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Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a mitochondrial disorder characterized by myoclonus epilepsy,generalized seizures,ataxia and myopathy. MERRF syndrome is primarily due to an A to G mutation at mtDNA 8344 that disrupts the mitochondrial gene for tRNA(Lys). However,the detailed mechanism by which this tRNA(Lys) mutation causes mitochondrial dysfunction in cardiomyocytes or neurons remains unclear. In this study,we generated human induced pluripotent stem cells (hiPSCs) that carry the A8344G genetic mutation from patients with MERRF syndrome. Compared with mutation-free isogenic hiPSCs,MERRF-specific hiPSCs (MERRF-hiPSCs) exhibited reduced oxygen consumption,elevated reactive oxygen species (ROS) production,reduced growth,and fragmented mitochondrial morphology. We sought to investigate the induction ability and mitochondrial function of cardiomyocyte-like cells differentiated from MERRF-hiPSCs. Our data demonstrate that that cardiomyocyte-like cells (MERRF-CMs) or neural progenitor cells (MERRF-NPCs) differentiated from MERRF-iPSCs also exhibited increased ROS levels and altered antioxidant gene expression. Furthermore,MERRF-CMs or -NPCs contained fragmented mitochondria,as evidenced by MitoTracker Red staining and transmission electron microscopy. Taken together,these findings showed that MERRF-hiPSCs and MERRF-CM or -NPC harboring the A8344G genetic mutation displayed contained mitochondria with an abnormal ultrastructure,produced increased ROS levels,and expressed upregulated antioxidant genes. View Publication

Silver nanoparticles (AgNPs) are used extensively as anti-microbial agents in various products,but little is known about their potential neurotoxic effects. In this study,we used glutamatergic neurons derived from human embryonic stem cells as a cellular model to study 20nm citrate-coated AgNPs (AgSCs) and Polyvinylpyrrolidone-coated AgNPs (AgSPs) induced neurotoxicity. AgSCs significantly damaged neurite outgrowths; increased the production of reactive oxygen species and Ca(2+) influxes; reduced the expression of MAP2,PSD95,vGlut1 and NMDA receptor proteins at concentrations as low as 0.1μg/ml. In contrast,AgSPs exhibited neurotoxicity only at higher concentration. Furthermore,our results showed that AgSCs induced glutamate excitotoxicity by the activation of calmodulin and the induction of nitric oxide synthase; increased the phosphorylation of glycogen synthase kinase-3 α/β at Tyr(216) and Tau at Ser(396) and reduced the expression of Tau46,which are typically observed in Alzheimer's disease. This study indicated that stem cells can provide an excellent platform for studying nanoparticle induced neurotoxicity. 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

Targeting of myeloid-dendritic cell receptor DC-SIGN by numerous chronic infectious agents,including Porphyromonas gingivalis,is shown to drive-differentiation of monocytes into dysfunctional mDCs. These mDCs exhibit alterations of their fine-tuned homeostatic function and contribute to dysregulated immune-responses. Here,we utilize P. gingivalis mutant strains to show that pathogen-differentiated mDCs from primary human-monocytes display anti-apoptotic profile,exhibited by elevated phosphorylated-Foxo1,phosphorylated-Akt1,and decreased Bim-expression. This results in an overall inhibition of DC-apoptosis. Direct stimulation of complex component CD40 on DCs leads to activation of Akt1,suggesting CD40 involvement in anti-apoptotic effects observed. Further,these DCs drove dampened CD8(+) T-cell and Th1/Th17 effector-responses while inducing CD25(+)Foxp3(+)CD127(-) Tregs. In vitro Treg induction was mediated by DC expression of indoleamine 2,3-dioxygenase,and was confirmed in IDO-KO mouse model. Pathogen-infected &CMFDA-labeled MoDCs long-lasting survival was confirmed in a huMoDC reconstituted humanized mice. In conclusion,our data implicate PDDCs as an important target for resolution of chronic infection. View Publication

The transcription factor NF-kappaB has anti-apoptotic properties and may confer chemoresistance to cancer cells. Here,we describe human pancreatic carcinoma cell lines that differ in the responsiveness to the topoisomerase-2 inhibitors VP16 (20 microM) and doxorubicin (0.3 microM): Highly sensitive T3M4 [corrected] and PT45-P1 cells,and Capan-1 and A818-4 cells that were almost resistant to both anti cancer drugs. VP16,but not doxorubicin,transiently induced NF-kappaB activity in all cell lines,whereas basal NF-kappaB binding was nearly undetectable in T3M4 [corrected] and PT45-P1 cells,but rather high in Capan-1 and A818-4 cells,as demonstrated by gel-shift and luciferase assays. Treatment with various NF-kappaB inhibitors (Gliotoxin,MG132 and Sulfasalazine),or transfection with the IkappaBalpha super-repressor,strongly enhanced the apoptotic effects of VP16 or doxorubicin on resistant Capan-1 and 818-4 cells. Our results indicate that under certain conditions the resistance of pancreatic carcinoma cells to chemotherapy is due to their constitutive NF-kappaB activity rather than the transient induction of NF-kappaB by some anti-cancer drugs. Blockade of basal NF-kappaB activity by well established drugs efficiently reduces chemoresistance of pancreatic cancer cells and offers the potential for improved therapeutic strategies. View Publication

