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The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However,the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis,the non-canonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a two-day window showed significantly enhanced osteogenic yield. Mechanistically,rWNT5a supplementation up-regulated PKC,CamKII and JNK activity while antagonizing the key effector of canonical Wnt signaling: beta-catenin. Conversely,when recombinant WNT3a (rWNT3a) or other positive regulators of �?�-catenin were employed during this same time-window there was a decrease in osteogenic marker expression. However,if rWNT3a was supplemented during a time-window following rWNT5a treatment,osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine. View Publication

Coronary arteriogenesis is a central step in cardiogenesis,requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present,it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro,and contribute extensively to coronary-like vessels in vivo,forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates,and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo. View Publication

Defining immunological mechanisms underlying NK cell biology is crucial for the treatment and prevention of immune deficiency and malignancy. The limited availability of human biological specimens presents a challenge to the study of human immunobiology. The use of high throughput,multi-parametric assays will not only aid in the definition and diagnosis of complex human immune disorders affecting NK cell function but also advance NK cell biology through population-based assessment of molecular signaling. In an effort to garner the most information from limited numbers of human cells,we designed a quantitative method to study NK cell function using imaging flow cytometry (IFC),which combines multiparametric flow cytometry and fluorescence microscopy. Specifically,we developed IFC as a tool to measure polarization and secretion of lytic granules at the immunological synapse formed between an NK cell and a susceptible target. We have further validated our approach through quantitative comparison with high-resolution confocal microscopy. We show that IFC can be used as a quantitative,high throughput measure of NK cell biological function possessing greater dimensionality than standard flow cytometry. View Publication

Insulin resistance,a critical component of type 2 diabetes (T2D),precedes and predicts T2D onset. T2D is also associated with mitochondrial dysfunction. To define the cause-effect relationship between insulin resistance and mitochondrial dysfunction,we compared mitochondrial metabolism in induced pluripotent stem cells (iPSC) from 5 healthy individuals and 4 patients with genetic insulin resistance due to insulin receptor mutations. Insulin-resistant iPSC had increased mitochondrial number and decreased mitochondrial size. Mitochondrial oxidative function was impaired,with decreased citrate synthase activity and spare respiratory capacity. Simultaneously,expression of multiple glycolytic enzymes was decreased,while lactate production increased 80%. These perturbations were accompanied by an increase in ADP/ATP ratio and 3-fold increase in AMPK activity,indicating energetic stress. Insulin-resistant iPSC also showed reduced catalase activity and increased susceptibility to oxidative stress. Thus,insulin resistance can lead to mitochondrial dysfunction with reduced mitochondrial size,oxidative activity,and energy production. View Publication

Conventional generation of stem cells from human blastocysts produces a developmentally advanced,or primed,stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However,whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here,we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration,global gene expression,and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. View Publication

The human naive pluripotent stem cell (PSC) state,corresponding to a pre-implantation stage of development,has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate,a normal karyotype,the ability to form teratomas,transcriptional similarities to human pre-implantation embryos,reduced heterochromatin levels,and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP-/- cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state,with implications for early human embryology. View Publication

Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However,precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs,and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase,which blocks entry into mitosis by phosphorylating CDK1 at Y15,was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal,but not ectodermal,lineages. Functionally,WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775. View Publication

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays,as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here,we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders,such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs,yielding electrophysiologically active neurons within just 3 wk. Using this platform,we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus,this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types. View Publication

BACKGROUND: Dystrophin-deficient cardiomyopathy is a growing clinical problem without targeted treatments. We investigated whether nicorandil promotes cardioprotection in human dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes and the muscular dystrophy mdx mouse heart. METHODS AND RESULTS: Dystrophin-deficient iPSC-derived cardiomyocytes had decreased levels of endothelial nitric oxide synthase and neuronal nitric oxide synthase. The dystrophin-deficient cardiomyocytes had increased cell injury and death after 2 hours of stress and recovery. This was associated with increased levels of reactive oxygen species and dissipation of the mitochondrial membrane potential. Nicorandil pretreatment was able to abolish these stress-induced changes through a mechanism that involved the nitric oxide-cyclic guanosine monophosphate pathway and mitochondrial adenosine triphosphate-sensitive potassium channels. The increased reactive oxygen species levels in the dystrophin-deficient cardiomyocytes were associated with diminished expression of select antioxidant genes and increased activity of xanthine oxidase. Furthermore,nicorandil was found to improve the restoration of cardiac function after ischemia and reperfusion in the isolated mdx mouse heart. CONCLUSION: Nicorandil protects against stress-induced cell death in dystrophin-deficient cardiomyocytes and preserves cardiac function in the mdx mouse heart subjected to ischemia and reperfusion injury. This suggests a potential therapeutic role for nicorandil in dystrophin-deficient cardiomyopathy. View Publication

Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However,natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that,on conjugation with CTL,human melanoma cells undergo an active late endosome/lysosome trafficking,which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking,pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance,we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. View Publication

Genome instability is a potential limitation to the research and therapeutic application of induced pluripotent stem cells (iPSCs). Observed genomic variations reflect the combined activities of DNA damage,cellular DNA damage response (DDR),and selection pressure in culture. To understand the contribution of DDR on the distribution of copy number variations (CNVs) in iPSCs,we mapped CNVs of iPSCs with mutations in the central DDR gene ATM onto genome organization landscapes defined by genome-wide replication timing profiles. We show that following reprogramming the early and late replicating genome is differentially affected by CNVs in ATM deficient iPSCs relative to wild type iPSCs. Specifically,the early replicating regions had increased CNV losses during retroviral reprogramming. This differential CNV distribution was not present after later passage or after episomal reprogramming. Comparison of different reprogramming methods in the setting of defective DNA damage response reveals unique vulnerability of early replicating open chromatin to retroviral vectors. View Publication

Human urine cells (HUCs) can be reprogrammed into neural progenitor cells (NPCs) or induced pluripotent stem cells (iPSCs) with defined factors and a small molecule cocktail,but the underlying fate choice remains unresolved. Here,through sequential removal of individual compound from small molecule cocktail,we showed that A8301,a TGF$$ signaling inhibitor,is sufficient to switch the cell fate from iPSCs into NPCs in OSKM-mediated HUCs reprogramming. However,TGF$$ exposure at early stage inhibits HUCs reprogramming by promoting EMT. Base on these data,we developed an optimized approach for generation of NPCs or iPSCs from HUCs with significantly improved efficiency by regulating TGF$$ activity at different reprogramming stages. This approach provides a simplified and improved way for HUCs reprogramming,thus would be valuable for banking human iPSCs or NPCs from people with different genetic background. View Publication