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BACKGROUND Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage,differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally,current differentiation strategies are hampered by inefficiency,lack of reproducibility,and use of animal-derived products. METHODS To direct the differentiation of hESCs to endothelial subtypes,H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3),basic fibroblast growth factor (bFGF),bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation,the endothelial progenitor cells (CD34(+)CD31(+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore,the safety and functionality of these cells upon in vivo transplantation were characterized. RESULTS Sequential modulation of hESCs with GSK-3 inhibitor,bFGF,BMP4 and VEGF resulted in stages reminiscent of primitive streak,early mesoderm/lateral plate mesoderm,and endothelial progenitors under feeder- and serum-free conditions. Furthermore,these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically,the endothelial progenitors differentiated to venous ECs in the absence of VEGF,and to arterial phenotype under low concentrations of VEGF. Additionally,these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore,these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse. CONCLUSIONS We report a simple,rapid,and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery,disease modeling and regenerative medicine in the future. View Publication

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells,there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression,we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype,including haematopoietic and stromal cells as well as a neuronal niche. Collectively,our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues. View Publication

BACKGROUND: Many retinal degenerative diseases are caused by the loss of retinal ganglion cells (RGCs). Autosomal dominant optic atrophy is the most common hereditary optic atrophy disease and is characterized by central vision loss and degeneration of RGCs. Currently,there is no effective treatment for this group of diseases. However,stem cell therapy holds great potential for replacing lost RGCs of patients. Compared with embryonic stem cells,induced pluripotent stem cells (iPSCs) can be derived from adult somatic cells,and they are associated with fewer ethical concerns and are less prone to immune rejection. In addition,patient-derived iPSCs may provide us with a cellular model for studying the pathogenesis and potential therapeutic agents for optic atrophy.backslashnbackslashnMETHODS: In this study,iPSCs were obtained from patients carrying an OPA1 mutation (OPA1 (+/-) -iPSC) that were diagnosed with optic atrophy. These iPSCs were differentiated into putative RGCs,which were subsequently characterized by using RGC-specific expression markers BRN3a and ISLET-1.backslashnbackslashnRESULTS: Mutant OPA1 (+/-) -iPSCs exhibited significantly more apoptosis and were unable to efficiently differentiate into RGCs. However,with the addition of neural induction medium,Noggin,or estrogen,OPA1 (+/-) -iPSC differentiation into RGCs was promoted.backslashnbackslashnCONCLUSIONS: Our results suggest that apoptosis mediated by OPA1 mutations plays an important role in the pathogenesis of optic atrophy,and both noggin and β-estrogen may represent potential therapeutic agents for OPA1-related optic atrophy. View Publication

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons,but little has been done to characterize these at cellular resolution. In particular,it is unclear to what extent in vitro two-dimensional,relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally,93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And,68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly,a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However,this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers,although effective,may not be able to disambiguate cortical layer identity in all cells. View Publication

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known,in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1,a large gene with at least 38 exons,and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1,as well as a neural-specific TAF1 isoform,N-TAF1,which showed decreased expression in post-mortem XDP brain compared with control tissue. Here,we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model,we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells,XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36,a region spanning the SVA insertion site. N-TAF1,which incorporates an alternative exon (exon 34'),was not expressed in fibroblasts,but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP,but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration. View Publication

A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function,the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore,under this stimulatory condition,CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10,which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation,which correlates with lower CD86 expression compared to patients with chronic rejection. Hence,the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production. View Publication

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL),HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and,in turn,is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis,we found that in CLL cells,HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma,reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models,and prolongs survival in mice. Of interest,we found that in CLL cells,HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore,HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes,including CXCR4,thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis. View Publication

Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1β,a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis,we performed metabolic and functional studies on human alveolar macrophages,human monocyte-derived macrophages,and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1β and decreased transcription of PTGS2,increased levels of anti-inflammatory IL-10,and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival,demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1β. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies. View Publication

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently,the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed,respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last,screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together,our results highlight the potential of hiPSCs for studying human tumourigenesis. 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

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

Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis,the animal model for MS,myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby,the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently,B cells were found to participate in the pathogenesis of CNS autoimmunity,with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood-brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore,myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue. View Publication