RESUMEN
The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.
Asunto(s)
Cápside , Quirópteros , Susceptibilidad a Enfermedades , Retroviridae , Animales , Humanos , Ratones , Australia , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Quirópteros/virología , Retroviridae/clasificación , Retroviridae/metabolismo , Infecciones por Retroviridae/metabolismo , Infecciones por Retroviridae/virología , Susceptibilidad a Enfermedades/metabolismo , Susceptibilidad a Enfermedades/virología , Línea Celular , Especificidad de la Especie , Factores de Restricción Antivirales/metabolismoRESUMEN
The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targeting ATP5B (rAAV-L-AD-shATP5B) and found that it caused a significant decrease in ATP5B mRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.
Asunto(s)
Dependovirus , Vectores Genéticos , Empaquetamiento del ADN , Dependovirus/genética , Vectores Genéticos/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Secuencias Repetidas Terminales/genética , TransgenesRESUMEN
Oncolytic herpes simplex viruses (oHSV) are under development for the treatment of a variety of human cancers, including breast cancer, a leading cause of cancer mortality among women worldwide. Here we report the design of a fully retargeted oHSV for preferential infection of breast cancer cells through virus recognition of GFRα1, the cellular receptor for glial cell-derived neurotrophic factor (GDNF). GFRα1 displays a limited expression profile in normal adult tissue, but is upregulated in a subset of breast cancers. We generated a recombinant HSV expressing a completely retargeted glycoprotein D (gD), the viral attachment/entry protein, that incorporates pre-pro-GDNF in place of the signal peptide and HVEM binding domain of gD and contains a deletion of amino acid 38 to eliminate nectin-1 binding. We show that GFRα1 is necessary and sufficient for infection by the purified recombinant virus. Moreover, this virus enters and spreads in GFRα1-positive breast cancer cells in vitro and caused tumor regression upon intratumoral injection in vivo. Given the heterogeneity observed between and within individual breast cancers at the molecular level, these results expand our ability to deliver oHSV to specific tumors and suggest opportunities to enhance drug or viral treatments aimed at other receptors.
Asunto(s)
Neoplasias de la Mama/terapia , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Nectinas/genética , Simplexvirus/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Chlorocebus aethiops , Femenino , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Humanos , Células MCF-7 , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Unión Proteica/genética , Células VeroRESUMEN
Inactivation of all herpes simplex virus (HSV) immediate early (IE) genes to eliminate vector cytotoxicity results in rapid silencing of the viral genome, similar to the establishment of HSV latency. We recently reported that silencing of a nonviral reporter cassette could be overcome in nonneuronal cells by positioning the cassette in the viral latency (LAT) locus between resident chromatin boundary elements. Here, we tested the abilities of the chicken hypersensitive site 4 insulator and the human ubiquitous chromatin opening element A2UCOE to promote transgene expression from an IE-gene-inactivated HSV vector. We found that A2UCOE was particularly active in nonneuronal cells and reduced reporter promoter occupancy by a repressive histone mark. We determined whether multiple transgenes could be expressed under the control of different promoters from different loci of the same virus. The results showed abundant coexpression of LAT-embedded and A2UCOE-flanked genes in nonneuronal cells. In addition, a third reporter gene without known protective elements was active in cultured rat sensory neurons. These findings indicate that cellular antisilencing sequences can contribute to the expression of multiple genes from separate promoters in fully IE gene-disabled HSV vectors, providing an opportunity for therapeutic applications requiring mutually independent expression of different gene products from a single vector.IMPORTANCE Gene therapy has now entered a phase of development in which a growing number of recessive single gene defects can be successfully treated by vector-mediated introduction of a wild-type copy of the gene into the appropriate tissue. However, many disease conditions, such as neurodegeneration, cancer, and inflammatory processes, are more complex, requiring either multiple gene corrections or provision of coordinated gene activities to achieve a therapeutic outcome. Although herpes simplex virus (HSV) vectors have the capacity to meet this need, the challenge has been to genetically engineer the HSV genome in a manner to prevent expression of any viral genes while retaining the ability to express multiple therapeutic transgenes under independent transcriptional control. Here, we show that non-HSV insulator elements can be applied to retain at least transient transgene activity from multiple viral loci, thereby opening the door for more complex gene therapy applications in the future.
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Genes Inmediatos-Precoces/genética , Genes Virales/genética , Vectores Genéticos , Herpesvirus Humano 1/genética , Transgenes/genética , Animales , Pollos , ADN Viral/genética , Terapia Genética , Genoma Viral , Herpes Simple/virología , Humanos , Regiones Promotoras Genéticas , Inactivación de Virus , Latencia del VirusRESUMEN
The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.
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Regulación de la Expresión Génica , Vectores Genéticos , Células Musculares/citología , Simplexvirus/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Distrofina/genética , Silenciador del Gen , Genes Reporteros , Terapia Genética/métodos , Genoma , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Lentivirus/metabolismo , Ratones , Músculos/citología , Neuronas , Regiones Promotoras Genéticas , Ratas , Transducción Genética , Células VeroRESUMEN
Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable ß-catenin, and in vitro studies, we show a role of Wnt signaling through ß-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a ß-catenin-independent manner. CONCLUSION: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both ß-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666).
Asunto(s)
Colestasis Intrahepática , Regeneración Hepática/fisiología , Proteínas Wnt/fisiología , Animales , Línea Celular Tumoral , Transdiferenciación Celular , Hepatocitos , Humanos , Ratones , Transducción de Señal , beta Catenina/fisiologíaRESUMEN
Glioblastoma multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSVs) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered four copies of the recognition sequence for miR-124 into the 3'UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.
Asunto(s)
Regiones no Traducidas 3' , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , MicroARNs/genética , Viroterapia Oncolítica/métodos , Animales , Secuencia de Bases , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Cromosomas Artificiales Bacterianos/química , Cromosomas Artificiales Bacterianos/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Células HEK293 , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/metabolismo , Inyecciones Intraventriculares , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Datos de Secuencia Molecular , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Ganglios Espinales/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/tratamiento farmacológico , Manejo del Dolor/métodos , Células Receptoras Sensoriales/efectos de los fármacos , Nervios Espinales/metabolismo , Factores de Transcripción/metabolismo , Animales , Conducta Animal , Regulación hacia Abajo , Hibridación in Situ , Inyecciones Espinales , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Dimensión del Dolor , ARN Interferente Pequeño/metabolismo , Ratas , Receptores de Prostaglandina E/metabolismoRESUMEN
Embryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.
Asunto(s)
Materiales Biomiméticos/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Nanofibras/química , Animales , Membrana Basal/química , Materiales Biomiméticos/síntesis química , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endodermo/citología , Endodermo/efectos de los fármacos , Endodermo/crecimiento & desarrollo , Células Nutrientes/citología , Regulación del Desarrollo de la Expresión Génica , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Neuropéptidos/genética , Neuropéptidos/metabolismo , Transducción de Señal , Andamios del Tejido , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Redirecting differentiation of somatic cells by over-expression of transcription factors is a promising approach for regenerative medicine, elucidation of pathogenesis and development of new therapies. We have previously defined a transcription factor combination, that is, CRX, RAX and NEUROD, that can generate photosensitive photoreceptor cells from human iris cells. Here, we show that human dermal fibroblasts are differentiated to photoreceptor cells by the same transcription factor combination as human iris cells. Transduction of a combination of the CRX, RAX and NEUROD genes up-regulated expression of the photoreceptor-specific genes, recoverin, blue opsin and PDE6C, in all three strains of human dermal fibroblasts that were tested. Additional OTX2 gene transduction increased up-regulation of the photoreceptor-specific genes blue opsin, recoverin, S-antigen, CNGB3 and PDE6C. Global gene expression data by microarray analysis further showed that photoreceptor-related functional genes were significantly increased in induced photoreceptor cells. Functional analysis, that is, patch-clamp recordings, clearly revealed that induced photoreceptor cells from fibroblasts responded to light. Both the NRL gene and the NR2E3 gene were endogenously up-regulated in induced photoreceptor cells, implying that exogenous CRX, RAX, OTX2 and NEUROD, but not NRL, are sufficient to generate rod photoreceptor cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas del Ojo/genética , Fibroblastos/citología , Proteínas de Homeodominio/genética , Factores de Transcripción Otx/genética , Células Fotorreceptoras de Vertebrados/citología , Transactivadores/genética , Factores de Transcripción/genética , Diferenciación Celular , Dermis/citología , Dermis/metabolismo , Fibroblastos/metabolismo , Humanos , Iris/citología , Iris/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/citología , Retina/metabolismo , Transducción GenéticaRESUMEN
Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), which leads to neutrophil dysfunction. However, the underlying causes of these dysfunctions and their relationship with glucose homeostasis are unclear. Induced pluripotent stem cells (iPSCs) hold a great promise for advances in developmental biology, cell-based therapy and modeling of human disease. Here, we examined the use of iPSCs as a model for GSDIb. In this study, one 2-year-old patient was genetically screened and diagnosed with GSDIb. We established iPSCs and differentiated these cells into hepatocytes and neutrophils, which comprise the main pathological components of GSDIb. Cells that differentiated into hepatocytes exhibited characteristic albumin secretion and indocyanine green uptake. Moreover, iPSC-derived cells generated from patients with GSDIb metabolic abnormalities recapitulated key pathological features of the diseases affecting the patients from whom they were derived, such as glycogen, lactate, pyruvate and lipid accumulation. Cells that were differentiated into neutrophils also showed the GSDIb pathology. In addition to the expression of neutrophil markers, we showed increased superoxide anion production, increased annexin V binding and activation of caspase-3 and caspase-9, consistent with the GSDIb patient's neutrophils. These results indicate valuable tools for the analysis of this pathology and the development of future treatments.
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Enfermedad del Almacenamiento de Glucógeno Tipo I/patología , Células Madre Pluripotentes Inducidas/patología , Diferenciación Celular , Células Cultivadas , Preescolar , Enfermedad del Almacenamiento de Glucógeno Tipo I/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patología , Estrés OxidativoRESUMEN
We previously identified zinc finger (ZF) protein ZNF385B as a molecule specifically expressed in Burkitt's lymphoma (BL) among hematologic malignancies. Here, we investigated ZNF385B expression in healthy B cells in a variety of hematological tissues by RT-PCR and immunohistochemistry. ZNF385B expression was found to be limited to a subset of GC B cells, the healthy counterpart to BL B cells. To elucidate the function of ZNF385B in healthy B cells, we established a tetracycline-controlled protein-inducible system in B-cell lines and observed that ectopic expression of the longest transcript variant of ZNF385B, possessing four ZF domains, induced upregulation of PERP and FAS/CD95, a downstream target of p53, and activation of caspase, resulting in apoptosis induction. However, a ZNF385B deletion mutant with three ZF domains corresponding to shorter isoforms, did not induce upregulation; rather it inhibited apoptosis induced by CD20 cross-linking and BCR stimulation. The direct binding of ZNF385B with p53 has suggested the involvement of ZNF385B in B-cell apoptosis via modulation of p53 transactivation; our data indicate that ZNF385B characteristically expressed in GC B cells has both proapoptotic and antiapoptotic activities depending on the type of isoform and should be a novel player in GC B-cell selection.
Asunto(s)
Apoptosis/inmunología , Linfocitos B/inmunología , Centro Germinal/inmunología , Proteína p53 Supresora de Tumor/inmunología , Dedos de Zinc/inmunología , Antígenos CD20/genética , Antígenos CD20/inmunología , Apoptosis/genética , Linfocitos B/metabolismo , Línea Celular , Femenino , Eliminación de Gen , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Centro Germinal/metabolismo , Humanos , Masculino , Activación Transcripcional/genética , Activación Transcripcional/inmunología , Proteína p53 Supresora de Tumor/genética , Dedos de Zinc/genéticaRESUMEN
BACKGROUND: hiPSCs are generated through epigenetic reprogramming of somatic tissue. Genomic imprinting is an epigenetic phenomenon through which monoallelic gene expression is regulated in a parent-of-origin-specific manner. Reprogramming relies on the successful erasure of marks of differentiation while maintaining those required for genomic imprinting. Loss of imprinting (LOI), which occurs in many types of malignant tumors, would hinder the clinical application of hiPSCs. RESULTS: We examined the imprinting status, expression levels and DNA methylation status of eight imprinted genes in five independently generated hiPSCs. We found a low frequency of LOI in some lines. Where LOI was identified in an early passage cell line, we found that this was maintained through subsequent passages of the cells. Just as normal imprints are maintained in long-term culture, this work suggests that abnormal imprints are also stable in culture. CONCLUSIONS: Analysis of genomic imprints in hiPSCs is a necessary safety step in regenerative medicine, with relevance both to the differentiation potential of these stem cells and also their potential tumorigenic properties.
