Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs.

Combination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases.

Applications of quantitative polymerase chain reaction protein assays during reprogramming.

The capability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) has opened a new area of biology and provides unprecedented access to patient-specific iPSCs for drug screening, disease models, and transplantation therapies. Although the process of obtaining iPSC lines is technically simple, reprogramming is a slow and inefficient process consisting of a largely uncharacterized chain of molecular events. To date, researchers have reported a wide range of reprogramming efficiencies, from <0.

Fcγ receptor profile of monocytes and macrophages from rheumatoid arthritis patients and their response to immune complexes formed with autoantibodies to citrullinated proteins.

Objective: To analyse Fcγ receptor (FcγR) expression on monocytes and macrophages from rheumatoid arthritis (RA) patients versus healthy controls (HC), and to compare their responses to immune complexes containing RA-specific anti-citrullinated proteins auto antibodies (ACPA).

Methods: Monocytes and monocyte-derived macrophages were obtained from the peripheral blood of 34 RA patients and 69 HC. FcγR expression was studied by flow cytometry.

A bioinformatic assay for pluripotency in human cells.

Pluripotent stem cells (PSCs) are defined by their potential to generate all cell types of an organism. The standard assay for pluripotency of mouse PSCs is cell transmission through the germline, but for human PSCs researchers depend on indirect methods such as differentiation into teratomas in immunodeficient mice. Here we report PluriTest, a robust open-access bioinformatic assay of pluripotency in human cells based on their gene expression profiles.

Equivalence of conventionally-derived and parthenote-derived human embryonic stem cells.

Background: As human embryonic stem cell (hESC) lines can be derived via multiple means, it is important to determine particular characteristics of individual lines that may dictate the applications to which they are best suited. The objective of this work was to determine points of equivalence and differences between conventionally-derived hESC and parthenote-derived hESC lines (phESC) in the undifferentiated state and during neural differentiation.

Methodology/principal Findings: hESC and phESC were exposed to the same expansion conditions and subsequent neural and retinal pigmented epithelium (RPE) differentiation protocols.

Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.

Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions.

Friedreich's ataxia induced pluripotent stem cells model intergenerational GAA⋅TTC triplet repeat instability.

The inherited neurodegenerative disease Friedreich's ataxia (FRDA) is caused by GAA⋅TTC triplet repeat hyperexpansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA⋅TTC repeats cause heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming.

Towards computational prediction of microRNA function and activity.

While it has been established that microRNAs (miRNAs) play key roles throughout development and are dysregulated in many human pathologies, the specific processes and pathways regulated by individual miRNAs are mostly unknown. Here, we use computational target predictions in order to automatically infer the processes affected by human miRNAs. Our approach improves upon standard statistical tools by addressing specific characteristics of miRNA regulation.

Dynamic changes in the human methylome during differentiation.

DNA methylation is a critical epigenetic regulator in mammalian development. Here, we present a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic differentiated derivative of the hESCs, and neonatal fibroblasts. As a reference, we compared our maps with a methylome map of a fully differentiated adult cell type, mature peripheral blood mononuclear cells (monocytes).

Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system.

We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer.

DNA methylation in embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent, self-renewing cells. These cells can be used in applications such as cell therapy, drug development, disease modeling, and the study of cellular differentiation. Investigating the interplay of epigenetics, genetics, and gene expression in control of pluripotence and differentiation could give important insights on how these cells function.

MicroRNAs in embryonic stem cells and early embryonic development.

MicroRNAs (miRNAs) are involved in the regulation of a broad range of biological processes. There is an extensive literature on the roles of miRNAs in metazoan development, from early embryogenesis to lineage commitment, and formation and maturation of cellular subtypes and complex tissues. This review will present the evidence that miRNAs are essential for the earliest stages of metazoan development, including establishment and maintenance of the pluripotent embryonic stem cells (ESCs) from which all foetal tissues arise.

Regulatory networks define phenotypic classes of human stem cell lines.

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cells, even though they range from pluripotent cells-typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation-to adult stem cell lines, which can generate a far more limited repertoire of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine has highlighted the need for a general, reproducible method for classification of these cells.

Comprehensive microRNA profiling reveals a unique human embryonic stem cell signature dominated by a single seed sequence.

