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Differentiation of pluripotent cells into endoderm-related cell types initially requires in vitro gastrulation into the definitive endoderm (DE). Most differentiation protocols are initiated from colonies of pluripotent cells complicating their adaption due to insufficiently defined starting conditions. The protocol described here was initiated from a defined cell number of dispersed single cells and tested on three different human embryonic stem cell lines and one human induced pluripotent stem cell line. Combined activation of ActivinA/Nodal signaling and GSK3 inhibition for the first 24 h,followed by ActivinA/Nodal signaling efficiently induced the DE state. Activation of ActivinA/Nodal signaling alone was not effective. Efficient GSK3 inhibition allowed the reduction of the ActivinA concentration during the entire protocol. A feeder-independent cultivation of pluripotent cells was preferred to achieve the high efficiency and robustness since feeder cells hindered the differentiation process. Additionally,inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway was not required,nonetheless yielding high cell numbers efficiently committed toward the DE. Finally,the endoderm generated could be differentiated further into PDX1-positive pan-pancreatic cells and NGN3-positive endocrine progenitors. Thus,this efficient and robust DE differentiation protocol is a step forward toward better reproducibility due to the well-defined conditions based on dispersed single cells from feeder-free-cultivated human pluripotent cells. View Publication

Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders,and /`a disease of synapses/' is the major hypothesis for the biological basis of schizophrenia. Although this hypothesis has gained indirect support from human post-mortem brain analyses and genetic studies,little is known about the pathophysiology of synapses in patient neurons and how susceptibility genes for mental disorders could lead to synaptic deficits in humans. Genetics of most psychiatric disorders are extremely complex due to multiple susceptibility variants with low penetrance and variable phenotypes. Rare,multiply affected,large families in which a single genetic locus is probably responsible for conferring susceptibility have proven invaluable for the study of complex disorders. Here we generated induced pluripotent stem (iPS) cells from four members of a family in which a frameshift mutation of disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders and we further produced different isogenic iPS cell lines via gene editing. We showed that mutant DISC1 causes synaptic vesicle release deficits in iPS-cell-derived forebrain neurons. Mutant DISC1 depletes wild-type DISC1 protein and,furthermore,dysregulates expression of many genes related to synapses and psychiatric disorders in human forebrain neurons. Our study reveals that a psychiatric disorder relevant mutation causes synapse deficits and transcriptional dysregulation in human neurons and our findings provide new insight into the molecular and synaptic etiopathology of psychiatric disorders. View Publication

The complement activation product,C5a,is a pivotal member of the innate immune response; however,a diverse number of nonimmune functions are now being ascribed to C5a signaling,including roles during embryonic development. Here,we identify the expression of the C5a precursor protein,C5,as well as the C5a receptors,C5aR and C5L2,in both human embryonic stem cells and human-induced pluripotent stem cells. We show that administration of a physiologically relevant dose of purified human C5a (1 nM) stimulates activation of ERK1/2 and AKT signaling pathways,and is able to promote maintenance of the pluripotent state in the absence of FGF2. C5a also reduced cell loss following dissociation of human pluripotent stem cells. Our results reveal that complement C5a signaling supports human stem cell pluripotency and survival,and thus may play a key role in shaping early human embryonic development. Stem Cells 2014;32:3278-3284. View Publication

Alopecia areata (AA) is a common autoimmune disease resulting from damage of the hair follicle by T cells. The immune pathways required for autoreactive T cell activation in AA are not defined limiting clinical development of rational targeted therapies. Genome-wide association studies (GWAS) implicated ligands for the NKG2D receptor (product of the KLRK1 gene) in disease pathogenesis. Here,we show that cytotoxic CD8(+)NKG2D(+) T cells are both necessary and sufficient for the induction of AA in mouse models of disease. Global transcriptional profiling of mouse and human AA skin revealed gene expression signatures indicative of cytotoxic T cell infiltration,an interferon-γ (IFN-γ) response and upregulation of several γ-chain (γc) cytokines known to promote the activation and survival of IFN-γ-producing CD8(+)NKG2D(+) effector T cells. Therapeutically,antibody-mediated blockade of IFN-γ,interleukin-2 (IL-2) or interleukin-15 receptor β (IL-15Rβ) prevented disease development,reducing the accumulation of CD8(+)NKG2D(+) T cells in the skin and the dermal IFN response in a mouse model of AA. Systemically administered pharmacological inhibitors of Janus kinase (JAK) family protein tyrosine kinases,downstream effectors of the IFN-γ and γc cytokine receptors,eliminated the IFN signature and prevented the development of AA,while topical administration promoted hair regrowth and reversed established disease. Notably,three patients treated with oral ruxolitinib,an inhibitor of JAK1 and JAK2,achieved near-complete hair regrowth within 5 months of treatment,suggesting the potential clinical utility of JAK inhibition in human AA. View Publication

Improvement of methods to produce endoderm-derived cells from pluripotent stem cells is important to realize high-efficient induction of endodermal tissues such as pancreas and hepatocyte. Difficulties hampering such efforts include the low efficiency of definitive endoderm cell induction and establishing appropriate defined culture conditions to ensure a safe cell source for human transplantation. Based on previous studies,we revised the experimental condition of definitive endoderm induction in feeder- and serum-free culture. Our results suggested that CHIR99021 is more effective than Wnt3A ligand in feeder- and serum-free conditions. In addition,keeping cell density low during endoderm induction is important for the efficiency. On the other hand,we showed that overtreatment with CHIR99021 converted the cells into BRACHYURY-expressing posterior mesoderm cells rather than endoderm,indicating strict CHIR99021 treatment requirements for endoderm differentiation. Nevertheless,these results should enable better control in the production of definitive endoderm-derived cells. View Publication

