1,720,965 research outputs found
Improved transduction efficiency of human amniotic mesenchymal stem cells using optimized lentiviral vectors
No full textBackground: Amniotic mesenchymal stem cells (A-MSC)
are excellent candidates for regenerative medicine since they
are multipotent, easy to isolate, expandable and seem to be
immunoprivileged. In rodents, stable genetic modification with
viral vectors improves A-MSC function. Unfortunately, a single
cycle transduction with standard viral vectors does not achieve
high efficiency in human A-MSC. On the other hand, multiple
transduction cycles or antibiotic-based selection methods may alter the cell phenotype. We hypothesized that the use of lentiviral vectors containing specific regulatory sequences may result in improved transduction efficiency in human A-MSC. Accordingly, we compared two types of third generation lentiviral vectors, one of which containing the optimized sequences for Polypurine Tract (cPPT) and Woodchuck Posttranscriptional Regulatory Element (WPRE).
Methods: Human A-MSC were isolated from amniotic membranes
of human term placenta. The immunophenotype of the cells was determined by fluorescence-activated cell sorting (FACS). To confirm their mesenchymal origin, the ability of A-MSC to
differentiate into adipocytes and osteocytes was tested by RT-PCR
and cytochemistry. The production of both lentiviral vectors was carried out following standard protocols. The cPPT and WPRE sequences were cloned between the LTR sequences. It is known that the cPPT enhances the lentivirus production, whereas the WPRE stabilizes the unspliced RNAs during the transfection. In both vectors, a fluorescent tag (green fluorescent protein – GFP) and a gene encoding for a protein of interest (Insulin-like growth factor I – IGF-I) were also cloned. The number of GFP positive cells was quantified by FACS following a single cycle of transduction. Finally, the expression of IGF-I was assessed with Western blot analysis.
Results: A-MSC were successfully isolated and displayed the
antigen profile typical of bone marrow-derived MSC. Specifically,
they were positive for CD90, CD105, CD73, HLA-ABC e CD117
and negative for HLA-DR, CD34, CD45, CD14, CD80, CD31 and
CD133. Furthermore, A-MSC efficiently differentiated into osteocytes and adipocytes. In particular, at the end of the differentiation protocol into osteocytes, the A-MSC expressed the typical osteogenic genes secreted phosphoprotein 10, cathepsin K and bone sialoprotein and stained positive for Alkaline Phosphatase activity and Von Kossa. After the differentiation protocol into adipocytes, the A-MSC expressed the typical adipogenic genes adipocyte differentiation related protein and peroxisome proliferator-activated receptor gamma and stained positive for Oil Red-O. The transduction efficiency with the standard lentivirus was only 15%. Conversely, with the optimized vector we obtained transduction efficiency as high as 50%. Accordingly, the A-MSC transduced with the optimized vector expressed higher levels of IGF-I protein compared with the A-MSC genetically modified with the standard vector.
Conclusions: We have demonstrated for the first time that, using
lentiviral vectors containing the cPPT and WPRE sequences, it is
possible to efficiently transduce human A-MSC with a single cycle
of transduction
The cardioprotective paracrine effects exerted by human mesenchymal stem cells are negatively influenced by donor age
No full textBackground: Mesenchymal stem cells (MSC) repair infarcted hearts mainly through the release of cytoprotective factors. We compared fetal MSC (F-MSC) with adult MSC from young and elderly patients to establish if donor age influences their cardioprotective paracrine properties.
Methods: F-MSC were isolated from human placenta while adult MSC were isolated from bone marrow samples of young (yBM-MSC; age 65 ) donors. Rat neonatal cardiomyocytes (H9c2) were exposed to hypoxia/reoxygenation (6/18 hours) 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 Caspase 3 activation (colorimetric assay and Western Blot). We used RT-PCR to verify the expression of known cytoprotective factors.
Results: The hypoxia/reoxygenation protocol reduced H9c2 viability by 55% compared with basal condition (p<0.001). F-CM and Y-CM increased cell viability by 45% and 33% (p<0.017), respectively; O-CM had no significant effect on H9c2 viability (p=n.s. vs CTRL-M). Compared with CTRL-M and O-CM, F-CM significantly reduced the number of TUNEL positive nuclei by 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.). Both colorimetric assay and Western Blot analysis showed that Caspase-3 activation was prevented by F-CM and Y-CM but not by O-CM. F-MSC expressed PDGF-β, BMP2, EPO, FGF2 and VEGF at significantly higher level compared with oBM-MSC (p<0.05); VEGF, FGF2 and HGF transcripts were significantly higher in yBM-MSC than in oBM-MSC (p<0.05).
