molecule for the expression of pro-inflammatory molecules. This latter role is dependent on the concentration of AAT, rather than on its proteinase inhibitory activity.
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| 7. |
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| 8. |
- Jarmalaviciute, Akvile, et al.
(författare)
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A New Experimental Model for Neuronal and Glial Differentiation Using Stem Cells Derived from Human Exfoliated Deciduous Teeth
- 2013
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Ingår i: Journal of Molecular Neuroscience. - : Humana Press. - 0895-8696 .- 1559-1166. ; 51:2, s. 307-317
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Tidskriftsartikel (refereegranskat)abstract
- Stem cells isolated from human adult tissues represent a promising source for neural differentiation studies in vitro. We have isolated and characterized stem cells from human exfoliated deciduous teeth (SHEDs). These originate from the neural crest and therefore particularly suitable for induction of neural differentiation. We here established a novel three-stage protocol for neural differentiation of SHEDs cells. After adaptation to a serum-free and neurogenic environment, SHEDs were induced to differentiate. This resulted in the formation of stellate or bipolar round-shaped neuron-like cells with subpopulations expressing markers of sensory neurons (Brn3a, peripherin) and glia (myelin basic protein). Commercial PCR array analyses addressed the expression profiles of genes related to neurogenesis and cAMP/calcium signalling. We found distinct evidence for the upregulation of genes regulating the specification of sensory (MAF), sympathetic (midkine, pleitrophin) and dopaminergic (tyrosine hydroxylase, Nurr1) neurons and the differentiation and support of myelinating and non-myelinating Schwann cells (Krox24, Krox20, apolipoprotein E). Moreover, for genes controlling major developmental signalling pathways, there was upregulation of BMP (TGF beta-3, BMP2) and Notch (Notch 2, DLL1, HES1, HEY1, HEY2) in the differentiating SHEDs. SHEDs treated according to our new differentiation protocol gave rise to mixed neuronal/glial cell cultures, which opens new possibilities for in vitro studies of neuronal and glial specification and broadens the potential for the employment of such cells in experimental models and future treatment strategies.
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