1,720,966 research outputs found
Reprogramming of primary human fetal fibroblasts towards cardiomyoctes
No full textThe mammalian heart is primarily composed of fibroblasts, cardiomyocytes, endothelial and smooth muscle cells. Cardiomyocytes show little regenerative capabilities following damage (e.g. myocardial infarction, MI) and subsequently cardiac fibroblasts migrate to the site of injury and secrete extracellular matrix proteins to form scar tissue which supports the damaged myocardium. Thus, there is a therapeutic need for cell-based therapies for the generation of functional cardiomyocytes. One method to achieve this is to directly reprogram fibroblasts into cardiomyocytes, which has previously been achieved in mice through overexpression of the transcription factors: Gata4, Mef2c and Tbx5. The present study has hypothesised that fibroblast cells, isolated from human fetal heart and skin, can be reprogrammed into cardiomyocytes by overexpressing GATA4, MEF2C and TBX5 (GMT).Initial experiments aimed to characterise the phenotype of cardiac cell types within the human fetal heart using protein markers. Flow cytometry data revealed that the human fetal heart is composed of approximately 75-80% cardiomyocytes and 20-25% non-myocytes. The results also showed that Thy-1 and vimentin, considered to be fibroblast markers, localise in a proportion of cells that also express sarcomeric proteins (cardiomyocyte markers), confirming that they are not specific markers of fibroblasts. Isolation of fibroblasts from primary human fetal tissue was achieved through explant migration and phenotyped by RT-PCR, immunocytochemistry and flow cytometry. These cells were transfected with two vectors that allowed bicistronic expression of GATA4 with GFP and MEF2C with TBX5. Three transfection methods were compared: JetPEI, FuGENE6 and nucleofection. Nucleofection was found to be the best method of vector delivery into primary fibroblasts and enabled the selection of G418- resistant cells that were viable for 8 weeks.Flow cytometry analysis showed upregulation of NKX2.5, cardiac troponin I (TnI) and α-actinin in skin fibroblasts transfected with GMT and treated with G418, however, expression was not sustained beyond two weeks, implying that these cells were not stably expressing GMT. Treatment with the signalling molecule TGFβ-1 increased the percentage of cells expressing NKX2.5, TnI and α-actinin. This study has demonstrated the viability of a non-viral system for the delivery of GMT into primary human fetal fibroblasts and has shown that overexpression of GMT in these cells appears to initiate the early stages of direct cardiomyocyte reprogramming
Defining cardiac cell populations and relative cellular composition of the early fetal human heart
No full textWhile the adult human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells, the cellular composition during early development remains largely unknown. Reliable identification of fetal cardiac cell types using protein markers is critical to understand cardiac development and delineate the cellular composition of the developing human heart. This is the first study to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8–12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and cardiac troponin I (cTnI), fibroblasts (DDR2, THY1, Vimentin), endothelial cells (CD31) and smooth muscle cells (α-SMA) were assessed. Two distinct populations of cTnI positive cells were identified through flow cytometry, with MHC positive cardiomyocytes showing high cTnI expression (cTnI(High)) while MHC negative non-myocytes showed lower cTnI expression (cTnI(Low)). cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin (VIM) was expressed in cultured ventricular fibroblast populations and flow cytometry revealed VIM(High) and VIM(Low) cell populations in the fetal heart. MHC positive cardiomyocytes were VIM(Low) whilst CD31 positive endothelial cells were VIM(High). Using markers investigated within this study, we characterised fetal human cardiac populations and estimate that 75–80% of fetal cardiac cells are cardiomyocytes and are MHC(+)/cTnI(High)/VIM(Low), whilst non-myocytes comprise 20–25% of total cells and are MHC(-)/cTnI(Low)/VIM(High), with CD31(+) endothelial cells comprising ~9% of this population. These findings show distinct differences from those reported for adult heart
In vitro stem cell modelling demonstrates a proof-of-concept for excess functional mutant TIMP3 as the cause of Sorsby Fundus Dystrophy
No full textSorsby Fundus Dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting mutant TIMP3 dimerization does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of extracellular matrix proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-iPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, platelet-derived growth factor, and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis related changes in SFD
The diverse roles of TIMP-3: Insights into degenerative diseases of the senescent retina and brain