Embryonic stem cells are unique among cultured cells in their ability to self-renew and differentiate into a wide diversity of cell types, suggesting that a specific molecular control network underlies these features. Human embryonic stem cells (hESCs) are known to have distinct mRNA expression, global DNA methylation, and chromatin profiles, but the involvement of high-level regulators, such as microRNAs (miRNA), in the hESC-specific molecular network is poorly understood. We report that global miRNA expression profiling of hESCs and a variety of stem cell and differentiated cell types using a novel microarray platform revealed a unique set of miRNAs differentially regulated in hESCs, including numerous miRNAs not previously linked to hESCs.

Unraveling epigenetic regulation in embryonic stem cells.

Embryonic stem (ES) cells can replicate indefinitely while retaining the capacity to differentiate into functionally distinct cell types. ES cells proliferate and differentiate without detectable genetic changes, indicating that these processes are controlled by epigenetic factors. Here we describe what is known about the epigenetics of ES cells and speculate that a dynamic balance among at least three epigenetic elements (chromatin structure, DNA methylation, and microRNAs), in conjunction with transcription factors, contributes to the maintenance of pluripotence.

Induction of macrophage secretion of tumor necrosis factor alpha through Fcgamma receptor IIa engagement by rheumatoid arthritis-specific autoantibodies to citrullinated proteins complexed with fibrinogen.

Objective: Macrophage-derived tumor necrosis factor alpha (TNFalpha) is a dominant mediator of synovitis in rheumatoid arthritis (RA). This study was undertaken to assess whether and how immune complexes (ICs) formed by the interaction of disease-specific autoantibodies to citrullinated proteins (ACPAs) with their main synovial target antigen, citrullinated fibrin, contribute to TNFalpha production by macrophages.

Methods: An in vitro human model was developed in which monocyte-derived macrophages were stimulated with ACPA-containing ICs that were generated by capturing ACPAs from RA sera on immobilized citrullinated fibrinogen.

[The "footscan" in sport medicine].

Clinical examination of the foot in a patient or sportsman requires a detailed analysis of walking (and running). Current technology allows to study temporal fluctuations of plantar pressures and to detect the anomalies responsible for sport specific pathologies or pathologies encountered in sick predisposed people.

WHIM syndromes with different genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12.

The WHIM syndrome is a rare immunodeficiency disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. Dominant heterozygous mutations of the gene encoding CXCR4, a G-protein-coupled receptor with a unique ligand, CXCL12, have been associated with this pathology. We studied patients belonging to 3 different pedigrees.

Microdisplacements induced by a local perturbation inside a granular packing.

The microdisplacements generated by a small localized overload at the free surface are visualized experimentally inside a packing of steel beads. For a triangular packing, beads rearrangements remain confined in two inverted triangles on both sides of the applied overload. This pattern disappears for stronger disorder.

[Hereditary neuropathy with pressure hypersensitivity or tomaculous neuropathy].

Hereditary neuropathy liability to pressure palsies is characterized by recurring accesses of painless paralysis at the level of various nerves likely to be compressed. This affection remains underdiagnosed because of its usually benign course, sometimes without any symptom. The diagnosis is supported by clinical and electrophysiological data associated with, in the majority of patients, a deletion of one of the alleles coding for protein PMP 22 on the level of the locus 17p11.

Multiscale clustering in granular surface flows.

We investigate steady granular surface flows in a rotating drum and demonstrate the existence of rigid clusters of grains embedded in the flowing layer. These clusters appear to be fractal and their size is power law distributed from the grain size scale up to the thickness of the flowing layer. The implications of the absence of a characteristic length scale on available theoretical models of dense granular flows are discussed.

Distinctive alterations of invasiveness, drug resistance and cell-cell organization in 3D-cultures of MCF-7, a human breast cancer cell line, and its multidrug resistant variant.

Growth of human tumor cells as three-dimensional (3D) multicellular spheroids modifies their invasive properties. Here we study the differences in the biological features of MCF-7, a human breast cancer cell line, and its multidrug resistant variant (MDR-MCF-7) cultured as spheroids or as monolayers. Three-dimensional culture decreased the proliferative rate of both cell lines, reduced the drug sensitivity of MCF-7 cells and did not affect the resistance of MDR-MCF-7 cells.

Comprehensive mutational analysis of the Moloney murine leukemia virus envelope protein.

The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues.

Human cytomegalovirus infection of bone marrow myofibroblasts enhances myeloid progenitor adhesion and elicits viral transmission.

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells.