1 3839 149 IPSC-DERIVED NEURAL PRECURSOR CELLS: POTENTIAL FOR CELL TRANSPLANTATION THERAPY IN SPINAL CORD INJURY. A NUMBER OF STUDIES HAVE DEMONSTRATED THAT TRANSPLANTATION OF NEURAL PRECURSOR CELLS (NPCS) PROMOTES FUNCTIONAL RECOVERY AFTER SPINAL CORD INJURY (SCI). HOWEVER, THE NPCS HAD BEEN MOSTLY HARVESTED FROM EMBRYONIC STEM CELLS OR FETAL TISSUE, RAISING THE ETHICAL CONCERN. YAMANAKA AND HIS COLLEAGUES ESTABLISHED INDUCED PLURIPOTENT STEM CELLS (IPSCS) WHICH COULD BE GENERATED FROM SOMATIC CELLS, AND THIS INNOVATIVE DEVELOPMENT HAS MADE RAPID PROGRESSION IN THE FIELD OF SCI REGENERATION. WE AND OTHER GROUPS SUCCEEDED IN PRODUCING NPCS FROM IPSCS, AND DEMONSTRATED BENEFICIAL EFFECTS AFTER TRANSPLANTATION FOR ANIMAL MODELS OF SCI. IN PARTICULAR, EFFICACY OF HUMAN IPSC-NPCS IN NON-HUMAN PRIMATE SCI MODELS FOSTERED MOMENTUM OF CLINICAL APPLICATION FOR SCI PATIENTS. AT THE SAME TIME, HOWEVER, ARTIFICIAL INDUCTION METHODS IN IPSC TECHNOLOGY CREATED ALTERNATIVE ISSUES INCLUDING GENETIC AND EPIGENETIC ABNORMALITIES, AND TUMORIGENICITY AFTER TRANSPLANTATION. TO OVERCOME THESE PROBLEMS, IT IS CRITICALLY IMPORTANT TO SELECT ORIGINS OF SOMATIC CELLS, USE INTEGRATION-FREE SYSTEM DURING TRANSFECTION OF REPROGRAMMING FACTORS, AND THOROUGHLY INVESTIGATE THE CHARACTERISTICS OF IPSC-NPCS WITH RESPECT TO QUALITY MANAGEMENT. MOREOVER, SINCE MOST OF THE PREVIOUS STUDIES HAVE FOCUSED ON SUBACUTE PHASE OF SCI, ESTABLISHMENT OF EFFECTIVE NPC TRANSPLANTATION SHOULD BE EVALUATED FOR CHRONIC PHASE HEREAFTER. OUR GROUP IS CURRENTLY PREPARING CLINICAL-GRADE HUMAN IPSC-NPCS, AND WILL MOVE FORWARD TOWARD CLINICAL STUDY FOR SUBACUTE SCI PATIENTS SOON IN THE NEAR FUTURE. 2018 2 893 30 CHRONIC ETHANOL EXPOSURE AND WITHDRAWAL IMPAIR SYNAPTIC GABA(A) RECEPTOR-MEDIATED NEUROTRANSMISSION IN DEEP-LAYER PREFRONTAL CORTEX. BACKGROUND: THE PREFRONTAL CORTEX (PFC) ACTS AS AN INTEGRATIVE HUB FOR THE PROCESSING OF CORTICAL AND SUBCORTICAL INPUT INTO MEANINGFUL EFFERENT SIGNALING, PERMITTING COMPLEX ASSOCIATIVE BEHAVIORS. PFC DYSFUNCTION IS CONSISTENTLY OBSERVED WITH ETHANOL (ETOH) DEPENDENCE AND IS A CORE COMPONENT OF THE PATHOLOGY OF ALCOHOL USE DISORDERS IN CURRENT MODELS OF ADDICTION. WHILE INTRACORTICAL GAMMA-AMINOBUTRYRIC ACID (GABA)ERGIC NEUROTRANSMISSION IS UNDERSTOOD TO BE ESSENTIAL FOR MAINTAINING COORDINATED NETWORK ACTIVITY WITHIN THE CORTEX, RELATIVELY LITTLE IS KNOWN REGARDING FUNCTIONAL GABAERGIC ADAPTATIONS IN PFC DURING ETOH DEPENDENCE. METHODS: IN THE PRESENT STUDY, MALE AND FEMALE (> POSTNATAL DAY 60) SPRAGUE-DAWLEY RATS WERE ADMINISTERED ETOH (5.0 G/KG; INTRAGASTRIC GAVAGE) FOR 14 TO 15 CONSECUTIVE DAYS. TWENTY-FOUR HOURS AFTER THE FINAL ADMINISTRATION, ANIMALS WERE SACRIFICED AND BRAINS EXTRACTED FOR ELECTROPHYSIOLOGICAL RECORDINGS OF ISOLATED GABA(A) RECEPTOR-MEDIATED CURRENTS OR ANALYSIS OF GABA(A) RECEPTOR SUBUNIT PROTEIN EXPRESSION IN DEEP-LAYER PFC NEURONS. RESULTS: CHRONIC ETOH EXPOSURE SIGNIFICANTLY ATTENUATED ACTIVITY-DEPENDENT SPONTANEOUS GABA(A) RECEPTOR-MEDIATED INHIBITORY POSTSYNAPTIC CURRENT (IPSC) FREQUENCY WITH NO EFFECT ON AMPLITUDE. FURTHERMORE, ANALYSIS OF IPSC DECAY KINETICS REVEALED A SIGNIFICANT ENHANCEMENT OF IPSC DECAY TIME THAT WAS ASSOCIATED WITH DECREMENTS IN EXPRESSION OF THE ALPHA1 GABA(A) RECEPTOR SUBUNIT, INDICATIVE OF FURTHER IMPAIRED PHASIC INHIBITION. THESE PHENOMENA OCCURRED IRRESPECTIVE OF NEURON PROJECTION DESTINATION AND SEX. BASED ON PREVIOUS OBSERVATIONS BY OUR LABORATORY OF AN EPIGENETIC MECHANISM FOR ETOH-INDUCED CHANGES IN CORTICAL GABA(A) RECEPTOR SUBUNIT EXPRESSION, THE HISTONE DEACETYLASE INHIBITOR TRICHOSTATIN A WAS ADMINISTERED TO WATER- AND ETOH-EXPOSED ANIMALS, AND PREVENTED ETOH-INDUCED CHANGES IN SPONTANEOUS IPSC FREQUENCY, IPSC DECAY KINETICS, AND GABA(A) RECEPTOR SUBUNIT EXPRESSION. CONCLUSIONS: TAKEN TOGETHER, THESE RESULTS DEMONSTRATE THAT CHRONIC ETOH EXPOSURE IMPAIRS SYNAPTIC INHIBITORY NEUROTRANSMISSION IN DEEP-LAYER PYRAMIDAL NEURONS OF THE MEDIAL PFC IN BOTH MALE AND FEMALE RATS. THESE MALADAPTATIONS OCCUR IN NEURONS PROJECTING TO NUMEROUS REGIONS IMPLICATED IN THE SEQUELAE OF ETOH DEPENDENCE, OFFERING A MECHANISTIC LINK BETWEEN THE MANIFESTATION OF PFC DYSFUNCTION AND NEGATIVE AFFECTIVE STATES OBSERVED WITH EXTENDED CONSUMPTION. 2019 3 89 41 A PILOT INVESTIGATION OF DIFFERENTIAL HYDROXYMETHYLATION LEVELS IN PATIENT-DERIVED NEURAL STEM CELLS IMPLICATES ALTERED CORTICAL DEVELOPMENT IN BIPOLAR DISORDER. INTRODUCTION: BIPOLAR DISORDER (BD) IS A CHRONIC MENTAL ILLNESS CHARACTERIZED BY RECURRENT EPISODES OF MANIA AND DEPRESSION AND ASSOCIATED WITH SOCIAL AND COGNITIVE DISTURBANCES. ENVIRONMENTAL FACTORS, SUCH AS MATERNAL SMOKING AND CHILDHOOD TRAUMA, ARE BELIEVED TO MODULATE RISK GENOTYPES AND CONTRIBUTE TO THE PATHOGENESIS OF BD, SUGGESTING A KEY ROLE IN EPIGENETIC REGULATION DURING NEURODEVELOPMENT. 5-HYDROXYMETHYLCYTOSINE (5HMC) IS AN EPIGENETIC VARIANT OF PARTICULAR INTEREST, AS IT IS HIGHLY EXPRESSED IN THE BRAIN AND IS IMPLICATED IN NEURODEVELOPMENT, AND PSYCHIATRIC AND NEUROLOGICAL DISORDERS. METHODS: INDUCED PLURIPOTENT STEM CELLS (IPSCS) WERE GENERATED FROM THE WHITE BLOOD CELLS OF TWO ADOLESCENT PATIENTS WITH BIPOLAR DISORDER AND THEIR SAME-SEX AGE-MATCHED UNAFFECTED SIBLINGS (N = 4). FURTHER, IPSCS WERE DIFFERENTIATED INTO NEURONAL STEM CELLS (NSCS) AND CHARACTERIZED FOR PURITY USING IMMUNO-FLUORESCENCE. WE USED REDUCED REPRESENTATION HYDROXYMETHYLATION PROFILING (RRHP) TO PERFORM GENOME-WIDE 5HMC PROFILING OF IPSCS AND NSCS, TO MODEL 5HMC CHANGES DURING NEURONAL DIFFERENTIATION AND ASSESS THEIR IMPACT ON BD RISK. FUNCTIONAL ANNOTATION AND ENRICHMENT TESTING OF GENES HARBORING DIFFERENTIATED 5HMC LOCI WERE PERFORMED WITH THE ONLINE TOOL DAVID. RESULTS: APPROXIMATELY 2 MILLION SITES WERE MAPPED AND QUANTIFIED, WITH THE MAJORITY (68.8%) LOCATED IN GENIC REGIONS, WITH ELEVATED 5HMC LEVELS PER SITE OBSERVED FOR 3' UTRS, EXONS, AND 2-KB SHORELINES OF CPG ISLANDS. PAIRED T-TESTS OF NORMALIZED 5HMC COUNTS BETWEEN IPSC AND NSC CELL LINES REVEALED GLOBAL HYPO-HYDROXYMETHYLATION IN NSCS AND ENRICHMENT OF DIFFERENTIALLY HYDROXYMETHYLATED SITES WITHIN GENES ASSOCIATED WITH PLASMA MEMBRANE (FDR = 9.1 X 10(-12)) AND AXON GUIDANCE (FDR = 2.1 X 10(-6)), AMONG OTHER NEURONAL PROCESSES. THE MOST SIGNIFICANT DIFFERENCE WAS OBSERVED FOR A TRANSCRIPTION FACTOR BINDING SITE FOR THE KCNK9 GENE (P = 8.8 X 10(-6)), ENCODING A POTASSIUM CHANNEL PROTEIN INVOLVED IN NEURONAL ACTIVITY AND MIGRATION. PROTEIN-PROTEIN-INTERACTION (PPI) NETWORKING SHOWED SIGNIFICANT CONNECTIVITY (P = 3.2 X 10(-10)) BETWEEN PROTEINS ENCODED BY GENES HARBORING HIGHLY DIFFERENTIATED 5HMC SITES, WITH GENES INVOLVED IN AXON GUIDANCE AND ION TRANSMEMBRANE TRANSPORT FORMING DISTINCT SUB-CLUSTERS. COMPARISON OF NSCS OF BD CASES AND UNAFFECTED SIBLINGS REVEALED ADDITIONAL PATTERNS OF DIFFERENTIATION IN HYDROXYMETHYLATION LEVELS, INCLUDING SITES IN GENES WITH FUNCTIONS RELATED TO SYNAPSE FORMATION AND REGULATION, SUCH AS CUX2 (P = 2.4 X 10(-5)) AND DOK-7 (P = 3.6 X 10(-3)), AS WELL AS AN ENRICHMENT OF GENES INVOLVED IN THE EXTRACELLULAR MATRIX (FDR = 1.0 X 10(-8)). DISCUSSION: TOGETHER, THESE PRELIMINARY RESULTS LEND EVIDENCE TOWARD A POTENTIAL ROLE FOR 5HMC IN BOTH EARLY NEURONAL DIFFERENTIATION AND BD RISK, WITH VALIDATION AND MORE COMPREHENSIVE CHARACTERIZATION TO BE ACHIEVED THROUGH FOLLOW-UP STUDY. 2023 4 3380 39 HIV-1 INFECTION OF GENETICALLY ENGINEERED IPSC-DERIVED CENTRAL NERVOUS SYSTEM-ENGRAFTED MICROGLIA IN A HUMANIZED MOUSE MODEL. THE CENTRAL NERVOUS SYSTEM (CNS) IS A MAJOR HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 RESERVOIR. MICROGLIA ARE THE PRIMARY TARGET CELL OF HIV-1 INFECTION IN THE CNS. CURRENT MODELS HAVE NOT ALLOWED THE PRECISE MOLECULAR PATHWAYS OF ACUTE AND CHRONIC CNS MICROGLIAL INFECTION TO BE TESTED WITH IN VIVO GENETIC METHODS. HERE, WE DESCRIBE A NOVEL HUMANIZED MOUSE MODEL UTILIZING HUMAN-INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO XENOGRAFT INTO MURINE HOSTS. THESE MICE ARE ADDITIONALLY ENGRAFTED WITH HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS THAT SERVED AS A MEDIUM TO ESTABLISH A PERIPHERAL INFECTION THAT THEN SPREAD TO THE CNS MICROGLIA XENOGRAFT, MODELING A TRANS-BLOOD-BRAIN BARRIER ROUTE OF ACUTE CNS HIV-1 INFECTION WITH HUMAN TARGET CELLS. THE APPROACH IS COMPATIBLE WITH IPSC GENETIC ENGINEERING, INCLUDING INSERTING TARGETED TRANSGENIC REPORTER CASSETTES TO TRACK THE XENOGRAFTED HUMAN CELLS, ENABLING THE TESTING OF NOVEL TREATMENT AND VIRAL TRACKING STRATEGIES IN A COMPARATIVELY SIMPLE AND COST-EFFECTIVE WAY VIVO MODEL FOR NEUROHIV. IMPORTANCE: OUR MOUSE MODEL IS A POWERFUL TOOL FOR INVESTIGATING THE GENETIC MECHANISMS GOVERNING CNS HIV-1 INFECTION AND LATENCY IN THE CNS AT A SINGLE-CELL LEVEL. A MAJOR ADVANTAGE OF OUR MODEL IS THAT IT USES IPSC-DERIVED MICROGLIA, WHICH ENABLES HUMAN GENETICS, INCLUDING GENE FUNCTION AND THERAPEUTIC GENE MANIPULATION, TO BE EXPLORED IN VIVO , WHICH IS MORE CHALLENGING TO STUDY WITH CURRENT HEMATOPOIETIC STEM CELL-BASED MODELS FOR NEUROHIV. OUR TRANSGENIC TRACING OF XENOGRAFTED HUMAN CELLS WILL PROVIDE A QUANTITATIVE MEDIUM TO DEVELOP NEW MOLECULAR AND EPIGENETIC STRATEGIES FOR REDUCING THE HIV-1 LATENT RESERVOIR AND TO TEST THE IMPACT OF THERAPEUTIC INFLAMMATION-TARGETING DRUG INTERVENTIONS ON CNS HIV-1 LATENCY. 2023 5 1359 31 DEVELOPMENT REFRACTORINESS OF MLL-REARRANGED HUMAN B CELL ACUTE LEUKEMIAS TO REPROGRAMMING INTO PLURIPOTENCY. INDUCED PLURIPOTENT STEM CELLS (IPSCS) ARE A POWERFUL TOOL FOR DISEASE MODELING. THEY ARE ROUTINELY GENERATED FROM HEALTHY DONORS AND PATIENTS FROM MULTIPLE CELL TYPES AT DIFFERENT DEVELOPMENTAL STAGES. HOWEVER, REPROGRAMMING LEUKEMIAS IS AN EXTREMELY INEFFICIENT PROCESS. FEW STUDIES GENERATED IPSCS FROM PRIMARY CHRONIC MYELOID LEUKEMIAS, BUT IPSC GENERATION FROM ACUTE MYELOID OR LYMPHOID LEUKEMIAS (ALL) HAS NOT BEEN ACHIEVED. WE ATTEMPTED TO GENERATE IPSCS FROM DIFFERENT SUBTYPES OF B-ALL TO ADDRESS THE DEVELOPMENTAL IMPACT OF LEUKEMIC FUSION GENES. OKSM(L)-EXPRESSING MONO/POLYCISTRONIC-, RETROVIRAL/LENTIVIRAL/EPISOMAL-, AND SENDAI VIRUS VECTOR-BASED REPROGRAMMING STRATEGIES FAILED TO RENDER IPSCS IN VITRO AND IN VIVO. ADDITION OF TRANSCRIPTOMIC-EPIGENETIC REPROGRAMMING "BOOSTERS" ALSO FAILED TO GENERATE IPSCS FROM B CELL BLASTS AND B-ALL LINES, AND WHEN IPSCS EMERGED THEY LACKED LEUKEMIC FUSION GENES, DEMONSTRATING NON-LEUKEMIC MYELOID ORIGIN. CONVERSELY, MLL-AF4-OVEREXPRESSING HEMATOPOIETIC STEM CELLS/B PROGENITORS WERE SUCCESSFULLY REPROGRAMMED, INDICATING THAT B CELL ORIGIN AND LEUKEMIC FUSION GENE WERE NOT REPROGRAMMING BARRIERS. GLOBAL TRANSCRIPTOME/DNA METHYLOME PROFILING SUGGESTED A DEVELOPMENTAL/DIFFERENTIATION REFRACTORINESS OF MLL-REARRANGED B-ALL TO REPROGRAMMING INTO PLURIPOTENCY. 2016 6 5870 42 SUSTAINED KNOCKDOWN OF A DISEASE-CAUSING GENE IN PATIENT-SPECIFIC INDUCED PLURIPOTENT STEM CELLS USING LENTIVIRAL VECTOR-BASED GENE THERAPY. PATIENT-SPECIFIC INDUCED PLURIPOTENT STEM CELLS (IPSCS) HOLD GREAT PROMISE FOR STUDIES ON DISEASE-RELATED DEVELOPMENTAL PROCESSES AND MAY SERVE AS AN AUTOLOGOUS CELL SOURCE FOR FUTURE TREATMENT OF MANY HEREDITARY DISEASES. NEW GENETIC ENGINEERING TOOLS SUCH AS ZINC FINGER NUCLEASES AND TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE ALLOW TARGETED CORRECTION OF MONOGENETIC DISORDERS BUT ARE VERY CUMBERSOME TO ESTABLISH. AIMING AT STUDIES ON THE KNOCKDOWN OF A DISEASE-CAUSING GENE, LENTIVIRAL VECTOR-MEDIATED EXPRESSION OF SHORT HAIRPIN RNAS (SHRNAS) IS A VALUABLE OPTION, BUT IT IS LIMITED BY SILENCING OF THE KNOCKDOWN CONSTRUCT UPON EPIGENETIC REMODELING DURING DIFFERENTIATION. HERE, WE PROPOSE AN APPROACH FOR THE EXPRESSION OF A THERAPEUTIC SHRNA IN DISEASE-SPECIFIC IPSCS USING THIRD-GENERATION LENTIVIRAL VECTORS. TARGETING SEVERE ALPHA-1-ANTITRYPSIN (A1AT) DEFICIENCY, WE OVEREXPRESSED A HUMAN MICRORNA 30 (MIR30)-STYLED SHRNA DIRECTED AGAINST THE PIZ VARIANT OF A1AT, WHICH IS KNOWN TO CAUSE CHRONIC LIVER DAMAGE IN AFFECTED PATIENTS. THIS KNOCKDOWN CASSETTE IS TRACEABLE FROM CLONAL IPSC LINES TO DIFFERENTIATED HEPATIC PROGENY VIA AN ENHANCED GREEN FLUORESCENCE PROTEIN REPORTER EXPRESSED FROM THE SAME RNA-POLYMERASE II PROMOTER. IMPORTANTLY, THE CYTOMEGALOVIRUS I/E ENHANCER CHICKEN BETA ACTIN (CAG) PROMOTER-DRIVEN EXPRESSION OF THIS CONSTRUCT IS SUSTAINED WITHOUT TRANSGENE SILENCING DURING HEPATIC DIFFERENTIATION IN VITRO AND IN VIVO. AT LOW LENTIVIRAL COPY NUMBERS PER GENOME WE CONFIRMED A FUNCTIONAL RELEVANT REDUCTION (-66%) OF INTRACELLULAR PIZ PROTEIN IN HEPATIC CELLS AFTER DIFFERENTIATION OF PATIENT-SPECIFIC IPSCS. IN CONCLUSION, WE HAVE DEMONSTRATED THAT LENTIVIRAL VECTOR-MEDIATED EXPRESSION OF SHRNAS CAN BE EFFICIENTLY USED TO KNOCK DOWN AND FUNCTIONALLY EVALUATE DISEASE-RELATED GENES IN PATIENT-SPECIFIC IPSCS. 2013 7 454 83 APPLICATIONS OF INDUCED PLURIPOTENT STEM CELL TECHNOLOGIES IN SPINAL CORD INJURY. NUMEROUS BASIC RESEARCH STUDIES HAVE SUGGESTED THE POTENTIAL EFFICACY OF NEURAL PRECURSOR CELL (NPC) TRANSPLANTATION IN SPINAL CORD INJURY (SCI). HOWEVER, IN MOST SUCH STUDIES, THE ORIGIN OF THE CELLS USED WAS MAINLY FETAL TISSUE OR EMBRYONIC STEM CELLS, BOTH OF WHICH CARRY POTENTIAL ETHICAL CONCERNS WITH RESPECT TO CLINICAL USE. THE DEVELOPMENT OF INDUCED PLURIPOTENT STEM CELLS (IPSCS) OPENED A NEW PATH TOWARD REGENERATIVE MEDICINE FOR SCI. IPSCS CAN BE GENERATED FROM SOMATIC CELLS BY INDUCTION OF TRANSCRIPTION FACTORS, AND INDUCED TO DIFFERENTIATE INTO NPCS WITH CHARACTERISTICS OF CELLS OF THE CENTRAL NERVOUS SYSTEM. THE BENEFICIAL EFFECT OF IPSC-DERIVED NPC TRANSPLANTATION HAS BEEN REPORTED FROM OUR GROUP AND OTHERS WORKING IN RODENT AND NON-HUMAN PRIMATE MODELS. THESE PROMISING RESULTS FACILITATE THE APPLICATION OF IPSCS FOR CLINICAL APPLICATIONS IN SCI PATIENTS. HOWEVER, IPSCS ALSO HAVE ISSUES, SUCH AS GENETIC/EPIGENETIC ABNORMALITIES AND TUMORIGENESIS BECAUSE OF THE ARTIFICIAL INDUCTION METHOD, THAT MUST BE ADDRESSED PRIOR TO CLINICAL USE. THE SELECTION OF SOMATIC CELLS, GENERATION OF INTEGRATION-FREE IPSCS, AND CHARACTERIZATION OF DIFFERENTIATED NPCS WITH THOROUGH QUALITY MANAGEMENT ARE ALL NEEDED TO ADDRESS THESE POTENTIAL RISKS. TO ENHANCE THE EFFICACY OF THE TRANSPLANTED IPSC-NPCS, ESPECIALLY AT CHRONIC PHASE OF SCI, ADMINISTRATION OF A CHONDROITINASE OR SEMAPHORIN3A INHIBITOR REPRESENTS A POTENTIALLY IMPORTANT MEANS OF PROMOTING AXONAL REGENERATION THROUGH THE LESION SITE. THE COMBINED USE OF REHABILITATION WITH SUCH CELL THERAPY APPROACHES IS ALSO IMPORTANT, AS REPETITIVE TRAINING ENHANCES NEURITE OUTGROWTH OF TRANSPLANTED CELLS AND STRENGTHENS NEURAL CIRCUITS AT CENTRAL PATTERN GENERATORS. OUR GROUP HAS ALREADY EVALUATED CLINICAL GRADE IPSC-DERIVED NPCS, AND WE LOOK FORWARD TO INITIATING CLINICAL TESTING AS THE NEXT STEP TOWARD DETERMINING WHETHER THIS APPROACH IS SAFE AND EFFECTIVE FOR CLINICAL USE. THIS ARTICLE IS PART OF THE MINI REVIEW SERIES "60TH ANNIVERSARY OF THE JAPANESE SOCIETY FOR NEUROCHEMISTRY". 