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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

Abnormal glucose metabolism and enhanced oxidative stress accelerate cardiovascular disease,a chronic inflammatory condition causing high morbidity and mortality. Here,we report that in monocytes and macrophages of patients with atherosclerotic coronary artery disease (CAD),overutilization of glucose promotes excessive and prolonged production of the cytokines IL-6 and IL-1β,driving systemic and tissue inflammation. In patient-derived monocytes and macrophages,increased glucose uptake and glycolytic flux fuel the generation of mitochondrial reactive oxygen species,which in turn promote dimerization of the glycolytic enzyme pyruvate kinase M2 (PKM2) and enable its nuclear translocation. Nuclear PKM2 functions as a protein kinase that phosphorylates the transcription factor STAT3,thus boosting IL-6 and IL-1β production. Reducing glycolysis,scavenging superoxide and enforcing PKM2 tetramerization correct the proinflammatory phenotype of CAD macrophages. In essence,PKM2 serves a previously unidentified role as a molecular integrator of metabolic dysfunction,oxidative stress and tissue inflammation and represents a novel therapeutic target in cardiovascular disease. View Publication

Background & Aims Hepatocytes differentiated from human embryonic stem cells (hESCs) have the potential to overcome the shortage of primary hepatocytes for clinical use and drug development. Many strategies for this process have been reported,but the functionality of the resulting cells is incomplete. We hypothesize that the functionality of hPSC-derived hepatocytes might be improved by making the differentiation method more similar to normal in vivo hepatic development. Methods We tested combinations of growth factors and small molecules targeting candidate signaling pathways culled from the literature to identify optimal conditions for differentiation of hESCs to hepatocytes,using qRT-PCR for stage-specific markers to identify the best conditions. Immunocytochemistry was then used to validate the selected conditions. Finally,induction of expression of metabolic enzymes in terminally differentiated cells was used to assess the functionality of the hESC-derived hepatocytes. Results Optimal differentiation of hESCs was attained using a 5-stage protocol. After initial induction of definitive endoderm (stage 1),we showed that inhibition of the WNT/??-catenin pathway during the 2nd and 3rd stages of differentiation was required to specify first posterior foregut,and then hepatic gut cells. In contrast,during the 4th stage of differentiation,we found that activation of the WNT/??-catenin pathway allowed generation of proliferative bipotent hepatoblasts,which then were efficiently differentiated into hepatocytes in the 5th stage by dual inhibition of TGF-?? and NOTCH signaling. Conclusion Here,we show that stage-specific regulation of the WNT/??-catenin pathway results in improved differentiation of hESCs to functional hepatocytes. View Publication

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes,respectively. Using patient-derived induced pluripotent stem cells (iPSC),we corrected the dysferlin nonsense mutation c.5713CtextgreaterT; p.R1905X and the most common alpha-sarcoglycan mutation,missense c.229CtextgreaterT; p.R77C,by single-stranded oligonucleotide-mediated gene editing,using the CRISPR/Cas9 gene editing system to enhance the frequency of homology-directed repair. We demonstrated seamless,allele-specific correction at efficiencies of 0.7-1.5%. As an alternative,we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22,using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination,and DICE also utilized site-specific recombinases. With DICE and THRIP,we obtained targeting efficiencies after selection of ˜20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization,as shown by immunoblot and immunocytochemistry. In summary,we demonstrate for the first time precise correction of LGMD iPSC and validation of expression,opening the possibility of cell therapy utilizing these corrected iPSC.Molecular Therapy (2016); doi:10.1038/mt.2016.40. View Publication

Ab-mediated lymphoablation is commonly used in solid organ and hematopoietic cell transplantation. However,these strategies fail to control pathogenic memory T cells efficiently and to improve long-term transplant outcomes significantly. Understanding the mechanisms of T cell reconstitution is critical for enhancing the efficacy of Ab-mediated depletion in sensitized recipients. Using a murine analog of anti-thymocyte globulin (mATG) in a mouse model of cardiac transplantation,we previously showed that peritransplant lymphocyte depletion induces rapid memory T cell proliferation and only modestly prolongs allograft survival. We now report that T cell repertoire following depletion is dominated by memory CD4 T cells. Additional depletion of these residual CD4 T cells severely impairs the recovery of memory CD8 T cells after mATG treatment. The CD4 T cell help during CD8 T cell recovery depends on the presence of B cells expressing CD40 and intact CD40/CD154 interactions. The requirement for CD4 T cell help is not limited to the use of mATG in heart allograft recipients,and it is observed in nontransplanted mice and after CD8 T cell depletion with mAb instead of mATG. Most importantly,limiting helper signals increases the efficacy of mATG in controlling memory T cell expansion and significantly extends heart allograft survival in sensitized recipients. Our findings uncover the novel role for helper memory CD4 T cells during homeostatic CD8 T cell proliferation and open new avenues for optimizing lymphoablative therapies in allosensitized patients. View Publication

