Flow cytometry analyses of calcification propensity

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Xenogeneic M2-polarization of Macrophages by undifferentiated and differentiated adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) are considered to be â â immunologically privileged.â â In a previous work when human adipose tissue-derived stem cells (hASCs) subcutaneously implanted in mice we did not identify an adverse host response1. Recently, it was shown that tissue regeneration could benefit from the polarization of M2 macrophages subpopulations 2. In this study we hypothesised that undifferentiated hASCs and derived osteoblasts and chondrocytes are able to switch murine bone marrow-derived macrophages (mBMMÃ s) into M2 phenotype, aiding tissue regeneration. Murine BMMÃ s were plated in direct contact with undifferentiated and osteo or chondro-differentiated hASCs for 4 h, 10 h, 24 h and 72 h. The cytokine profile was analysed by qRT-PCR and the surface markers were detected by flow cytometry. The direct interaction of both cell types was observed by time lapse microscopy. The results showed that mBMMÃ s polarized after contacting tissue culture polystyrene. This M2 phenotype was maintained along the experiment in direct contact with both undifferentiated and osteo or chondro-differentiated hASCs. This was confirmed by the expression of IL-1, IL-10, IL-4, TNF-a and IFN-g (genetic profile) and surface markers (CD206 + + , CD336 + + , MHC II + and CD86 + + ) detection. These data suggest the potential of hASCs in contemporary xenogenic tissue engineering and regenerative medicine strategies, as well as host immune system modulation in autoimmune diseases. 

Establishing guidelines for obtaining optimal cell sources to use in tendon tissue engineering strategies

Cell-based approaches in tissue engineering (TE) have been barely explored for the treatment of tendon and ligament (T/L) tissues, requiring the establishment of a widely available cell source with tenogenic potential. As T/L cells are scarce, stem cells may provide a good alternative. Understanding how resident cells behave in vitro, might be useful for recapitulating the tenogenic potential of stem cells for tendon TE applications. Therefore, we propose to isolate and characterize human T/L-derived cells (hTDCs and hLDCs) and compare their regenerative potential with stem cells from adipose tissue (hASCs) and amniotic fluid (hAFSCs)(1). T/L cells were isolated using different procedures and stem cells isolated as described elsewhere(1). Moreover, T/L cells were stimu- lated into the three mesenchymal lineages, using standard differentia- tion media. Cells were characterized for the typical stem cell markers as well as T/L related markers, namely tenascin-C, collagen I and III, decorin and scleraxis, using different complementary techniques such as real time RT-PCR, immunocytochemistry and flow cytometry. No differences were observed between T/L in gene expression and protein deposition. T/L cells were mostly positive for stem ness markers (CD73/CD90/CD105), and have the potential to differentiate towards osteogenesis, chondrogenesis and adipogenesis, demonstrated by the positive staining for AlizarinRed, SafraninO, ToluidineBlue and OilRed. hASCs and hAFSCs exhibit positive expression of all tenogenic mark- ers, although at lower levels than hTDCs and hLDCs. Nevertheless, stem cells availability is key factor in TE strategies, despite that it’s still required optimization to direct their tenogenic phenotype.

Cryopreservation of Cell Sheets of Adipose Stem Cells: Limitations and Successes

Cell Sheets of hASCs (hASCs-CS) have been previously proposed for wound healing applications(1, 2) and despite the concern for production time reduction, the possibility of having these hASCs-CS off-the-shelf is appealing. The goal of this work was to define a cryopreservation methodology allowing to preserve cells viability and the properties CS matrix. hASCs-CS obtained from three different donors were created in UP-cell thermoresponsive dishes(Nunc, Germany) as previously reported(1,2). Different cryopreservation conditions were considered: i)FBS plus DMSO(5% and10%); ii)0.4M of Trehalose plus DMSO (5% and 10%); iii)cryosolution PLL (Akron Biotech, USA); and iv)vitrification. The cryopreservation effect was first assessed for cellular viability by flow cytometry using 7-AAD, and after dissociating the hASCs-CS with collagenase and trypsin-EDTA 0.25%. The expression (RT-PCR) and deposition (western blot and immunocytochemistry) of collagen type I, laminin and fibronectin, and the organization (TEM) of the extracellular matrix was further assessed before and after hASCs-CS cryopreservation to determine a potential effect of the method over matrix composition and integrity. The obtained results confirmed that cell viability is affected by the cryopreservation methodology, as shown before for different CS(3). Interestingly, the matrix properties were not significantly altered and the typical cell sheetâ s easiness of manipulation for transplantation was not lost.

Effector memory CD4+ T cells are associated with cognitive performance in a senior population

Objective: Immunosenescence and cognitive decline are common markers of the aging process. Taking into consideration the heterogeneity observed in aging processes and the recently described link between lymphocytes and cognition, we herein explored the possibility of an association between alterations in lymphocytic populations and cognitive performance. Methods: In a cohort of cognitively healthy adults (n = 114), previously characterized by diverse neurocognitive/psychological performance patterns, detailed peripheral blood immunophenotyping of both the innate and adaptive immune systems was performed by flow cytometry. Results: Better cognitive performance was associated with lower numbers of effector memory CD4(+) T cells and higher numbers of naive CD8(+) T cells and B cells. Furthermore, effector memory CD4(+) T cells were found to be predictors of general and executive function and memory, even when factors known to influence cognitive performance in older individuals (e.g., age, sex, education, and mood) were taken into account. Conclusions: This is the first study in humans associating specific phenotypes of the immune system with distinct cognitive performance in healthy aging.

Mesenchymal stem cells culture on poly(vinylidene fluoride) piezoelectric microspheres

Dissertação de mestrado em Biofísica e Bionanossistemas

Fluorescence-based approaches towards the assessment of structural and functional alterations in lysosomes and vacuoles induced by lactoferrin and acetic acid

Dissertação de mestrado em Biofísica e Bionanossistemas

Contributos para a elucidação do modo de ação de própolis português: o caso do própolis do Pereiro

Dissertação de mestrado em Genética Molecular

Evaluation of bioactivities of a propolis sample (Gerês) of Portuguese origin

Dissertação de mestrado em Genética Molecular

FISHji: new ImageJ macros for the quantification of fluorescence in epifluorescence images

Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software. This work presents FISHji, a set of new ImageJ methods for automated quantification of fluorescence in images obtained by epifluorescence microscopy. To validate the methods, results obtained by FISHji were compared with results obtained by flow cytometry. The mean correlation between FISHji and flow cytometry was high and significant, showing that the imaging methods are able to accurately assess the signal intensity of fluorescence images. FISHji are available for non-commercial use at http://paginas.fe.up.pt/nazevedo/.