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries,with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC,but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still,despite marked tumour shrinkage,the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D),herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function,autophagy and lysosome activity,as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly,surviving cells show high sensitivity to oxidative phosphorylation inhibitors,which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer. View Publication

Coxsackie virus and adenovirus receptor,CXADR (CAR),is present during embryogenesis and is involved in tissue regeneration,cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells,from the cleavage stage up to the precursor epiblast,and corresponded with the presence of soluble CXADR3/7 splice variant. CAR was displayed on the membrane,involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells,and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts,CAR was reduced in the membrane and concentrated in the nucleus,which correlated with the switch in RNA expression to the CXADR4/7 and CXADR2/7 splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus,corresponding with the transmembrane CXADR and CXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage,the expression of CXADR was downregulated. We concluded that CXADR is differentially expressed during human preimplantation development. We described various CAR expressions: i) soluble CXADR marking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types,such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated. View Publication

The ontogeny of human Langerhans cells (LCs) remains poorly characterized,in particular the nature of LC precursors and the factors that may drive LC differentiation. Here we report that thymic stromal lymphopoietin (TSLP),a keratinocyte-derived cytokine involved in epithelial inflammation,cooperates with transforming growth factor (TGF)-β for the generation of LCs. We show that primary human blood BDCA-1(+),but not BDCA-3(+),dendritic cells (DCs) stimulated with TSLP and TGF-β harbor a typical CD1a(+)Langerin(+) LC phenotype. Electron microscopy established the presence of Birbeck granules,an intracellular organelle specific to LCs. LC differentiation was not observed from tonsil BDCA-1(+) and BDCA-3(+) subsets. TSLP + TGF-β LCs had a mature phenotype with high surface levels of CD80,CD86,and CD40. They induced a potent CD4(+) T-helper (Th) cell expansion and differentiation into Th2 cells with increased production of tumor necrosis factor-α and interleukin-6 compared with CD34-derived LCs. Our findings establish a novel LC differentiation pathway from BDCA-1(+) blood DCs with potential implications in epithelial inflammation. Therapeutic targeting of TSLP may interfere with tissue LC repopulation from circulating precursors. View Publication

Differentiation of neural lineages from human pluripotent stem cells (hPSCs) raises the hope of generating functional cells for the treatment of neural diseases. However,current protocols for differentiating hPSCs into neural lineages remain inefficient and largely variable between different hPSC lines. We report that microRNA 376c (miR-376c) significantly enhanced neural differentiation of hPSCs in a defined condition by suppressing SMAD4,the co-SMAD for TGF-β signaling. Downstream,SMAD4 directly bound and suppressed PAX6,the critical neural lineage specification factor. Interestingly,we also found that SMAD4 binds and suppresses miR-376c clusters in undifferentiated hESCs. In summary,our findings revealed a reciprocal antagonism between miR-376c and SMAD signaling that regulates cell fate during human neural differentiation.-Liu,J.,Wang,L.,Su,Z.,Wu,W.,Cai,X.,Li,D.,Hou,J.,Pei,D.,Pan,G. A reciprocal antagonism between miR-376c and TGF-β signaling regulates neural differentiation of hPSCs. View Publication

Nature Neuroscience 17,1180 (2014). doi:10.1038/nn.3787 View Publication

BACKGROUND Acute respiratory distress syndrome (ARDS) is characterized by overwhelming inflammatory responses and lung remodeling. We hypothesized that leukocyte infiltration during the inflammatory response modulates epithelial remodeling through a mechanism of epithelial-mesenchymal transition (EMT). METHODS Human lung epithelial cells were treated for 30 min with hydrochloric acid (HCl). Human monocytes were then cocultured with the epithelial cells for up to 48 h,in the presence or absence of blocking peptides against lymphocyte function-associated antigen-1 (LFA-1),or tyrphostin A9,a specific inhibitor for platelet-derived growth factor (PDGF) receptor tyrosine kinase. RESULTS Exposure of lung epithelial cells to HCl resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and production of interleukin (IL)-8 at 24 h. The expression of the epithelial markers E-cadherin decreased while the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) increased at 24 h and remained high at 48 h. The addition of monocytes augmented the profiles of lower expression of epithelial markers and higher mesenchymal markers accompanied by increased collagen deposition. This EMT profile was associated with an enhanced production of IL-8 and PDGF. Treatment of the lung epithelial cells with the LAF-1 blocking peptides CD11a237-246 or/and CD18112-122 suppressed monocyte adhesion,production of IL-8,PDGF and hydroxyproline as well as EMT markers. Treatment with tyrphostin A9 prevented the EMT profile shift induced by HCl stimulation. CONCLUSIONS The interaction between epithelial cells and monocytes enhanced epithelial remodelling after initial injury through EMT signalling that is associated with the release of soluble mediators,including IL-8 and PDGF. View Publication

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency,and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3,H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers,such as OCT4 (POU5F1),TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors. View Publication