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Protocols for in vitro differentiation of human mesenchymal stem cells into osteogenic, chondrogenic and adipogenic lineages
No full textMesenchymal stem cells (MSC) possess high plasticity and the potential to differentiate into several different cell types; this characteristic has implications for cell therapy and reparative biotechnologies. MSC have been originally isolated from the bone marrow (BM-MSC), but they have been found also in other tissues such as adipose tissue, cord blood, synovium, skeletal muscle, and lung. MSC are able to differentiate in vitro and in vivo into several cell types such as bone, osteocytes, chondrocytes, adipocytes, and skeletal myocytes, just to name a few.During the last two decades, an increasing number of studies have proven the therapeutic potential of MSC for the treatment of neurodegenerative diseases, spinal cord and brain injuries, cardiovascular diseases, diabetes mellitus, and diseases of the skeleton. Their immuno-privileged profile allows both autologous and allogeneic use. For all these reasons, the scientific appeal of MSC is constantly on the rise.The identity of MSC is currently based on three main criteria: plastic-adherence capacity, defined epitope profile, and capacity to differentiate in vitro into osteocytes, chondrocytes, and adipocytes. Here, we describe standard protocols for the differentiation of BM-MSC into the osteogenic, chondrogenic, and adipogenic lineages
Donor age negatively influences the cytoprotective paracrine effects exerted by human mesenchymal stem cells
No full textBackground: In animal models mesenchymal stem cells (MSC) repair infarcted hearts mainly through cytoprotective paracrine mechanisms. For translational purposes, it would be important to verify if human MSC also mediate cardioprotection. In particular, since ischemic heart diseases occur mainly in elderly, it is essential to establish if donor age influences the production of cytoprotective factors. Accordingly, we compared the paracrine properties of fetal MSC (F-MSC) with adult MSC. Furthermore, we aimed to elucidate through which signalling pathway MSC lead to cytoprotection.
Methods: F-MSC were isolated from human placenta and adult MSC from the bone marrow of young (yBM-MSC; age65) donors. Rat neonatal cardiomyocytes (H9c2) were exposed to hypoxia (6 hrs)/reoxygenation (18 hrs) (H/R) in the presence of control medium (CTRL-M) or conditioned medium from F-MSC (F-CM), yBM-MSC (y-CM) or oBM-MSC (o-CM). H9c2 viability was evaluated by MTS assay. Apoptosis was measured by TUNEL staining and by cleaved Caspase 3 (colorimetric assay and Western blot). We evaluated SAPK/JNK and p38 MAPK activation by Western blot and the expression of pro- and anti-apoptotic genes by RT-PCR.
Results: The H/R protocol reduced H9c2 viability by 55% (p<0.001 CTRL-M vs basal condition). Compared with CTRL-M, both F-CM and y-CM increased cell viability (+45% and +33% respectively; p<0.017) while o-CM had no effect. F-CM significantly reduced the number of TUNEL positive cells (-91% vs CTRL-M and -89% vs o-CM; p<0.001). The y-CM also reduced H9c2 apoptotic nuclei (-67,5% vs CTRL-M, p<0.01; -64% vs o-CM, p<0.01). In contrast, o-CM did not prevent apoptosis (-11% vs CTRL-M, p=ns). Both colorimetric assay and Western blot showed that Caspase-3 activation was prevented by F-CM and y-CM but not by o-CM. The H/R protocol strongly activated both SAPK/JNK and p38 MAPK. This activation was markedly reduced by F-CM while y-CM and o-CM had modest effect on both pathways. Furthermore, compared with CTRL-M and both y-CM and o-CM, F-CM up-regulated the anti-apoptotic genes Bcl-2 and Stat3 and down-regulated the pro-apoptotic genes TNF-α and FasL.
Conclusions: We showed that human MSC mediate cardiomyocyte protection by releasing soluble anti-apoptotic factors. However, donor age negatively influences the cytoprotective properties of adult MSC. We also demonstrated that MSC of fetal origin exerts powerful cytoprotective effects via inhibition of different pro-apoptotic signalling pathways. Our data suggest that autologous MSC therapy for ischemic heart diseases may be less effective in elderly patients
The cardioprotective paracrine effects exerted by human mesenchymal stem cells are negatively influenced by donor age
No full textBackground: In experimental animal models mesenchymal stem cells (MSC) repair infarcted hearts mainly through paracrine mechanisms. In particular, MSC produce and release anti-apoptotic factors that lead to cytoprotection. For translational purposes, it would be important to verify if human MSC also mediate cardioprotection. In particular, since ischemic heart diseases occur mainly in elderly, it is essential to establish if donor age negatively influences the production of cytoprotective factors. Accordingly, we have compared the paracrine properties of fetal MSC with adult MSC from young and old patients.