Human induced pluripotent stem (iPS) cells potentially provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms and treatments. However,existing approaches to cardiomyocyte production from human iPS cells are inefficient,limiting the application of iPS cells in basic and translational cardiac research. Furthermore,strategies to accurately record changes in iPS cell-derived cardiomyocyte action potential duration (APD) are needed to monitor APD-related cardiac disease and for rapid drug screening. We examined whether modulation of the bone morphogenetic protein 4 (BMP-4) and Wnt/β-catenin signaling pathways could induce efficient cardiac differentiation of human iPS cells. We found that early treatment of human iPS cells with BMP-4 followed by late treatment with small molecule Wnt inhibitors led to a marked increase in production of cardiomyocytes compared to existing differentiation strategies. Using immunocytochemical staining and real-time intracellular calcium imaging,we showed that these induced cardiomyocytes expressed typical sarcomeric markers,exhibited normal rhythmic Ca(2+) transients,and responded to both β-adrenergic and electric stimulation. Furthermore,human iPS cell-derived cardiomyocytes demonstrated characteristic changes in action potential duration in response to cardioactive drugs procainamide and verapamil using voltage-sensitive dye-based optical recording. Thus,modulation of the BMP-4 and Wnt signaling pathways in human iPS cells leads to highly efficient production of cardiomyocytes with typical electrophysiological function and pharmacologic responsiveness. The use of human iPS cell-derived cardiomyocytes and the application of calcium- and voltage-sensitive dyes for the direct,rapid measurement of iPS cell-derived cardiomyocyte activity promise to offer attractive platforms for studying cardiac disease mechanisms and therapeutics. View Publication

Embryonic and induced pluripotent stem cells (IPSCs) derived from mammalian species are valuable tools for modeling human disease,including retinal degenerative eye diseases that result in visual loss. Restoration of vision has focused on transplantation of neural progenitor cells (NPCs) and retinal pigmented epithelium (RPE) to the retina. Here we used transgenic common marmoset (Callithrix jacchus) and human pluripotent stem cells carrying the enhanced green fluorescent protein (eGFP) reporter as a model system for retinal differentiation. Using suspension and subsequent adherent differentiation cultures,we observed spontaneous in vitro differentiation that included NPCs and cells with pigment granules characteristic of differentiated RPE. Retinal cells derived from human and common marmoset pluripotent stem cells provide potentially unlimited cell sources for testing safety and immune compatibility following autologous or allogeneic transplantation using nonhuman primates in early translational applications. View Publication

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or,alternatively,induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects,we focused on SNON (SKIL),a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast,enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus,our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment. View Publication

INTRODUCTION: The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming,however,represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA),an excisable lentiviral reprogramming vector,provides,in our hands,the most consistent reprogramming approach that addresses this safety concern. Nevertheless,most viral integrations occur in genes,and exactly how the integration,epigenetic reprogramming,and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. METHODS: In this study,we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry,karyotyping and teratoma formation,and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. RESULTS: We found that a STEMCCA-derived iPSC line that contains a single integration,found to be located in an intronic location in an actively transcribed gene,PRPF39,displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs,differentiated them into multiple clinically relevant cell types (including oligodendrocytes,hepatocytes,and cardiomyocytes),and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. CONCLUSION: For the first time,these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and,in light of the limited availability of current good manufacturing practice cellular manufacturing facilities,highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics. View Publication

Gain of 20q11.21 is a chromosomal abnormality that is recurrently found in human pluripotent stem cells and cancers,strongly suggesting that this mutation confers a proliferative or survival advantage to these cells. In this work we studied three human embryonic stem cell (hESC) lines that acquired a gain of 20q11.21 during in vitro culture. The study of the mRNA gene expression levels of the loci located in the common region of duplication showed that HM13,ID1,BCL2L1,KIF3B and the immature form of the micro-RNA miR-1825 were up-regulated in mutant cells. ID1 and BCL2L1 were further studied as potential drivers of the phenotype of hESC with a 20q11.21 gain. We found no increase in the protein levels of ID1,nor the downstream effects expected from over-expression of this gene. On the other hand,hESC with a gain of 20q11.21 had on average a 3-fold increase of Bcl-xL (the anti-apoptotic isoform of BCL2L1) protein levels. The mutant hESC underwent 2- to 3-fold less apoptosis upon loss of cell-to-cell contact and were ∼2-fold more efficient in forming colonies from a single cell. The key role of BCL2L1 in this mutation was further confirmed by transgenic over-expression of BCL2L1 in the wild-type cells,leading to apoptosis-resistant cells,and BCL2L1-knock-down in the mutant hESC,resulting in a restoration of the wild-type phenotype. This resistance to apoptosis supposes a significant advantage for the mutant cells,explaining the high frequency of gains of 20q11.21 in human pluripotent stem cells. View Publication

Astrocytes are instrumental to major brain functions,including metabolic support,extracellular ion regulation,the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental,psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes),we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP,S100$\$,NFIA and ALDH1L1. In addition,mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion,the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy. View Publication