Conclusions: 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
Prognostic significance of the interaction between abnormal umbilical and middle cerebral artery Doppler velocimetry in pregnancies complicated by fetal growth restriction
No full textPentraxin-3 and galectin-1 are key mediators of the cardioprotective paracrine effects exerted by fetal mesenchymal stem cells isolated from human placenta
No full textModel for Neurotoxicity Testing
No full textNeurotoxicity (NT) testing for regulatory purposes is based on in vivo animal testing. There is general consensus, however, about the need for the development of alternative methodologies to allow researchers to more rapidly and cost effectively screen large numbers of chemicals for their potential to cause NT, or to investigate their mode of action. In vitro assays are considered an important source of information for making regulatory decisions, and human cell–based systems are recommended as one of the most relevant models in toxicity testing, to reduce uncertainty in the extrapolation of results from animal-based models. Human neuronal models range from various neuroblastoma cell lines to stem cell–derived systems, including those derived from mesenchymal stem/stromal cells (hMSC). hMSCs exhibit numerous advantages, including the fact that they can be obtained in high yield from healthy human adult tissues, can be cultured with a minimal laboratory setup and without genetic manipulations, are able of continuous and repeated self-renewal, are nontumorigenic, and can form large populations of stably differentiated cells representative of different tissues, including neuronal cells. hMSCs derived from human umbilical cord (hUC) in particular possess several prominent advantages, including a painless, non-invasive, and ethically acceptable collection procedure, simple and convenient preparation, and high proliferation capacity. In addition, hMSCs can be efficiently differentiated into neuron-like cells (hNLCs), which can then be used for the assessment of neuronal toxicity of potential neurotoxic compounds in humans. Here, we describe a step-by-step procedure to use hMSCs from the umbilical cord for in vitro neurotoxicity testing. First, we describe how to isolate, amplify, and store hMSCs derived from the umbilical cord. We then outline the steps to transdifferentiate these cells into hNLCs, and then use the hNLCs for neurotoxicity testing by employing multiple common cytotoxicity assays after treatment with test compounds. The approach follows the most updated guidance on using human cell–based systems. These protocols will allow investigators to implement an alternative system for obtaining primary NLCs of human origin, and support advancement in neurotoxicity research. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and maintenance of human mesenchymal stem/stromal cells (hMSCs) obtained from the umbilical cord lining membrane. Basic Protocol 2: Transdifferentiation of hMSCs into neuron-like cells (hNLCs) and basic neurotoxicity assessment
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
Multiple human papillomavirus infection with or without type 16 and risk of cervical intraepithelial neoplasia among women with cervical cytological abnormalities.
No full textPURPOSE:
To evaluate the impact of multiple human papillomavirus (HPV) infections on the risk of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in subjects with cervical cytological abnormalities.
METHODS:
A cross-sectional study of 3,842 women attending a colposcopy service was carried out. Genotyping of 18 high-risk, seven low-risk, and two undefined-risk HPVs was carried out by the INNO-LiPA genotyping system.
RESULTS:
The final colposcopic/pathological diagnoses were as follows: 1,933 (50.3 %) subjects were negative; 1,041 (27.1 %) CIN1; 280 (7.3 %) CIN2; 520 (13.5 %) CIN3; and 68 (1.8 %) invasive cervical cancer. The prevalence of HPV infection was 75.8 % (2,911/3,842), whereas multiple HPVs were detected in 34.5 % of HPV-positive subjects (2,255/3,842). The adjusted risks of CIN3+ in the group with multiple compared to the group with single infection were 2.31 (95 % CI = 1.54-3.47), among HPV16-positive women, and 3.25 (95 % CI = 2.29-4.61, p = 0.21 compared with HPV16-positive subjects), in HPV16-negative subjects. Out of a total of 1,285 subjects with mild lesions, followed up for a median of 16.1 months (interquartile range = 8.9-36.8), the rate of progression to CIN2-3 was 0.6 % (5/541) among subjects negative or with low-risk HPVs, 1.7 % (8/463) among those with single high-risk HPV, and 5 % (14/281, p < 0.001 compared with HPV-negative/low-risk HPV and p = 0.038 compared with single high-risk HPV) among those with multiple high-risk HPVs.
CONCLUSIONS:
Among women with cervical cytological abnormalities, infection by multiple high-risk HPVs increased the risk of CIN3+ in both HPV16-positive and HPV16-negative subjects. These findings suggest a potential synergistic interaction between high-risk HPVs, favoring the progression of CIN lesions
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