Methods: MSC of fetal origin (F-MSC) were isolated from placental amniotic membranes processed immediately after delivery. Adult MSC were collected from bone marrow samples of young (yBM-MSC; donor age 65 years). Rat neonatal cardiomyocytes (H9c2 cells) were used to test cytoprotection. H9c2 cells were exposed to 6 hours of hypoxia followed by 18 hours of reoxygenation in the presence of control medium (CTRL-M) or conditioned medium (CM) from F-MSC (F-CM) or yBM-MSC (Y-CM) or oBM-MSC (O-CM). CM was obtained by growing MSC for 36 hours in the absence of serum. H9c2 viability was measured by MTS assay. The rate of apoptosis was quantified by TUNEL staining. We also evaluated cleaved Caspase 3 using both a colorimetric assay and Western blotting. Gene expression profile of several known cytoprotective factors was assessed by RT-PCR.
Results: The hypoxia/reoxygenation protocol reduced H9c2 viability by 55% compared with basal condition (p<0.001). Both F-CM and Y-CM prevented cell damage compared with CTRL-M resulting in a significant increase in cell viability by 45% (p<0.017) and 33% (p<0.017), respectively. At the contrary, O-CM had no significant effect on H9c2 viability (p=n.s. vs CTRL-M). After the induction of hypoxia/reoxygenation injury, 35 ± 8% of H9c2 cells tested positive for TUNEL staining. Compared with CTRL-M and O-CM, in the presence of F-CM we observed a relative reduction in the number of TUNEL positive nuclei of 91% (p<0.001) and 89% (p<0.001) respectively. The Y-CM also reduced H9c2 apoptotic nuclei (- 67,5% vs CTRL-M, p<0.01; - 64% vs O-CM, p<0.01). In contrast, O-CM did not prevent H9c2 apoptotic death (-11% vs CTRL-M, p=n.s.). The colorimetric assay documented that the F-CM significantly reduce the level of cleaved Caspase 3 by 50% vs CTRL-M (p< 0.017) and by 42% vs Y-CM (p< 0.017); furthermore, the Y-CM reduced the amount of cleaved Caspase 3 by 33% vs O-CM (p< 0.017). Finally, the oBM-MSC-CM did not reduce cleaved Caspase 3 compared with CTRL-M. In the presence of F-CM Western blot analysis confirmed a marked reduction in Caspase 3 activation, while no striking differences were present between CTRL-M, Y-CM and O-CM. The RT-PCR analysis documented that F-MSC express several known cytoprotective factors such as PDGF-β, BMP2, EPO, FGF2 and VEGF at significantly higher level compared with oBM-MSC (p<0.05) and that VEGF, FGF2 and HGF transcripts were significantly higher in yBM-MSC than in oBM-MSC (p<0.05).
Conclusions: We have shown that human MSC can mediate cardiomyocyte protection through the release of soluble anti-apoptotic factors. However, we documented that donor age negatively influences the paracrine cytoprotective properties of adult MSC. Our data suggest that autologous MSC theraphy for ischemic heart disease might be less effective in elderly patients
Human fetal mesenchymal stem cells protect cardiac myocytes against hypoxia/reoxygenation injury
No full textBackground: Myocardial reperfusion injury represents a major complication of the reperfusive therapies used to treat acute infarction. Consequently, the identification of new cytoprotective strategies able to prevent reperfusion injury is urgently needed. We and others have shown that adult mesenchymal stem cells (MSC) limit infarct size in rodents mainly through cytoprotective paracrine mechanisms. More recently, the existence of fetal MSC in the human placenta has been described but it is unknown whether these cells can mediate cardiomyocyte protection.
Methods: MSC were isolated from the placental amniotic membrane (A-MSC) of women delivering male newborns. We performed FACS analysis and FISH staining for the Y-chromosome to determine the immunophenotype and to confirm the fetal origin of the cells, respectively. The expression of several known cytoprotective factors was verified by RT-PCR. Rat neonatal cardiomyocytes (H9c2) were exposed to 6 hours of hypoxia followed by 18 hours of reoxygenation in the presence of control medium (CTRL-M) or conditioned medium (CM) from A-MSC. H9c2 viability was evaluated by MTS assay and cleaved Caspase 3 was quantified by colorimetric assay and Western blotting. The anti-apoptotic protein Bcl-2 was analyzed in H9c2 cells by Western blotting.
