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Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1,12,17,and 20. Thus,hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes,there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding,qPCR,fluorescence in situ hybridization,and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods,a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine. View Publication

Understanding the molecular mechanisms that underlie neurodegenerative disorders has been hampered by a lack of readily available model systems that replicate the complexity of the human disease. Recent advances in stem cell technology have facilitated the derivation of patient-specific stem cells from a variety of differentiated cell types. These induced pluripotent stem cells (iPSCs) are attractive disease models since they can be grown and differentiated to produce large numbers of disease-relevant cell types. However,most iPSC lines are derived in advance of,and without the benefit of,neuropathological confirmation of the donor - the gold standard for many disease classifications and measurement of disease severity. While others have reported the generation of autopsy-confirmed iPSC lines from patient explants,these methods require outgrowth of cadaver tissue,which require additional time and is often only successul 50% of the time. Here we report the rapid generation of autopsy-confirmed iPSC lines from peripheral blood mononuclear cells (PBMCs) drawn postmortem. Since this approach doesn't require the propagation of previously frozen cadaver tissue,iPSC can be rapidly and efficiently produced from patients with autopsy-confirmed pathology. These matched iPSC-derived patient-specific neurons and postmortem brain tissue will support studies of specific mechanisms that drive the pathogenesis of neurodegenerative diseases. View Publication

The influence of GABA receptor agonists on the terminal differentiation in vitro of dopaminergic (DA) neurons derived from IPS cells was investigated. GABA-A agonist muscimol induced transient elevation of intracellular Ca(2+) level ([Ca(2+)] i ) in the investigated cells at days 5 to 21 of differentiation. Differentiation of cells in the presence of muscimol reduced tyrosine hydroxylase expression. Thus,the presence of active GABA-A receptors,associated with phenotype determination via Ca(2+)-signalling was demonstrated in differentiating human DA neurons. View Publication

The $\beta$-haemoglobinopathies,such as sickle cell disease and $\beta$-thalassaemia,are caused by mutations in the $\beta$-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure $\beta$-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably,we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90{\%} targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that,after differentiation into erythrocytes,express adult $\beta$-globin (HbA) messenger RNA,which confirms intact transcriptional regulation of edited HBB alleles. Collectively,these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for $\beta$-haemoglobinopathies. View Publication

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease,including congenital birth defects during pregnancy(1). To develop candidate therapeutic agents against ZIKV,we isolated a panel of human monoclonal antibodies (mAbs) from subjects with prior ZIKV infection. A subset of mAbs recognized diverse epitopes on the envelope (E) protein and exhibited potently neutralizing activity. One of the most inhibitory mAbs,ZIKV-117,broadly neutralized infection of ZIKV strains corresponding to African,Asian,and American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. mAb treatment markedly reduced tissue pathology,placental and fetal infection,and mortality in mice. Thus,neutralizing human mAbs can protect against maternal-fetal transmission,infection and disease,and reveal important determinants for structure-based rational vaccine design efforts. View Publication

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are strongly associated with familial Parkinson's disease (PD). High expression levels in immune cells suggest a role of LRRK2 in regulating the immune system. In this study,we investigated the effect of the LRRK2 (G2019S) mutation in monocytes,using a human stem cell-derived model expressing LRRK2 at endogenous levels. We discovered alterations in the differentiation pattern of LRRK2 mutant,compared to non-mutant isogenic controls,leading to accelerated monocyte production and a reduction in the non-classical CD14+CD16+ monocyte subpopulation in the LRRK2 mutant cells. LPS-treatment of the iPSC-derived monocytes significantly increased the release of pro-inflammatory cytokines,demonstrating a functional response without revealing any significant differences between the genotypes. Assessment of the migrational capacity of the differentiated monocytes revealed moderate deficits in LRRK2 mutant cells,compared to their respective controls. Our findings indicate a pivotal role of LRRK2 in hematopoietic fate decision,endorsing the involvement of the immune system in the development of PD. View Publication

Imprime PGG (Imprime),an intravenously-administered,soluble $\beta$-glucan,has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically,Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells,triggering a coordinated anti-cancer immune response. Herein,using whole blood from healthy human subjects,we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring,anti-$\beta$ glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement,primarily via the classical complement pathway,and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa,eliciting phenotypic activation of,and enhanced chemokine production by,neutrophils and monocytes,enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly,these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together,these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy. View Publication

IL-25,a new member of the IL-17 cytokine family,is involved in type 2 immunity initiation and has been associated with the pathogenesis of rheumatoid arthritis (RA). However,its exact role remains unclear. Here,we aimed to analyse IL-25 expression in the serum and synovial fluid of RA patients and evaluated the correlations between serum IL-25 levels,clinical and laboratory values and inflammation cytokines. Additionally,we investigated whether IL-25 can suppress Th1/Th17 responses involved in RA pathogenesis. We further determined whether IL-25 can alleviate collagen-induced arthritis (CIA) development in mice and the underlying mechanisms using in vitro and in vivo experiments. Our results showed that IL-25 was upregulated in the serum and synovial fluid of RA patients. Increased serum IL-25 levels were associated with disease severity and inflammatory response in RA patients. Furthermore,IL-25 inhibited CD4(+) T-cell activation and differentiation into Th17 cells,without affecting Th1 cells in human RA and CIA models. Administration of IL-25 could attenuate CIA development by Th17 suppression in an IL-13-dependent manner. Our findings indicate that IL-25 plays a potent immunosuppressive role in the pathogenesis of RA and CIA by downregulating Th17 cell response,and thus,may be a potential therapeutic agent for RA. View Publication

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However,most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5),a known tumour suppressor and growth arrest-related lncRNA,is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal,but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis,we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore,we propose that the above pluripotency factors,GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific,our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms. View Publication

Human induced pluripotent stem cell (hiPSC) utility is limited by variations in the ability of these cells to undergo lineage-specific differentiation. We have undertaken a transcriptional comparison of human embryonic stem cell (hESC) lines and hiPSC lines and have shown that hiPSCs are inferior in their ability to undergo neuroectodermal differentiation. Among the differentially expressed candidates between hESCs and hiPSCs,we identified a mitochondrial protein,CHCHD2,whose expression seems to correlate with neuroectodermal differentiation potential of pluripotent stem cells. We provide evidence that hiPSC variability with respect to CHCHD2 expression and differentiation potential is caused by clonal variation during the reprogramming process and that CHCHD2 primes neuroectodermal differentiation of hESCs and hiPSCs by binding and sequestering SMAD4 to the mitochondria,resulting in suppression of the activity of the TGFβ signaling pathway. Using CHCHD2 as a marker for assessing and comparing the hiPSC clonal and/or line differentiation potential provides a tool for large scale differentiation and hiPSC banking studies. View Publication

Chromatin regulation is critical for differentiation and disease. However,features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches,we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably,we found that the chromatin environment of Ewing sarcoma,a mesenchymally derived tumor,is shared with primary mesenchymal stem cells (MSCs). Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements,a feature associated with differentiation and oncogenesis. View Publication

A novel cardiomimetic biohybrid material,termed as the human ventricular cardiac anisotropic sheet (hvCAS) is reported. Well-characterized human pluripotent stem-cell-derived ventricular cardiomyocytes are strategically aligned to reproduce key electrophysiological features of native human ventricle,which,along with specific selection criteria,allows for a direct visualization of arrhythmic spiral re-entry and represents a revolutionary tool to assess preclinical drug-induced arrhythmogenicity. View Publication