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VEXAS syndrome is a clonal hematopoietic disorder characterized by hyperinflammation,bone marrow failure,and high mortality. The molecular hallmark of VEXAS is somatic mutations at methionine 41 (M41) in the E1 ubiquitin enzyme,UBA1. These mutations induce a protein isoform switch,but the mechanisms underlying disease pathogenesis remain unclear. Here,we developed a human cell model of VEXAS syndrome by engineering the male monocytic THP1 cell line to express the common UBA1 M41V mutation. We found that mutant UBA1 M41V cells exhibit aberrant UBA1 isoform expression,increased vacuolization,and upregulation of the unfolded protein response,recapitulating key features of VEXAS. Moreover,proteomic analyses revealed dysregulated ubiquitination and proteotoxic stress in UBA1 M41V cells,with alterations in inflammatory and stress-response pathways. Functional studies demonstrated that UBA1 M41V cells were highly sensitive to genetic or pharmacological inhibition of E1 ubiquitin enzymes. Treatment with the E1 enzyme inhibitor TAK-243 preferentially suppressed colony formation of UBA1 M41V cells as compared to WT cells. Moreover,UBA1 M41V cells exhibited greater sensitivity to TAK-243 in competition assays and showed increased apoptosis. Interestingly,TAK-243 preferentially inhibited UBA6 activity over UBA1,suggesting that UBA6 may compensate for UBA1 dysfunction in UBA1 M41V cells. Targeting UBA6 using shRNA or the UBA6-specific inhibitor phytic acid further revealed an acquired dependency on UBA6 in UBA1 M41V cells. Phytic acid selectively impaired growth and colony formation in UBA1 M41V cells while sparing WT cells,highlighting a potential therapeutic vulnerability. Together,these findings establish a novel human model of VEXAS syndrome,identify key roles for UBA1 and UBA6 in disease pathogenesis,and demonstrate that UBA6 inhibition represents a promising therapeutic strategy for selectively targeting UBA1 mutant clones. Subject terms: Haematological cancer,Cell signalling View Publication

Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population,and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular,disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors,aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits,we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as globulomers"� View Publication

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation,whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans,or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs,suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci,characteristic of the inactive X. Moreover,analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs. View Publication

Transcription factors NANOG,OCT4,and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however,their expression profiles during early differentiation of hES cells are unclear. In this study,we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG,OCT4,and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells,characterized by specific expression patterns of NANOG,OCT4,and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time,indicating a gradual mode of developmental transition in these cells. Notably,distinct regulation of SOX2 during early differentiation events was detected,highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation. View Publication

Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival,self-renewal,and differentiation. Thus,hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture. Soluble angiopoeitin-1 (Ang-1)-derived peptide QHREDGS added to defined serum-free media increased hPSC colony cell number and size during long- and short-term culture when grown on feeder cell layers or Matrigel,i.e. on standard substrates,without affecting hPSC morphology,growth rate or the ability to differentiate into multiple lineages both invitro and invivo. Importantly,QHREDGS treatment decreased hPSC apoptosis during routine passaging and single-cell dissociation. Mechanistically,the interaction of QHREDGS with ??1-integrins increased expression of integrin-linked kinase (ILK),increased expression and activation of extracellular signal-regulated kinases 1/2 (ERK1/2),and decreased caspase-3/7 activity. QHREDGS immobilization to polyethylene glycol hydrogels significantly increased cell adhesion in a dose-dependent manner. We propose QHREDGS as a small molecule inhibitor of hPSC apoptosis and the basis of an affordable defined substrate for hPSC maintenance. ?? 2014 Elsevier Ltd. View Publication

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast,T cells with other antigen specificities from these patients,or autoreactive T cells from healthy individuals and disease controls,express low levels of Kv1.3 and are predominantly naïve or central-memory (T(CM)) cells. In T(EM) cells,Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2,SAP97,ZIP,p56(lck),and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation,they suppress Ca2+-signaling,cytokine production,and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted. View Publication

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence,which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here,we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity,simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. textcopyright 2013 Elsevier Inc. View Publication

Monoclonal antibodies (mAbs) have proven to be instrumental in the advancement of research,diagnostic,industrial vaccine,and therapeutic applications. The use of mAbs in laboratory protocols has been growing in an exponential fashion for the last four decades. Described herein are methods for the development of highly specific mAbs through traditional hybridoma fusion. For ultimate success,a series of simultaneously initiated protocols are to be undertaken with careful attention to cell health of both the myeloma fusion partner and immune splenocytes. Coordination and attention to detail will enable a researcher with basic tissue culture skills to generate mAbs from immunized rodents to a variety of antigens (including proteins,carbohydrates,DNA,and haptens) (see Note 1). Furthermore,in vivo and in vitro methods used for antigen sensitization of splenocytes prior to somatic fusion are described herein. View Publication

The anisotropic alignment of cardiomyocytes in native myocardium tissue is a functional feature that is absent in traditional in vitro cardiac cell culture. Microenvironmental factors cue structural organization of the myocardium,which promotes the mechanical contractile properties and electrophysiological patterns seen in mature cardiomyocytes. Current nano- and microfabrication techniques,such as photolithography,generate simplified cell culture topographies that are not truly representative of the multifaceted and multi-scale fibrils of the cardiac extracellular matrix. In addition,such technologies are costly and require a clean room for fabrication. This chapter offers an easy,fast,robust,and inexpensive fabrication of biomimetic multi-scale wrinkled surfaces through the process of plasma treating and shrinking prestressed thermoplastic. Additionally,this chapter includes techniques for culturing stem cells and their cardiac derivatives on these substrates. Importantly,this wrinkled cell culture platform is compatible with both fluorescence and bright-field imaging; real-time physiological monitoring of CM action potential propagation and contraction properties can elucidate cardiotoxicity drug effects. View Publication

Biochemical and biophysical stimuli of stem cell niches finely regulate the self-renewal/differentiation equilibrium. Replicating this in vitro is technically challenging,making the control of stem cell functions difficult. Cell derived matrices capture certain aspect of niches that influence fate decisions. Here,aligned fibrous matrices synthesized by MC3T3 cells were produced and the role of matrix orientation and stiffness on the maintenance of stem cell characteristics and adipo- or osteo-genic differentiation of murine mesenchymal stem cells (mMSCs) was investigated. Decellularized matrices promoted mMSC proliferation. Fibrillar alignment and matrix stiffness work in concert in defining cell fate. Soft matrices preserve stemness,whereas stiff ones,in presence of biochemical supplements,promptly induce differentiation. Matrix alignment impacts the homogeneity of the cell population,that is,soft aligned matrices ameliorate the spontaneous adipogenic differentiation,whereas stiff aligned matrices reduce cross-differentiation. We infer that mechanical signaling is a dominant factor in mMSC fate decision and the matrix alignment contributes to produce a more homogeneous environment,which results in a uniform response of cells to biophysical environment. Matrix thus produced can be obtained in vitro in a facile and consistent manner and can be used for homogeneous stem cell amplification or for mechanotransduction-related studies. View Publication