2017 8 2697 33 EX VIVO MODELS OF CHRONIC GRANULOMATOUS DISEASE. INDUCED PLURIPOTENT STEM CELLS (IPSCS) ARE PLURIPOTENT STEM CELLS THAT CAN BE ESTABLISHED FROM DEDIFFERENTIATION OF ALL SOMATIC CELL TYPES BY EPIGENETIC PHENOMENA. IPSCS CAN BE DIFFERENTIATED INTO ANY MATURE CELLS LIKE NEURONS, HEPATOCYTES, OR PANCREATIC CELLS THAT HAVE NOT BEEN EASILY AVAILABLE TO DATE. THUS, IPSCS ARE WIDELY USED FOR DISEASE MODELING, DRUG DISCOVERY, AND CELL THERAPY DEVELOPMENT. HERE, WE DESCRIBE A PROTOCOL TO OBTAIN HUMAN MATURE AND FUNCTIONAL NEUTROPHILS AND MACROPHAGES AS EX VIVO MODELS OF X-LINKED CHRONIC GRANULOMATOUS DISEASE (X-CGD). THIS METHOD CAN BE APPLIED TO MODEL THE OTHER GENETIC FORMS OF CGD. WE ALSO DESCRIBE METHODS FOR TESTING THE CHARACTERISTICS AND FUNCTIONS OF NEUTROPHILS AND MACROPHAGES BY MORPHOLOGY, PHAGOCYTOSIS ASSAY, RELEASE OF GRANULE MARKERS OR CYTOKINES, CELL SURFACE MARKERS, AND NADPH OXIDASE ACTIVITY. 2019 9 2928 32 GENERATION OF IPSCS FROM CULTURED HUMAN MALIGNANT CELLS. INDUCED PLURIPOTENT STEM CELLS (IPSCS) CAN BE GENERATED FROM VARIOUS DIFFERENTIATED CELL TYPES BY THE EXPRESSION OF A SET OF DEFINED TRANSCRIPTION FACTORS. SO FAR, IPSCS HAVE BEEN GENERATED FROM PRIMARY CELLS, BUT IT IS UNCLEAR WHETHER HUMAN CANCER CELL LINES CAN BE REPROGRAMMED. HERE WE DESCRIBE THE GENERATION AND CHARACTERIZATION OF IPSCS DERIVED FROM HUMAN CHRONIC MYELOID LEUKEMIA CELLS. WE SHOW THAT, DESPITE THE PRESENCE OF ONCOGENIC MUTATIONS, THESE CELLS ACQUIRED PLURIPOTENCY BY THE EXPRESSION OF 4 TRANSCRIPTION FACTORS AND UNDERWENT DIFFERENTIATION INTO CELL TYPES DERIVED OF ALL 3 GERM LAYERS DURING TERATOMA FORMATION. INTERESTINGLY, ALTHOUGH THE PARENTAL CELL LINE WAS STRICTLY DEPENDENT ON CONTINUOUS SIGNALING OF THE BCR-ABL ONCOGENE, ALSO TERMED ONCOGENE ADDICTION, REPROGRAMMED CELLS LOST THIS DEPENDENCY AND BECAME RESISTANT TO THE BCR-ABL INHIBITOR IMATINIB. THIS FINDING INDICATES THAT THE THERAPEUTIC AGENT IMATINIB TARGETS CELLS IN A SPECIFIC EPIGENETIC DIFFERENTIATED CELL STATE, AND THIS MAY CONTRIBUTE TO ITS INABILITY TO FULLY ERADICATE DISEASE IN CHRONIC MYELOID LEUKEMIA PATIENTS. 2010 10 892 36 CHRONIC ETHANOL EXPOSURE ALTERS DNA METHYLATION IN NEURAL STEM CELLS: ROLE OF MOUSE STRAIN AND SEX. PRENATAL ALCOHOL EXPOSURE (PAE) IS CONSIDERED AS A RISK FACTOR FOR THE DEVELOPMENT OF FETAL ALCOHOL SPECTRUM DISORDERS (FASD). EVIDENCE INDICATES THAT PAE AFFECTS EPIGENETIC MECHANISMS (SUCH AS DNA METHYLATION) AND ALTERS THE NORMAL DIFFERENTIATION AND DEVELOPMENT OF NEURAL STEM CELLS (NSC) IN THE FETAL BRAIN. HOWEVER, PAE EFFECTS DEPEND ON SEVERAL FACTORS SUCH AS SEX AND STRAIN OF THE STUDIED SUBJECTS. HERE, WE INVESTIGATED WHETHER MURINE SEX AND STRAIN CONTRIBUTE TO THE EFFECTS OF CHRONIC ETHANOL EXPOSURE ON DNA METHYLATION MACHINERY OF DIFFERENTIATING NSC. FURTHER, THE EFFECTS OF PAE ON GLIAL LINEAGE (INCLUDING BOTH ASTROCYTES AND OLIGODENDROCYTES) IN A SEX- AND STRAIN-DEPENDENT MANNER HAVE NOT BEEN STUDIED YET. TO EXAMINE THE EFFECTS OF CHRONIC ETHANOL EXPOSURE ON GLIOGENESIS, WE EXPOSED DIFFERENTIATING NSC TO GLIO-INDUCTIVE CULTURE CONDITIONS. APPLYING A STANDARD IN VITRO MODEL SYSTEM, WE TREATED MALE AND FEMALE DIFFERENTIATING NSC (OBTAINED FROM THE FOREBRAIN OF CD1 AND C57BL/6 EMBRYOS AT EMBRYONIC DAY 14.5) WITH CHRONIC ETHANOL EXPOSURE (70 MM) FOR 8 DAYS. WE SHOW THAT ETHANOL INDUCES GLOBAL DNA HYPOMETHYLATION, WHILE ALTERING THE EXPRESSION OF DNA METHYLATION-RELATED GENES IN A SEX- AND STRAIN-SPECIFIC MANNER. THE OBSERVED CHANGE IN CELLULAR DNA METHYLATION LEVELS WAS ASSOCIATED WITH ALTERED EXPRESSION OF GLIAL MARKERS CNPASE, GFAP, AND OLIG2 IN CD1 (BUT NOT C57BL/6) CELLS. WE CONCLUDE THAT THE IMPACT OF ETHANOL EFFECT ON DNA METHYLATION IS DEPENDENT ON CELLULAR SEX AND STRAIN. ALSO, ETHANOL IMPACT ON NEURAL STEM CELL FATE COMMITMENT WAS ONLY DETECTED IN CELLS ISOLATED FROM CD1 MOUSE STRAIN, BUT NOT IN C57BL/6 CELLS. THE RESULTS OF THE CURRENT STUDY PROVIDE EVIDENCE THAT SEX AND STRAIN OF RODENTS (C57BL/6 AND CD1) DURING GESTATION ARE IMPORTANT FACTORS, WHICH AFFECT ALCOHOL EFFECTS ON NSC DIFFERENTIATION AND DNA METHYLATION. RESULTS OF THIS STUDY MAY ALSO HELP IN INTERPRETING DATA ON THE DEVELOPMENTAL TOXICITY OF MANY COMPOUNDS DURING THE GESTATIONAL PERIOD. 2020 11 5657 25 SEX-DEPENDENT PRONOCICEPTIVE ROLE OF SPINAL ALPHA(5) -GABA(A) RECEPTOR AND ITS EPIGENETIC REGULATION IN NEUROPATHIC RODENTS. EXTRASYNAPTIC ALPHA(5) -SUBUNIT CONTAINING GABA(A) (ALPHA(5) -GABA(A) ) RECEPTORS PARTICIPATE IN CHRONIC PAIN. PREVIOUSLY, WE REPORTED A SEX DIFFERENCE IN THE ACTION OF ALPHA(5) -GABA(A) RECEPTORS IN DYSFUNCTIONAL PAIN. HOWEVER, THE UNDERLYING MECHANISMS REMAIN UNKNOWN. THE AIM OF THIS STUDY WAS TO EXAMINE THIS SEXUAL DIMORPHISM IN NEUROPATHIC RODENTS AND THE MECHANISMS INVOLVED. FEMALE AND MALE WISTAR RATS OR ICR MICE WERE SUBJECTED TO NERVE INJURY FOLLOWED BY ALPHA(5) -GABA(A) RECEPTOR INVERSE AGONIST INTRATHECAL ADMINISTRATION, L-655,708. THE DRUG PRODUCED AN ANTIALLODYNIC EFFECT IN NERVE-INJURED FEMALE RATS AND MICE, AND A LOWER EFFECT IN MALES. WE HYPOTHESIZED THAT CHANGES IN ALPHA(5) -GABA(A) RECEPTOR, PROBABLY INFLUENCED BY HORMONAL AND EPIGENETIC STATUS, MIGHT UNDERLIE THIS SEX DIFFERENCE. THUS, WE PERFORMED QPCR AND WESTERN BLOT. NERVE INJURY INCREASED ALPHA(5) -GABA(A) MRNA AND PROTEIN IN FEMALE DORSAL ROOT GANGLIA (DRG) AND DECREASED THEM IN DRG AND SPINAL CORD OF MALES. TO INVESTIGATE THE HORMONAL INFLUENCE OVER ALPHA(5) -GABA(A) RECEPTOR ACTIONS, WE PERFORMED NERVE INJURY TO OVARIECTOMIZED RATS AND RECONSTITUTED THEM WITH 17BETA-ESTRADIOL (E2). OVARIECTOMY ABROGATED L-655,708 ANTIALLODYNIC EFFECT AND E2 RESTORED IT. OVARIECTOMY DECREASED ALPHA(5) -GABA(A) RECEPTOR AND ESTROGEN RECEPTOR ALPHA PROTEIN IN DRG OF NEUROPATHIC FEMALE RATS, WHILE E2 ENHANCED THEM. SINCE DNA METHYLATION MIGHT CONTRIBUTE TO ALPHA(5) -GABA(A) RECEPTOR DOWN-REGULATION IN MALES, WE EXAMINED CPG ISLAND DNA METHYLATION OF ALPHA(5) -GABA(A) RECEPTOR CODING GENE THROUGH PYROSEQUENCING. NERVE INJURY INCREASED METHYLATION IN MALE, BUT NOT FEMALE RATS. PHARMACOLOGICAL INHIBITION OF DNA METHYLTRANSFERASES INCREASED ALPHA(5) -GABA(A) RECEPTOR AND ENABLED L-655,708 ANTINOCICEPTIVE EFFECT IN MALE RATS. THESE RESULTS SUGGEST THAT ALPHA(5) -GABA(A) RECEPTOR IS A SUITABLE TARGET TO TREAT CHRONIC PAIN IN FEMALES. 