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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

Although promising new radiation therapy techniques such as hadrontherapy are currently being evaluated in the treatment of head and neck malignancies,local control of head and neck squamous cell carcinoma (HNSCC) remains low. Here,we investigated the involvement of cancer stem-like cells (CSCs) in a radioresistant HNSCC cell line (SQ20B). Stem-like cells SQ20B/SidePopulation(SP)/CD44(+)/ALDH(high) were more resistant to both photon and carbon ion irradiation compared with non-CSCs. This was confirmed by a BrdU labeling experiment,which suggests that CSCs were able to proliferate and to induce tumorigenicity after irradiation. SQ20B/SP/CD44(+)/ALDH(high) were capable of an extended G2/M arrest phase in response to photon or carbon ion irradiation compared with non-CSCs. Moreover,our data strongly suggest that resistance of CSCs may result from an imbalance between exacerbated self-renewal and proliferative capacities and the decrease in apoptotic cell death triggering. In order to modulate these processes,two targeted pharmacological strategies were tested. Firstly,UCN-01,a checkpoint kinase (Chk1) inhibitor,induced the relapse of G2/M arrest and radiosensitization of SQ20B-CSCs. Secondly,all-trans retinoic acid (ATRA) resulted in an inhibition of ALDH activity,and induction of the differentiation and radiosensitization of SQ20B/SP/CD44(+)/ALDH(high) cells. The combination of ATRA and UCN-01 treatments with irradiation drastically decreased the surviving fraction at 2Gy of SQ20B-CSCs from 0.85 to 0.38 after photon irradiation,and from 0.45 to 0.21 in response to carbon ions. Taken together,our results suggest that the combination of UCN-01 and ATRA represent a promising pharmacological-targeted strategy that significantly sensitizes CSCs to photon or carbon ion radiation. 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

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs),but little is known about SSEA-5 expression in other primitive tissues (e.g.,human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors,cell lines,teratomas,and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors,such as embryonal carcinoma. In tumor cell lines,SSEA-5 was highly immunoreactive in Capan-1 cells,while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs,with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use. View Publication

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies,we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system,which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor,yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures,revealing that the reaction engenders both MBC subsets with different immune effector function and,ultimately,LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity,for vaccine development,and for understanding long-term pathogen resistance. View Publication

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

It has been proposed that the ancestral fungus was mating competent and homothallic. However,many mating-competent fungi were initially classified as asexual because their mating capacity was hidden behind layers of regulation. For efficient in vitro mating,the essentially obligate diploid ascomycete pathogen Candida albicans has to change its mating type locus from heterozygous MTL a /α to homozygous MTL a / a or MTL α/α and then undergo an environmentally controlled epigenetic switch to the mating-competent opaque form. These requirements greatly reduce the potential for C. albicans mating. Deletion of the Yci1 domain gene OFR1 bypasses the need for C. albicans cells to change the mating type locus from heterozygous to homozygous prior to switching to the opaque form and mating and allows homothallic mating of MTL heterozygous strains. This bypass is carbon source dependent and does not occur when cells are grown on glucose. Transcriptional profiling of ofr1 mutant cells shows that in addition to regulating cell type and mating circuitry,Ofr1 is needed for proper regulation of histone and chitin biosynthesis gene expression. It appears that OFR1 is a key regulator in C. albicans and functions in part to maintain the cryptic mating phenotype of the pathogen. View Publication

Netrin-1 is a neuronal guidance cue that regulates cellular activation,migration,and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed within tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However,the ability of Netrin-1 to interact with lymphocytes and its in-depth effects on leukocyte migration are poorly understood. In this study,we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4(+) T cells. Neogenin,uncoordinated-5 (UNC5)A,and UNC5B were expressed at low levels in unstimulated cells,but they increased following mitogen-dependent activation. By immunofluorescence,we observed a cytoplasmic staining pattern of neogenin and UNC5A/B that also increased following activation. Using a novel microfluidic assay,we found that Netrin-1 stimulated bidirectional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4(+) T cells,but it had no demonstrable effects on the migration of purified CD4(+)CD25(+)CD127(dim) T regulatory cells. Furthermore,using a short hairpin RNA knockdown approach,we observed that the promigratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse,local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3(+) T cell infiltrates. Neogenin was also observed on CD3(+) T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively,our findings demonstrate that Netrin-1/neogenin interactions augment CD4(+) T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo. View Publication

An attractive target for therapeutic intervention is constitutively activated,mutant FLT3,which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing,and which is currently in late-stage clinical trials. However,the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel,structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here,we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487,which selectively targets mutant FLT3 protein kinase activity,is also shown to override PKC412 resistance in vitro,and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally,the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus,we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target,and which could potentially be used to override drug resistance in AML. View Publication

During persistent murine cytomegalovirus (MCMV) infection,the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells,which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation,MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead,we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported,at least in part,by memory T cells primed early in infection,as well as by recruitment of naive T cells at late times. Thus,these data show that memory inflation is maintained by a continuous replacement of short-lived,functional cells during chronic MCMV infection. View Publication

BACKGROUND Valproic acid (VPA),a commonly used mood stabilizer that promotes neuronal differentiation,regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK) and glycogen synthase kinase3beta (GSK3beta). However,the mechanism by which VPA promotes differentiation is not understood. RESULTS We report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF)-treated embryonic day 14 (E14) rat cerebral cortex neural progenitor cells (NPCs). The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21Cip/WAF1 pathway. These effects,however,are not mediated by the pathway involving the epidermal growth factor receptor (EGFR) but via the pathway which stabilizes Ras through beta-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos. CONCLUSION We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects,as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos. View Publication