No full textTissue inhibitor of metalloproteinase-3 (TIMP-3) is a component of the extracellular environment, where it mediates diverse processes including matrix regulation/turnover, inflammation and angiogenesis. Rare TIMP-3 risk alleles and mutations are directly linked with retinopathies such as age-related macular degeneration (AMD) and Sorsby fundus dystrophy, and potentially, through indirect mechanisms, with Alzheimer's disease. Insights into TIMP-3 activities may be gleaned from studying Sorsby-linked mutations. However, recent findings do not fully support the prevailing hypothesis that a gain of function through the dimerisation of mutated TIMP-3 is responsible for retinopathy. Findings from Alzheimer's patients suggest a hitherto poorly studied relationship between TIMP-3 and the Alzheimer's-linked amyloid-beta (Ab) proteins that warrant further scrutiny. This may also have implications for understanding AMD as aged/diseased retinae contain high levels of Ab. Findings from TIMP-3 knockout and mutant knock-in mice have not led to new treatments, particularly as the latter does not satisfactorily recapitulate the Sorsby phenotype. However, recent advances in stem cell and in vitro approaches offer novel insights into understanding TIMP-3 pathology in the retina-brain axis, which has so far not been collectively examined. We propose that TIMP-3 activities could extend beyond its hitherto supposed functions to cause age-related changes and disease in these organs
AMMECR1: a single point mutation causes developmental delay, midface hypoplasia and elliptocytosis
No full textBackground: Deletions in the Xq22.3–Xq23 region, inclusive of COL4A5, have been associated with a contiguous gene deletion syndrome characterised by Alport syndrome with intellectual disability (Mental retardation), Midface hypoplasia and Elliptocytosis (AMME). The extrarenal biological and clinical significance of neighbouring genes to the Alport locus has been largely speculative. We sought to discover a genetic cause for two half-brothers presenting with nephrocalcinosis, early speech and language delay and midface hypoplasia with submucous cleft palate and bifid uvula.Methods: Whole exome sequencing was undertaken on maternal half-siblings. In-house genomic analysis included extraction of all shared variants on the X chromosome in keeping with X-linked inheritance. Patient-specific mutants were transfected into three cell lines and microscopically visualised to assess the nuclear expression pattern of the mutant protein.Results: In the affected half-brothers, we identified a hemizygous novel non-synonymous variant of unknown significance in AMMECR1 (c.G530A; p.G177D), a gene residing in the AMME disease locus. Transfected cell lines with the p.G177D mutation showed aberrant nuclear localisation patterns when compared with the wild type. Blood films revealed the presence of elliptocytes in the older brother.Conclusions: Our study shows that a single missense mutation in AMMECR1 causes a phenotype of midface hypoplasia, mild intellectual disability and the presence of elliptocytes, previously reported as part of a contiguous gene deletion syndrome. Functional analysis confirms mutant-specific protein dysfunction. We conclude that AMMECR1 is a critical gene in the pathogenesis of AMME, causing midface hypoplasia and elliptocytosis and contributing to early speech and language delay, infantile hypotonia and hearing loss, and may play a role in dysmorphism, nephrocalcinosis and submucous cleft palate.<br/
Modelling Sorsby’s Fundus Dystrophy using patient-derived iPSC-RPE
No full textPurpose : Sorsby’s fundus dystrophy (SFD) leads to bilateral loss of central vision and is caused by mutations in the gene TIMP3. The mechanisms by which TIMP3 mutations cause SFD are poorly understood. iPSC-retinal pigmented epithelial (RPE) cells were generated from SFD patients and controls (ctrl) and cultured on transwell inserts to develop a RPE cell model which was used to study effects of specific TIMP3 mutations on cellular structure and physiology.Methods : Human fibroblasts were isolated from SFD (TIMP3 S204C) and ctrl subject skin biopsies by explant migration and reprogrammed into iPSCs. These iPSC colonies were cultured in RPE differentiation medium for 4 weeks until sufficient pigmentation was achieved before being transferred to transwells. Transmission electron microscopy (TEM) was used to analyse morphological differences between SFD and ctrl RPE cells. Transepithelial electrical resistance (TEER) was used to measure RPE barrier integrity. To assess the ability of TIMP3 to inhibit MMP activity, protein lysates from SFD and ctrl RPE cells were tested in an EnzCheck collagenase assay.Results : iPSC-RPE cells were successfully derived from SFD and ctrl patients. TEM analysis revealed significant differences in RPE containing mutant TIMP3 with respect to cell height (SFD 10.3µm ±0.23 SEM, ctrl 13.23µm ±0.43 SEM, p<0.0001) and basal laminar area (SFD 6µm2 ± 0.446 SEM, ctrl 14µm2 ±0.65 SEM, p<0.0001). Striated sub RPE deposits were observed, consistent with long-spacing collagen. A significantly higher number of focal collagen deposits were seen per cell in ctrl-RPE compared to SFD-RPE (SFD 1.444 ±0.44 SEM, ctrl 3.286 ± 0.52 SEM, p<0.05). The number of melanosomes per cell was also significantly higher in ctrl-RPE compared to SFD-RPE (SFD 40.2 ±2.34 SEM, ctrl 57.54 ± 3.74 SEM, p<0.001). However, there was no difference in their basal/apical distribution. SFD-RPE cells showed significantly lower average TEER over 90 days in culture compared to ctrl-RPE (SFD1 161.1 ± 15.9 SEM, ctrl 499.4 ± 49.79 SEM, p<0.001). The enzyme assay showed TIMP3 S204C mutation does not significantly affect the ability of TIMP3 to inhibit collagenase.Conclusions : The S204C TIMP3 mutation reduces the barrier integrity of RPE cells and results in key morphological alterations to the RPE, including cell height, not previously described. However, our findings indicate that the S204C mutation does not affect TIMP3’s ability to inhibit MMP-9 activity
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
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