2021 12 6697 33 VARICELLA-ZOSTER VIRUS HUMAN GANGLIONIC LATENCY: A CURRENT SUMMARY. VARICELLA-ZOSTER VIRUS (VZV) IS A UBIQUITOUS HUMAN HERPES VIRUS TYPICALLY ACQUIRED IN CHILDHOOD WHEN IT CAUSES VARICELLA (CHICKENPOX), FOLLOWING WHICH THE VIRUS ESTABLISHES A LATENT INFECTION IN TRIGEMINAL AND DORSAL ROOT GANGLIA THAT LASTS FOR THE LIFE OF THE INDIVIDUAL. VZV SUBSEQUENTLY REACTIVATES, SPONTANEOUSLY OR AFTER SPECIFIC TRIGGERING FACTORS, TO CAUSE HERPES ZOSTER (SHINGLES), WHICH MAY BE COMPLICATED BY POSTHERPETIC NEURALGIA AND SEVERAL OTHER NEUROLOGICAL COMPLICATIONS INCLUDING VASCULOPATHY. OUR UNDERSTANDING OF VZV LATENCY LAGS BEHIND OUR KNOWLEDGE OF HERPES SIMPLEX VIRUS TYPE 1 (HSV-1) LATENCY PRIMARILY DUE TO THE DIFFICULTY IN PROPAGATING THE VIRUS TO HIGH TITERS IN A CELL-FREE STATE, AND THE LACK OF A SUITABLE SMALL-ANIMAL MODEL FOR STUDYING VIRUS LATENCY AND REACTIVATION. IT IS NOW ESTABLISHED BEYOND DOUBT THAT LATENT VZV IS PREDOMINANTLY LOCATED IN HUMAN GANGLIONIC NEURONS. VIRUS GENE TRANSCRIPTION DURING LATENCY IS EPIGENETICALLY REGULATED, AND APPEARS TO BE RESTRICTED TO EXPRESSION OF AT LEAST SIX GENES, WITH EXPRESSION OF GENE 63 BEING THE HALLMARK OF LATENCY. HOWEVER, VIRAL GENE TRANSCRIPTION MAY BE MORE EXTENSIVE THAN PREVIOUSLY THOUGHT. THERE IS ALSO EVIDENCE FOR SEVERAL VZV GENES BEING EXPRESSED AT THE PROTEIN LEVEL, INCLUDING VZV GENE 63-ENCODED PROTEIN, BUT RECENT EVIDENCE SUGGESTS THAT THIS MAY NOT BE A COMMON EVENT. THE NATURE AND EXTENT OF THE CHRONIC INFLAMMATORY RESPONSE IN LATENTLY INFECTED GANGLIA IS ALSO OF CURRENT INTEREST. THERE REMAIN SEVERAL QUESTIONS CONCERNING THE VZV LATENCY PROCESS THAT STILL NEED TO BE RESOLVED UNAMBIGUOUSLY AND IT IS LIKELY THAT THIS WILL REQUIRE THE USE OF NEWLY DEVELOPED MOLECULAR TECHNOLOGIES, SUCH AS GEXPS MULTIPLEX POLYMERASE CHAIN REACTION (PCR) FOR VIRUS TRANSCRIPTIONAL ANALYSIS AND CHIP-SEQ TO STUDY THE EPIGENETIC OF LATENT VIRUS GENOME ( LIU ET AL, 2010 , BMC BIOL 8: 56). 2010 13 2827 28 FLUOXETINE INCREASES HIPPOCAMPAL NEUROGENESIS AND INDUCES EPIGENETIC FACTORS BUT DOES NOT IMPROVE FUNCTIONAL RECOVERY AFTER TRAUMATIC BRAIN INJURY. THE SELECTIVE SEROTONIN REUPTAKE INHIBITOR FLUOXETINE INDUCES HIPPOCAMPAL NEUROGENESIS, STIMULATES MATURATION AND SYNAPTIC PLASTICITY OF ADULT HIPPOCAMPAL NEURONS, AND REDUCES MOTOR/SENSORY AND MEMORY IMPAIRMENTS IN SEVERAL CNS DISORDERS. IN THE SETTING OF TRAUMATIC BRAIN INJURY (TBI), ITS EFFECTS ON NEUROPLASTICITY AND FUNCTION HAVE YET TO BE THOROUGHLY INVESTIGATED. HERE WE EXAMINED THE EFFICACY OF FLUOXETINE AFTER A MODERATE TO SEVERE TBI, PRODUCED BY A CONTROLLED CORTICAL IMPACT. THREE DAYS AFTER TBI OR SHAM SURGERY, MICE WERE TREATED WITH FLUOXETINE (10 MG/KG/D) OR VEHICLE FOR 4 WEEKS. TO EVALUATE THE EFFECTS OF FLUOXETINE ON NEUROPLASTICITY, HIPPOCAMPAL NEUROGENESIS AND EPIGENETIC MODIFICATION WERE STUDIED. STEREOLOGIC ANALYSIS OF THE DENTATE GYRUS REVEALED A SIGNIFICANT INCREASE IN DOUBLECORTIN-POSITIVE CELLS IN BRAIN-INJURED ANIMALS TREATED WITH FLUOXETINE RELATIVE TO CONTROLS, A FINDING CONSISTENT WITH ENHANCED HIPPOCAMPAL NEUROGENESIS. EPIGENETIC MODIFICATIONS, INCLUDING AN INCREASE IN HISTONE 3 ACETYLATION AND INDUCTION OF METHYL-CPG-BINDING PROTEIN, A TRANSCRIPTION FACTOR INVOLVED IN DNA METHYLATION, WERE LIKEWISE SEEN BY IMMUNOHISTOCHEMISTRY AND QUANTITATIVE WESTERN IMMUNOBLOTS, RESPECTIVELY, IN BRAIN-INJURED ANIMALS TREATED WITH FLUOXETINE. TO DETERMINE IF FLUOXETINE IMPROVES NEUROLOGICAL OUTCOMES AFTER TBI, GAIT FUNCTION AND SPATIAL LEARNING AND MEMORY WERE ASSESSED BY THE CATWALK-ASSISTED GAIT TEST AND BARNES MAZE TEST, RESPECTIVELY. NO DIFFERENCES IN THESE PARAMETERS WERE SEEN BETWEEN FLUOXETINE- AND VEHICLE-TREATED ANIMALS. THUS WHILE FLUOXETINE ENHANCED NEUROPLASTICITY IN THE HIPPOCAMPUS AFTER TBI, ITS CHRONIC ADMINISTRATION DID NOT RESTORE LOCOMOTOR FUNCTION OR AMELIORATE MEMORY DEFICITS. 