Systemic epigenetic response to recombinant lentiviral vectors independent of proviral integration

Background: Lentiviral vectors (LV) are widely used for various gene transfer or gene therapy applications. The effects of LV on target cells are expected to be limited to gene delivery. Yet, human hematopoietic CD34+ cells respond to functional LVs as well as several types of non-integrating LVs by genome-wide DNA methylation changes. Results: A new algorithm for the analysis of 450K Illumina data showed that these changes were marked by de novo methylation. The same 4126 cytosines located in islands corresponding to 1059 genes were systematically methylated. This effect required cellular entry of the viral particle in the cells but not the genomic integration of the vector cassette. Some LV preparations induced only mild sporadic changes while others had strong effects suggesting that LV batch heterogeneity may be related to the extent of the epigenetic response. Conclusion: These findings identify a previously uncharacterized but consistent cellular response to viral components and provide a novel example of environmentally modified epigenome. © 2016 The Author(s)

Bistable Cell Fate Specification as a Result of Stochastic Fluctuations and Collective Spatial Cell Behaviour

BACKGROUND: In culture, isogenic mammalian cells typically display enduring phenotypic heterogeneity that arises from fluctuations of gene expression and other intracellular processes. This diversity is not just simple noise but has biological relevance by generating plasticity. Noise driven plasticity was suggested to be a stem cell-specific feature. RESULTS: Here we show that the phenotypes of proliferating tissue progenitor cells such as primary mononuclear muscle cells can also spontaneously fluctuate between different states characterized by the either high or low expression of the muscle-specific cell surface molecule CD56 and by the corresponding high or low capacity to form myotubes. Although this capacity is a cell-intrinsic property, the cells switch their phenotype under the constraints imposed by the highly heterogeneous microenvironment created by their own collective movement. The resulting heterogeneous cell population is characterized by a dynamic equilibrium between "high CD56" and "low CD56" phenotype cells with distinct spatial distribution. Computer simulations reveal that this complex dynamic is consistent with a context-dependent noise driven bistable model where local microenvironment acts on the cellular state by encouraging the cell to fluctuate between the phenotypes until the low noise state is found. CONCLUSIONS: These observations suggest that phenotypic fluctuations may be a general feature of any non-terminally differentiated cell. The cellular microenvironment created by the cells themselves contributes actively and continuously to the generation of fluctuations depending on their phenotype. As a result, the cell phenotype is determined by the joint action of the cell-intrinsic fluctuations and by collective cell-to-cell interactions

The Origin of Phenotypic Heterogeneity in a Clonal Cell Population In Vitro

BACKGROUND: The spontaneous emergence of phenotypic heterogeneity in clonal populations of mammalian cells in vitro is a rule rather than an exception. We consider two simple, mutually non-exclusive models that explain the generation of diverse cell types in a homogeneous population. In the first model, the phenotypic switch is the consequence of extrinsic factors. Initially identical cells may become different because they encounter different local environments that induce adaptive responses. According to the second model, the phenotypic switch is intrinsic to the cells that may occur even in homogeneous environments. PRINCIPAL FINDINGS: We have investigated the “extrinsic” and the “intrinsic” mechanisms using computer simulations and experimentation. First, we simulated in silico the emergence of two cell types in a clonal cell population using a multiagent model. Both mechanisms produced stable phenotypic heterogeneity, but the distribution of the cell types was different. The “intrinsic” model predicted an even distribution of the rare phenotype cells, while in the “extrinsic” model these cells formed small clusters. The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line. CONCLUSIONS: The observations emphasize the importance of the “ecological” context and suggest that, consistently with the “extrinsic” model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch. Nevertheless, the “intrinsic” model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells

Robust physical methods that enrich genomic regions identical by descent for linkage studies: confirmation of a locus for osteogenesis imperfecta

<p>Abstract</p> <p>Background</p> <p>The monogenic disease osteogenesis imperfecta (OI) is due to single mutations in either of the collagen genes ColA1 or ColA2, but within the same family a given mutation is accompanied by a wide range of disease severity. Although this phenotypic variability implies the existence of modifier gene variants, genome wide scanning of DNA from OI patients has not been reported. Promising genome wide marker-independent physical methods for identifying disease-related loci have lacked robustness for widespread applicability. Therefore we sought to improve these methods and demonstrate their performance to identify known and novel loci relevant to OI.</p> <p>Results</p> <p>We have improved methods for enriching regions of identity-by-descent (IBD) shared between related, afflicted individuals. The extent of enrichment exceeds 10- to 50-fold for some loci. The efficiency of the new process is shown by confirmation of the identification of the Col1A2 locus in osteogenesis imperfecta patients from Amish families. Moreover the analysis revealed additional candidate linkage loci that may harbour modifier genes for OI; a locus on chromosome 1q includes COX-2, a gene implicated in osteogenesis.</p> <p>Conclusion</p> <p>Technology for physical enrichment of IBD loci is now robust and applicable for finding genes for monogenic diseases and genes for complex diseases. The data support the further investigation of genetic loci other than collagen gene loci to identify genes affecting the clinical expression of osteogenesis imperfecta. The discrimination of IBD mapping will be enhanced when the IBD enrichment procedure is coupled with deep resequencing.</p

Contribution à l'étude de la physiologie et physiopathologie moléculaire de la calpaïne 3

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

International audienc

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

International audienc

Artificial intelligence workflow quantifying muscle features on Hematoxylin–Eosin stained sections reveals dystrophic phenotype amelioration upon treatment

International audienceAbstract Cell segmentation is a key step for a wide variety of biological investigations, especially in the context of muscle science. Currently, automated methods still struggle to perform skeletal muscle fiber quantification on Hematoxylin-Eosin (HE) stained histopathological whole slide images due to low contrast. On the other hand, the Deep Learning algorithm Cellpose offers new perspectives considering its increasing adoption for segmentation of a wide range of cells. Combining two open-source tools, Cellpose and QuPath, we developed MyoSOTHES, an automated Myofibers Segmentation wOrkflow Tuned for HE Staining. MyoSOTHES enables solving segmentation inconsistencies encountered by default Cellpose model in presence of large range size cells and provides information related to muscle Feret’s diameter distribution and Centrally Nucleated Fibers, thus depicting muscle health and treatment effects. MyoSOTHES achieves high quality segmentation compared to baseline workflow with a detection F1-score increasing from 0.801 to 0.919 and a Root Mean Square Error (RMSE) on diameter improved by 31%. MyoSOTHES was validated on an animal study featuring gene transfer in $$\gamma$$ γ -Sarcoglycanopathy, for which dose-response effect is visible and conclusions drawn are consistent with those previously published. MyoSOTHES thus paves the way for wide quantification of HE stained muscle sections and retrospective analysis of HE labeled slices used in laboratories for decades