Encapsulation of donor islets using a hydrogel material is a well-studied strategy for islet transplantation,which protects donor islets from the host immune response. Replacement of donor islets by human embryonic stem cell (hESC) derived islets will also require a means of immune-isolating hESCs by encapsulation. However,a critical consideration of hESC differentiation is the effect of surrounding biophysical environment,in this case capsule biophysical properties,on differentiation. The objective of this study,thus,was to evaluate the effect of capsule properties on growth,viability,and differentiation of encapsulated hESCs throughout pancreatic induction. It was observed that even in the presence of soluble chemical cues for pancreatic induction,substrate properties can significantly modulate pancreatic differentiation,hence necessitating careful tuning of capsule properties. Capsules in the range of 4-7. kPa supported cell growth and viability,whereas capsules of higher stiffness suppressed cell growth. While an increase in capsule stiffness enhanced differentiation at the intermediate definitive endoderm (DE) stage,increased stiffness strongly suppressed pancreatic progenitor (PP) induction. Signaling pathway analysis indicated an increase in pSMAD/pAKT levels with substrate stiffness likely the cause of enhancement of DE differentiation. In contrast,sonic hedgehog inhibition was more efficient under softer gel conditions,which is necessary for successful PP differentiation. Statement of Significance: Cell replacement therapy for type 1 diabetes (T1D),affecting millions of people worldwide,requires the immunoisolation of insulin-producing islets by encapsulation with a semi-impermeable material. Due to the shortage of donor islets,human pluripotent stem cell (hPSC) derived islets are an attractive alternative. However,properties of the encapsulating substrate are known to influence hPSC cell fate. In this work,we determine the effect of substrate stiffness on growth and pancreatic fate of encapsulated hPSCs. We precisely identify the range of substrate properties conducive for pancreatic cell fate,and also the mechanism by which substrate properties modify the cell signaling pathways and hence cell fate. Such information will be critical in driving regenerative cell therapy for long term treatment of T1D. View Publication

Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells,however,the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase,a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues. View Publication

Bone is the most common site for metastasis in patients with solid tumours. Bisphosphonates are an effective treatment for preventing skeletal related events and preserving quality of life in these patients. Zoledronic acid (ZA) is the most potent osteoclast inhibitor and is licensed for the treatment of bone metastases. Clodronate and pamidronate are also licensed for this indication. In addition,ZA has been demonstrated to exhibit antitumour effect. Direct and indirect mechanisms of anti-tumour effect have been postulated and at many times proven. Evidence exists that ZA antitumour effect is mediated through inhibition of tumour cells proliferation,induction of apoptosis,synergistic/additive to inhibitory effect of cytotoxic agents,inhibition of angiogenesis,decrease tumour cells adhesion to bone,decrease tumour cells invasion and migration,disorganization of cell cytoskeleton and activation of specific cellular antitumour immune response. There is also clinical evidence from clinical trials that ZA improved long term survival outcome in cancer patients with and without bone metastases. In this review we highlight the preclinical and clinical studies investigating the antitumour effect of bisphosphonates with particular reference to ZA. View Publication

Although hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) are considered a promising model for predicting hepatotoxicity,their application has been restricted because of the low activity of drug metabolizing enzymes (DMEs). Here we found that the low expression of xenobiotic receptors (constitutive androstane receptor,CAR; and pregnane X receptor,PXR) contributes to the low activity of DMEs in hPSC-HLCs. Most CAR- and PXR-regulated DMEs and transporters were transcriptionally down-regulated in hPSC-HLC. Transcriptional expression of CAR and PXR was highly repressed in hPSC-HLCs,whereas mRNA levels of aryl hydrocarbon receptor (AHR) were comparable to those of adult liver. Furthermore,ligand-induced transcriptional activation was observed only at AHR in hPSC-HLCs. Bisulfite sequencing analysis demonstrated that promoter hypermethylation of CAR and PXR was associated with diminished transcriptional activity in hPSC-HLCs. Treatment with AHR-selective ligands increased the transcription of AHR-dependent target genes by direct AHR-DNA binding at the xenobiotic response element. In addition,an antagonist of AHR significantly inhibited AHR-dependent target gene expression. Thus,AHR may function intrinsically as a xenosensor as well as a ligand-dependent transcription factor in hPSC-HLCs. Our results indicate that hPSC-HLCs can be used to screen toxic substances related to AHR signaling and to identify potential AHR-targeted therapeutics. View Publication

Immune recognition of pathogen-associated ligands leads to assembly and activation of inflammasomes,resulting in the secretion of inflammatory cytokines IL-1β and IL-18 and an inflammatory cell death called pyroptosis. Inflammasomes are important for protection against many pathogens,but their role during chronic infectious disease is poorly understood. Pseudomonas aeruginosa is an opportunistic pathogen that persists in the lungs of cystic fibrosis (CF) patients and may be responsible for the repeated episodes of pulmonary exacerbation characteristic of CF. P. aeruginosa is capable of inducing potent inflammasome activation during acute infection. We hypothesized that to persist within the host during chronic infection,P. aeruginosa must evade inflammasome activation,and pulmonary exacerbations may be the result of restoration of inflammasome activation. We therefore isolated P. aeruginosa from chronically infected CF patients during stable infection and exacerbation and evaluated the impact of these isolates on inflammasome activation in macrophages and neutrophils. P. aeruginosa isolates from CF patients failed to induce inflammasome activation,as measured by the secretion of IL-1β and IL-18 and by pyroptotic cell death,during both stable infection and exacerbation. Inflammasome evasion likely was due to reduced expression of inflammasome ligands and reduced motility and was not observed in environmental isolates or isolates from acute,non-CF infection. These results reveal a novel mechanism of pathogen adaptation by P. aeruginosa to avoid detection by inflammasomes in CF patients and indicate that P. aeruginosa-activated inflammasomes are not involved in CF pulmonary exacerbations. View Publication

The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types,namely human umbilical cord vein endothelial cells (HUVECs),endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs),to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However,only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance. View Publication