2011 14 3971 35 LONG-LASTING NEUROTOXIC EFFECTS OF EXPOSURE TO METHYLMERCURY DURING DEVELOPMENT. AMONGST ENVIRONMENTAL CHEMICAL CONTAMINANTS, METHYLMERCURY (MEHG) REMAINS A MAJOR CONCERN BECAUSE OF ITS DETRIMENTAL EFFECTS ON DEVELOPING ORGANISMS, WHICH APPEAR TO BE PARTICULARLY SUSCEPTIBLE TO ITS TOXICITY. HERE, WE INVESTIGATED THE EFFECTS OF LOW MEHG LEVELS ON THE DEVELOPMENT OF THE NERVOUS SYSTEM USING BOTH IN VITRO AND IN VIVO EXPERIMENTAL MODELS. IN NEURAL STEM CELLS (NSCS), MEHG DECREASED PROLIFERATION AND NEURONAL DIFFERENTIATION AND INDUCED CELLULAR SENESCENCE ASSOCIATED WITH IMPAIRMENT IN MITOCHONDRIAL FUNCTION AND A CONCOMITANT DECREASE IN GLOBAL DNA METHYLATION. INTERESTINGLY, THE EFFECTS WERE HERITABLE AND COULD BE OBSERVED IN DAUGHTER NSCS NEVER DIRECTLY EXPOSED TO MEHG. BY CHRONICALLY EXPOSING PREGNANT/LACTATING MICE TO MEHG, WE FOUND PERSISTENT BEHAVIOURAL CHANGES IN THE MALE OFFSPRING, WHICH EXHIBITED DEPRESSION-LIKE BEHAVIOUR THAT COULD BE REVERSED BY CHRONIC TREATMENT WITH THE ANTIDEPRESSANT FLUOXETINE. THE BEHAVIOURAL ALTERATIONS WERE ASSOCIATED WITH A DECREASED NUMBER OF PROLIFERATING CELLS AND LOWER EXPRESSION OF BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) MRNA IN THE HIPPOCAMPAL DENTATE GYRUS. MEHG EXPOSURE ALSO INDUCED LONG-LASTING DNA HYPERMETHYLATION, INCREASED HISTONE H3-K27 TRI-METHYLATION AND DECREASED H3 ACETYLATION AT THE BDNF PROMOTER IV, INDICATING THAT EPIGENETIC MECHANISMS PLAY A CRITICAL ROLE IN MEDIATING THE LONG-LASTING EFFECTS OF PERINATAL EXPOSURE TO MEHG. FLUOXETINE TREATMENT RESTORED THE BDNF MRNA EXPRESSION LEVELS, AS WELL AS THE NUMBER OF PROLIFERATING CELLS IN THE GRANULE CELL LAYER OF THE DENTATE GYRUS, WHICH FURTHER SUPPORTS THE HYPOTHESIS THAT LINKS DEPRESSION TO IMPAIRED NEUROGENESIS. ALTOGETHER, OUR FINDINGS HAVE SHOWN THAT LOW CONCENTRATIONS OF MEHG INDUCE LONG-LASTING EFFECTS IN NSCS THAT CAN POTENTIALLY PREDISPOSE INDIVIDUALS TO DEPRESSION, WHICH WE HAVE REPORTED EARLIER TO OCCUR IN EXPERIMENTAL ANIMALS EXPOSED TO MEHG DURING PRENATAL AND EARLY POSTNATAL DEVELOPMENT. 2013 15 3493 25 IDENTIFICATION OF KEY GENES, PATHWAYS, AND MIRNA/MRNA REGULATORY NETWORKS OF CUMS-INDUCED DEPRESSION IN NUCLEUS ACCUMBENS BY INTEGRATED BIOINFORMATICS ANALYSIS. INTRODUCTION: MAJOR DEPRESSIVE DISORDER (MDD) IS A RECURRENT, DEVASTATING MENTAL DISORDER, WHICH AFFECTS >350 MILLION PEOPLE WORLDWIDE, AND EXERTS SUBSTANTIAL PUBLIC HEALTH AND FINANCIAL COSTS TO SOCIETY. THUS, THERE IS A SIGNIFICANT NEED TO DISCOVER INNOVATIVE THERAPEUTICS TO TREAT DEPRESSION EFFICIENTLY. STRESS-INDUCED DYSFUNCTION IN THE SUBTYPE OF NEURONAL CELLS AND THE CHANGE OF SYNAPTIC PLASTICITY AND STRUCTURAL PLASTICITY OF NUCLEUS ACCUMBENS (NAC) ARE IMPLICATED IN DEPRESSION SYMPTOMOLOGY. HOWEVER, THE MOLECULAR AND EPIGENETIC MECHANISMS AND STRESSES TO THE NAC PATHOLOGICAL CHANGES IN DEPRESSION REMAIN ELUSIVE. MATERIALS AND METHODS: IN THIS STUDY, TREATMENT GROUP MICE WERE TREATED CONTINUALLY WITH THE CHRONIC UNPREDICTABLE MILD STRESS (CUMS) UNTIL EXPRESSION OF DEPRESSION-LIKE BEHAVIORS WERE FOUND. DEPRESSION WAS CONFIRMED WITH SUCROSE PREFERENCE, NOVELTY-SUPPRESSED FEEDING, FORCED SWIMMING, AND TAIL SUSPENSION TESTS. WE APPLIED HIGH-THROUGHPUT RNA SEQUENCING TO ASSESS MICRORNA EXPRESSION AND TRANSCRIPTIONAL PROFILES IN THE NAC TISSUE FROM DEPRESSION-LIKE BEHAVIORS MICE AND CONTROL MICE. THE REGULATORY NETWORK OF MIRNAS/MRNAS WAS CONSTRUCTED BASED ON THE HIGH-THROUGHPUT RNA SEQUENCE AND BIOINFORMATICS SOFTWARE PREDICTIONS. RESULTS: A TOTAL OF 17 MIRNAS AND 10 MRNAS WERE SIGNIFICANTLY UPREGULATED IN THE NAC OF CUMS-INDUCED MICE WITH DEPRESSION-LIKE BEHAVIORS, AND 12 MIRNAS AND 29 MRNAS WERE DOWNREGULATED. A SERIES OF BIOINFORMATICS ANALYSES SHOWED THAT THESE ALTERED MIRNAS PREDICTED TARGET MRNA AND DIFFERENTIALLY EXPRESSED MRNAS WERE SIGNIFICANTLY ENRICHED IN THE MAPK SIGNALING PATHWAY, GABAERGIC SYNAPSE, DOPAMINERGIC SYNAPSE, CYTOKINE-CYTOKINE RECEPTOR INTERACTION, AXON GUIDANCE, REGULATION OF AUTOPHAGY, AND SO ON. FURTHERMORE, DUAL LUCIFERASE REPORT ASSAY AND QRT-PCR RESULTS VALIDATED THE MIRNA/MRNA REGULATORY NETWORK. CONCLUSION: THE DETERIORATIONS OF GABAERGIC SYNAPSES, DOPAMINERGIC SYNAPSES, NEUROTRANSMITTER SYNTHESIS, AS WELL AS AUTOPHAGY-ASSOCIATED APOPTOTIC PATHWAY ARE ASSOCIATED WITH THE MOLECULAR PATHOLOGICAL MECHANISM OF CUMS-INDUCED DEPRESSION. 2019 16 5453 29 REPROGRAMMING OF COPD LUNG FIBROBLASTS THROUGH FORMATION OF INDUCED PLURIPOTENT STEM CELLS. REPROGRAMMING SOMATIC CELLS TO INDUCED PLURIPOTENT STEM CELLS (IPSCS) ELIMINATES MANY EPIGENETIC MODIFICATIONS THAT CHARACTERIZE DIFFERENTIATED CELLS. IN THIS STUDY, WE TESTED WHETHER FUNCTIONAL DIFFERENCES BETWEEN CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) AND NON-COPD FIBROBLASTS COULD BE REDUCED UTILIZING THIS APPROACH. PRIMARY FIBROBLASTS FROM NON-COPD AND COPD PATIENTS WERE REPROGRAMMED TO IPSCS. REPROGRAMMED IPSCS WERE POSITIVE FOR OCT3/4, NANOG, AND SOX2, FORMED EMBRYOID BODIES IN VITRO, AND INDUCED TERATOMAS IN NONOBESE DIABETIC/SEVERE COMBINED IMMUNODEFICIENT MICE. REPROGRAMMED IPSCS WERE THEN DIFFERENTIATED INTO FIBROBLASTS (NON-COPD-I AND COPD-I) AND WERE ASSESSED EITHER FUNCTIONALLY BY CHEMOTAXIS AND GEL CONTRACTION OR FOR GENE EXPRESSION BY MICROARRAYS AND COMPARED WITH THEIR CORRESPONDING PRIMARY FIBROBLASTS. PRIMARY COPD FIBROBLASTS CONTRACTED THREE-DIMENSIONAL COLLAGEN GELS AND MIGRATED TOWARD FIBRONECTIN LESS ROBUSTLY THAN NON-COPD FIBROBLASTS. IN CONTRAST, REDIFFERENTIATED FIBROBLASTS FROM IPSCS DERIVED FROM THE NON-COPD AND COPD FIBROBLASTS WERE SIMILAR IN RESPONSE IN BOTH FUNCTIONAL ASSAYS. MICROARRAY