Results: A-MSC were successfully isolated from 15 amniotic membranes. At passage three, the A-MSC displayed the antigen profile typical of MSC and were positive for the Y chromosome. Furthermore, the A-MSC expressed several known cytoprotective factors, such as EPO, HGF, IGF-1, FGF2, VEGF, BMP2, PDGF-b, SFRP2, TGF-B, and thymosin B4. The hypoxia/reoxygenation protocol reduced by 68% the H9c2 viability (p<0.05 vs basal conditions). The A-MSC-CM remarkably increased cell viability by 62% compared with CTRL-M (p<0.05%). The colorimetric assay documented that in H9c2 fed with CRTL-M the amount of cleaved Caspase 3 was increased by 36% after hypoxia/reoxygenation (p<0.05) and that the A-MSC-CM significantly reduced the level of cleaved Caspase 3 (- 70% vs CTRL-M, p<0.05). Western blotting analysis confirmed the reduction of Caspase 3 in the presence of A-MSC-CM and showed an increase in Bcl-2 expression.
Conclusions: We documented that it is possible to consistently isolate MSC of fetal origin from human placenta. Furthermore, we showed that A-MSC express several cytoprotective factors and that A-MSC-CM remarkably protects cardiac myocytes against hypoxia/reoxygenation damage. The systematic analysis of A-MSC profile may lead to the identification of new powerful therapies to prevent myocardial reperfusion injury
Soluble factors released by human mesenchymal stem cells of fetal origin lead to cardiomyocyte protection through the inhibition of pro-apoptotic signaling
No full textBackground: It has been shown that human bone marrow-derived mesenchymal stem cells (BM-MSC) repair infarcted hearts mainly through the production and release of cytoprotective paracrine factors. However, donor age may negatively influence the paracrine properties of BM-MSC. Recently, MSC of fetal origin have been isolated from the amniotic membrane of human placenta (A-MSC). These cells showed promising results in experimental models of myocardial infarction and, due to their immunoprivileged phenotype, A-MSC may be used for heterologous transplantation. However, their cytoprotective properties have never been investigated. Our goal was to establish if A-MSC can mediate cytoprotection and which pathways are eventually involved.
Methods: A-MSC were isolated from placenta donated by healthy women undergoing cesarian section. Conditioned medium (CM) was obtained by growing A-MSC for 36 hours in serum starvation. Rat neonatal cardiomyocytes (H9c2) were exposed to 6 hours of hypoxia followed by 18 hours of reoxygenation in the presence of control medium (CTRL-M) or CM from A-MSC (A-MSC-CM). H9c2 viability was evaluated by MTS assay and the rate of apoptosis was quantified by TUNEL staining. Cleaved Caspase 3 was evaluated by colorimetric assay and Western blotting. SAPK/JNK, p38 MAPK and Akt activation was analyzed by Western blotting and the expression of standard pro- and anti-apoptotic genes were analyzed in H9c2 cells by RT-PCR. Finally we performed RT-PCR to compare the expression profile of several known cytoprotective factors in A-MSC vs BM-MSC.
Results: The hypoxia/reoxygenation protocol reduced the H9c2 viability by 55% (p<0.001 vs basal conditions); the A-MSC-CM remarkably increased cell viability by 45% compared with CTRL-M (p<0.001). In the presence of CTRL-M, TUNEL staining documented apoptotic death in 35 ± 8% of H9c2 cells. The A-MSC-CM significantly reduced the number of TUNEL positive nuclei by 91% vs CTRL-M (p<0.001). The colorimetric assay documented that, in H9c2 fed with CRTL-M, the amount of cleaved Caspase 3 was significantly increased by 60% after hypoxia/reoxygenation (p<0.001) while A-MSC-CM prevented Caspase 3 cleveage (p= n.s. vs normoxia). Western blot analysis confirmed the reduction of Caspase 3 in the presence of A-MSC-CM. As for the pathways involved, after hypoxia/reoxigenation we observed a marked activation of SAPK/JNK and p38 MAPK that was strongly reduced by A-MSC-CM; Akt phosphorilation did not show significant variation. Furthermore, the A-MSC-CM increased the expression of the anti-apoptotic genes Bcl-2 and Stat3 and inhibited the transcription of TNF-α and FasL compared with CTRL-M. Finally, the RT-PCR analysis documented that the A-MSC express several known cytoprotective factors such as PDGF-β , BMP2, EPO, IGF-1, FGF2 and VEGF at significantly higher levels compared with BM-MSC.
Conclusions: We demonstrated that A-MSC express high levels of several cytoprotective factors and that A-MSC-CM remarkably protects cardiac myocytes against hypoxia/reoxygenation damage. The cytoprotective effects exerted by the A-MSC-CM seem to be mediated through the inhibition of SAPK/JNK and p38 MAPK pro-apoptotic pathways and by a concomitant over-expression of anti-apoptotic genes Bcl-2 and Stat3 and by a reduction of pro-apoptotic factors TNF-α and FasL. A-MSC theraphy may represent a novel and powerful approach for cardioprotection in ischemic heart disease
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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