ANALYSIS IDENTIFIED 1,881 GENES THAT WERE DIFFERENTIALLY EXPRESSED BETWEEN PRIMARY COPD AND NON-COPD FIBROBLASTS, WITH 605 GENES DIFFERING BY MORE THAN TWOFOLD. AFTER REDIFFERENTIATION, 112 GENES WERE DIFFERENTIALLY EXPRESSED BETWEEN COPD-I AND NON-COPD-I WITH ONLY THREE GENES BY MORE THAN TWOFOLD. SIMILAR FINDINGS WERE OBSERVED WITH MICRORNA (MIRNA) EXPRESSION: 56 MIRNAS WERE DIFFERENTIALLY EXPRESSED BETWEEN NON-COPD AND COPD PRIMARY CELLS; AFTER REDIFFERENTIATION, ONLY 3 MIRNAS WERE DIFFERENTIALLY EXPRESSED BETWEEN NON-COPD-I AND COPD-I FIBROBLASTS. INTERESTINGLY, OF THE 605 GENES THAT WERE DIFFERENTIALLY EXPRESSED BETWEEN COPD AND NON-COPD FIBROBLASTS, 293 GENES WERE CHANGED TOWARD CONTROL AFTER REDIFFERENTIATION. IN CONCLUSION, FUNCTIONAL AND EPIGENETIC ALTERATIONS OF COPD FIBROBLASTS CAN BE REPROGRAMMED THROUGH FORMATION OF IPSCS. 2014 17 3331 26 HISTONE DEACETYLASE INHIBITOR SUBERANILOHYDROXAMIC ACID TREATMENT REVERSES HYPOSENSITIVITY TO GAMMA-AMINOBUTYRIC ACID IN THE VENTRAL TEGMENTAL AREA DURING ETHANOL WITHDRAWAL. BACKGROUND: THE VENTRAL TEGMENTAL AREA (VTA) IS IMPORTANT FOR ALCOHOL-RELATED REWARD AND REINFORCEMENT. MOUSE VTA NEURONS ARE HYPOSENSITIVE TO GAMMA-AMINOBUTYRIC ACID (GABA) DURING ETHANOL (ETOH) WITHDRAWAL, AND GABA RESPONSIVENESS IS NORMALIZED BY IN VITRO TREATMENT WITH HISTONE DEACETYLASE INHIBITORS (HDACI). THE PRESENT STUDY EXAMINED THE EFFECT OF A SYSTEMICALLY ADMINISTERED HDACI, SUBERANILOHYDROXAMIC ACID (SAHA) ON GABA SENSITIVITY, AND RELATED MOLECULAR CHANGES IN VTA NEURONS DURING WITHDRAWAL AFTER CHRONIC ETOH INTAKE IN RATS. METHODS: SPRAGUE DAWLEY MALE ADULT RATS WERE FED WITH LIEBER-DECARLI DIET (9% ETOH OR CONTROL DIET) FOR 16 DAYS. EXPERIMENTAL GROUPS INCLUDED CONTROL DIET-FED AND ETOH DIET-FED (0- OR 24-HOUR WITHDRAWAL) RATS TREATED WITH EITHER SAHA OR VEHICLE INJECTION. SINGLE-UNIT RECORDINGS WERE USED TO MEASURE THE RESPONSE OF VTA NEURONS TO GABA. IMMUNOHISTOCHEMISTRY WAS PERFORMED TO EXAMINE LEVELS OF HDAC2, ACETYLATED HISTONE H3 LYSINE 9 (ACH3K9), AND GABA(A) RECEPTOR ALPHA1 AND ALPHA5 SUBUNITS IN THE VTA; QUANTITATIVE POLYMERASE CHAIN REACTION WAS PERFORMED TO EXAMINE THE MRNA LEVELS OF HDAC2 AND GABA(A) RECEPTOR SUBUNITS. RESULTS: VTA NEURONS FROM THE WITHDRAWAL GROUP EXHIBITED GABA HYPOSENSITIVITY. IN VIVO SAHA TREATMENT 2 HOURS BEFORE SACRIFICE NORMALIZED THE SENSITIVITY OF VTA NEURONS TO GABA. ETOH WITHDRAWAL WAS ASSOCIATED WITH INCREASED HDAC2 AND DECREASED ACH3K9 PROTEIN LEVELS; SAHA TREATMENT NORMALIZED ACH3K9 LEVELS. INTERESTINGLY, NO SIGNIFICANT CHANGE WAS OBSERVED IN THE MRNA LEVELS OF HDAC2. THE MRNA LEVELS, BUT NOT PROTEIN LEVELS, OF GABA(A) RECEPTOR ALPHA1 AND ALPHA5 SUBUNITS WERE INCREASED DURING WITHDRAWAL. CONCLUSIONS: WITHDRAWAL FROM CHRONIC ETOH EXPOSURE RESULTS IN A DECREASE IN GABA-MEDIATED INHIBITION, AND THIS GABA HYPOSENSITIVITY IS NORMALIZED BY IN VIVO SAHA TREATMENT. DISRUPTION OF SIGNALING IN THE VTA PRODUCED BY ALTERATION OF GABA NEUROTRANSMISSION COULD BE 1 NEUROADAPTIVE PHYSIOLOGICAL PROCESS LEADING TO CRAVING AND RELAPSE. THESE RESULTS SUGGEST THAT HDACI PHARMACOTHERAPY WITH AGENTS LIKE SAHA MIGHT BE AN EFFECTIVE TREATMENT FOR ALCOHOLISM. 2018 18 4304 28 MICRORNA-223 PROTECTS NEURONS FROM DEGENERATION IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS. MULTIPLE SCLEROSIS IS A CHRONIC INFLAMMATORY, DEMYELINATING, AND NEURODEGENERATIVE DISEASE AFFECTING THE BRAIN, SPINAL CORD AND OPTIC NERVES. NEURONAL DAMAGE IS TRIGGERED BY VARIOUS HARMFUL FACTORS THAT ENGAGE DIVERSE SIGNALLING CASCADES IN NEURONS; THUS, THERAPEUTIC APPROACHES TO PROTECT NEURONS WILL NEED TO FOCUS ON AGENTS THAT CAN TARGET MULTIPLE BIOLOGICAL PROCESSES. WE HAVE THEREFORE FOCUSED OUR ATTENTION ON MICRORNAS: SMALL NON-CODING RNAS THAT PRIMARILY FUNCTION AS POST-TRANSCRIPTIONAL REGULATORS THAT TARGET MESSENGER RNAS AND REPRESS THEIR TRANSLATION INTO PROTEINS. A SINGLE MICRORNA CAN TARGET MANY FUNCTIONALLY RELATED MESSENGER RNAS MAKING MICRORNAS POWERFUL EPIGENETIC REGULATORS. DYSREGULATION OF MICRORNAS HAS BEEN DESCRIBED IN MANY NEURODEGENERATIVE DISEASES INCLUDING MULTIPLE SCLEROSIS. HERE, WE REPORT THAT TWO MICRORNAS, MIR-223-3P AND MIR-27A-3P, ARE UPREGULATED IN NEURONS IN THE EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS MOUSE MODEL OF CNS INFLAMMATION AND IN GREY MATTER-CONTAINING MULTIPLE SCLEROSIS LESIONS. PRIOR WORK HAS SHOWN PERIPHERAL BLOOD MONONUCLEAR CELL CONDITIONED MEDIA CAUSES SUBLETHAL DEGENERATION OF NEURONS IN CULTURE. WE FIND OVEREXPRESSION OF MIR-27A-3P OR MIR-223-3P PROTECTS DISSOCIATED CORTICAL NEURONS FROM CONDITION MEDIA MEDIATED DEGENERATION. INTRODUCTION OF MIR-223-3P IN VIVO IN MOUSE RETINAL GANGLION CELLS PROTECTS THEIR AXONS FROM DEGENERATION IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS. IN SILICO ANALYSIS REVEALED THAT MESSENGER RNAS INVOLVED IN GLUTAMATE RECEPTOR SIGNALLING ARE ENRICHED AS MIR-27A-3P AND MIR-223-3P TARGETS. WE OBSERVE THAT ANTAGONISM OF NMDA AND AMPA TYPE GLUTAMATE RECEPTORS PROTECTS NEURONS FROM CONDITION MEDIA DEPENDENT DEGENERATION. OUR RESULTS SUGGEST THAT MIR-223-3P AND MIR-27A-3P ARE UPREGULATED IN RESPONSE TO INFLAMMATION TO MEDIATE A COMPENSATORY NEUROPROTECTIVE GENE EXPRESSION PROGRAM THAT DESENSITIZES NEURONS TO GLUTAMATE BY TARGETING MESSENGER RNAS INVOLVED IN GLUTAMATE RECEPTOR SIGNALLING. 2019 19 6108 30 THE ENRICHED ENVIRONMENT AMELIORATES CHRONIC UNPREDICTABLE MILD STRESS-INDUCED DEPRESSIVE-LIKE BEHAVIORS AND COGNITIVE IMPAIRMENT BY ACTIVATING THE SIRT1/MIR-134 SIGNALING PATHWAY IN HIPPOCAMPUS. BACKGROUND: CHRONIC UNPREDICTABLE MILD STRESS (CUMS) IS AN IMPORTANT RISK FACTOR FOR DEPRESSION AND COGNITIVE DEFICITS IN HUMANS. ENRICHED ENVIRONMENT (EE) SHOWED A BENEFICIAL EFFECT ON DEPRESSION AND COGNITION BY ENHANCING BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF) EXPRESSION AND SYNAPTIC PLASTICITY. HOWEVER, IT IS STILL NOT CLEARLY UNDERSTOOD WHETHER AN EPIGENETIC MECHANISM IS INVOLVED IN THE BDNF MODULATION AND SYNAPTIC PLASTICITY THAT OCCURS AFTER EE TREATMENT FOR THE DEPRESSIVE-LIKE BEHAVIORS AND COGNITIVE DEFICITS ELICITED BY CUMS. IN THIS STUDY, WE INVESTIGATED THE POSSIBLE MECHANISM OF THE NEUROPROTECTIVE EFFECT OF EE. METHODS: ALL RATS WERE EXPOSED TO THE 5-WEEK CUMS PROCEDURE EXCEPT THE CONTROL GROUP. AFTER CUMS PROCEDURE, SOME RATS WERE STEREOTAXICALLY INJECTED WITH SIRT1 PHARMACOLOGIC INHIBITOR EX527 OR SIRT1 KNOCKING DOWN LENTIVIRUS (SH-SIRT1) IN THE HIPPOCAMPUS FOLLOWED BY EE TREATMENT FOR 3 WEEKS. OTHER RATS WERE DIRECTLY SUBJECTED TO EE TREATMENT WITHOUT STEREOTAXIC INJECTION. BEHAVIORAL TESTS WERE USED TO APPRAISE DEPRESSION AND COGNITION AFTER EE TREATMENT. THEN EPIGENETIC MOLECULES, SYNAPTIC PROTEINS, DENDRITIC SPINE DENSITY AND BRANCHES, AND SYNAPTIC MORPHOLOGY OF THE DORSAL HIPPOCAMPUS WERE DETERMINED. RESULTS: WE FOUND THAT CUMS INDUCED DEPRESSIVE-LIKE BEHAVIORS INCLUDING DECREASED SUCROSE PREFERENCE RATIO, PROLONGED IMMOBILITY AND REDUCED LOCOMOTOR AND EXPLORATORY ACTIVITY; COGNITIVE DEFICITS INCLUDING SPATIAL LEARNING AND MEMORY IMPAIRMENT; REDUCED DENDRITIC SPINE DENSITY AND NUMBER OF BRANCHES; THINNED POSTSYNAPTIC DENSITY; DOWNREGULATED SIRT1/MICRORNA-134 PATHWAY, DECREASED BDNF AND SYNAPTIC PROTEINS INCLUDING SYNAPTOPHYSIN (SYN) AND POSTSYNAPTIC DENSITY PROTEIN 95 (PSD95) EXPRESSION IN THE HIPPOCAMPUS. HOWEVER, THE CUMS-INDUCED DEPRESSIVE-LIKE BEHAVIORS, COGNITIVE DEFICITS, DENDRITIC SPINE DENSITY AND BRANCH NUMBER REDUCTION, POSTSYNAPTIC DENSITY THINNING, SIRT1/MICRORNA-134 PATHWAY DOWNREGULATION, BDNF AND SYNAPTIC PROTEINS REDUCTION, INCLUDING SYNAPTOPHYSIN (SYN) AND POSTSYNAPTIC DENSITY PROTEIN 95 (PSD95), WERE REVERSED BY EE TREATMENT. HOWEVER, DEPRESSIVE-LIKE BEHAVIORS AND COGNITIVE DEFICITS WERE OBSERVED AGAIN IN RATS SUBJECTED TO STEREOTAXIC INJECTION WITH EX527 OR SH-SIRT1. FURTHERMORE, THIS STUDY ALSO FOUND THAT SIRT1/MICRORNA-134 REGULATES THE DOWNSTREAM MOLECULES BDNF, AND THE SYNAPTIC PROTEINS SYN AND PSD95 IN PRIMARY CULTURED HIPPOCAMPAL NEURONS. CONCLUSIONS: THIS STUDY PROVIDES EVIDENCE FOR THE NEUROPROTECTIVE ROLE OF EE ON DEPRESSION AND COGNITIVE DEFICITS BY ACTIVATING THE SIRT1/MICRORNA-134 PATHWAY, WHICH ACCOUNTS FOR THE REGULATION OF SYNAPTIC PROTEINS, INCLUDING BDNF, PSD95 AND SYN, DENDRITIC REMODELING AND ULTRASTRUCTURE CHANGES OF SYNAPSES IN THE HIPPOCAMPUS. 2019 20 2072 33 EPIGENETIC CONVERSION OF HUMAN ADULT BONE MESODERMAL STROMAL CELLS INTO NEUROECTODERMAL CELL TYPES FOR REPLACEMENT THERAPY OF NEURODEGENERATIVE DISORDERS. TISSUE-SPECIFIC STEM CELLS, SUCH AS BONE MARROW-DERIVED MESODERMAL STROMAL CELLS (MSCS), ARE THOUGHT TO BE LINEAGE RESTRICTED AND, THEREFORE, COULD ONLY BE DIFFERENTIATED INTO CELL TYPES OF THE TISSUE OF ORIGIN. SEVERAL RECENT STUDIES, HOWEVER, SUGGEST THAT THESE TYPES OF STEM CELLS MIGHT BE ABLE TO BREAK BARRIERS OF GERM LAYER COMMITMENT AND DIFFERENTIATE IN VITRO AND/OR IN VIVO INTO CELLS OF DIFFERENT TISSUES, SUCH AS NEUROECTODERMAL CELL TYPES. RECENTLY, PROTOCOLS FOR HIGH-YIELD GENERATION OF UNDIFFERENTIATED NEURAL STEM CELL (NSC)-LIKE CELLS FROM MSCS OF PRIMATE AND HUMAN ORIGIN WERE REPORTED. UNDIFFERENTIATED NSCS ARE COMMONLY USED AND ARE MORE SUITABLE FOR NEUROTRANSPLANTATION COMPARED WITH FULLY DIFFERENTIATED NEURAL CELLS, AS DIFFERENTIATED NEURAL CELLS ARE WELL KNOWN TO POORLY SURVIVE DETACHMENT AND SUBSEQUENT TRANSPLANTATION PROCEDURES. THESE HUMAN MSC-DERIVED NSC-LIKE CELLS (MSC-NSCS) GROW IN NEUROSPHERE-LIKE STRUCTURES AND EXPRESS HIGH LEVELS OF EARLY NEUROECTODERMAL MARKERS, BUT LOSE CHARACTERISTICS OF MSCS. IN THE PRESENCE OF SELECTED GROWTH FACTORS, HUMAN MSC-NSCS CAN BE DIFFERENTIATED INTO THE THREE MAIN NEURAL PHENOTYPES: ASTROGLIA, OLIGODENDROGLIA AND NEURONS. COMPARED WITH DIRECT DIFFERENTIATION OF HUMAN MSCS INTO MATURE NEURAL CELLS, THE CONVERSION STEP SEEMS TO BE ESSENTIAL TO GENERATE MATURE FUNCTIONAL NEUROECTODERMAL CELLS. THIS REVIEW DESCRIBES THE TECHNIQUES FOR THE CONVERSION OF HUMAN MSCS INTO NSCS AND SUMMARISES THE DATA ON EPIGENETIC CONVERSION OF HUMAN MSCS INTO IMMATURE NEUROECTODERMAL CELLS. THESE CELLS PROVIDE A POWERFUL TOOL FOR INVESTIGATING THE MOLECULAR MECHANISMS OF NEURAL DIFFERENTIATION, AND MIGHT SERVE AS AN AUTOLOGOUS CELL SOURCE TO TREAT ACUTE AND CHRONIC NEURODEGENERATIVE DISEASES. 2006