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Impresión Genómica , Inestabilidad Genómica , Células Madre Pluripotentes Inducidas/metabolismo , Metilación de ADN , Regulación de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Pérdida de HeterocigocidadRESUMEN
Primary sensory neurons regulate inflammatory processes in innervated regions through neuro-immune communication. However, how their immune-modulating functions are regulated in concert remains largely unknown. Here, we show that Neat1 long non-coding RNA (lncRNA) organizes the proinflammatory gene expressions in the dorsal root ganglion (DRG) in chronic intractable neuropathic pain in rats. Neat1 was abundantly expressed in the DRG and was upregulated after peripheral nerve injury. Neat1 overexpression in primary sensory neurons caused mechanical and thermal hypersensitivity, whereas its knockdown alleviated neuropathic pain. Bioinformatics analysis of comprehensive transcriptome changes indicated the inflammatory response was the most relevant function of genes upregulated through Neat1. Consistent with this, upregulation of proinflammatory genes in the DRG following nerve injury was suppressed by Neat1 knockdown. Expression changes of these proinflammatory genes were regulated through Neat1-mRNA interaction-dependent and -independent mechanisms. Notably, Neat1 increased proinflammatory genes by stabilizing its interacting mRNAs in neuropathic pain. Finally, Neat1 in primary sensory neurons contributed to spinal inflammatory processes that mediated peripheral neuropathic pain. These findings demonstrate that Neat1 lncRNA is a key regulator of neuro-immune communication in neuropathic pain.
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Neuralgia , ARN Largo no Codificante , Traumatismos del Sistema Nervioso , Animales , Ratas , ARN Largo no Codificante/genética , Ganglios Espinales , Neuralgia/genética , ARN Mensajero , TranscriptomaRESUMEN
Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) hold great promise as a cell source for transplantation into injured tissues to alleviate inflammation. However, the therapeutic efficacy of iMSC transplantation for ischemic stroke remains unknown. In this study, we evaluated the therapeutic effects of iMSC transplantation on brain injury after ischemia-reperfusion using a rat transient middle cerebral artery occlusion model and compared its therapeutic efficacy with that of bone marrow mesenchymal stem cells (BMMSCs). We showed that iMSCs and BMMSCs reduced infarct volumes after reperfusion and significantly improved motor function on days 3, 7, 14, 28, and 56 and cognitive function on days 28 and 56 after reperfusion compared with the vehicle group. Furthermore, immunological analyses revealed that transplantation of iMSCs and BMMSCs inhibited microglial activation and expression of proinflammatory cytokines and suppressed oxidative stress and neuronal cell death in the cerebral cortex at the ischemic border zone. No difference in therapeutic effect was observed between the iMSC and BMMSC groups. Taken together, our results demonstrate that iMSC therapy can be a practical alternative as a cell source for attenuation of brain injury and improvement of neurological function because of the unlimited supply of uniform therapeutic cells.
RESUMEN
LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid-rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Metaloproteínas/genética , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Precursoras de Linfocitos B/fisiología , Factores de Transcripción/metabolismo , Translocación Genética , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/fisiología , Western Blotting , Línea Celular Tumoral , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 19 , Sangre Fetal/citología , Regulación Leucémica de la Expresión Génica , Humanos , Proteínas con Dominio LIM , Lentivirus/genética , Metaloproteínas/metabolismo , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Precursoras de Linfocitos B/citología , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas , ARN Interferente Pequeño , Factores de Transcripción/genética , Regulación hacia Arriba/genéticaRESUMEN
Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Genes Homeobox/fisiología , Hepatocitos/citología , Proteínas de Homeodominio/fisiología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción/fisiología , Adenoviridae/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Genes Homeobox/genética , Vectores Genéticos/genética , Proteínas de Homeodominio/genética , Humanos , Factores de Transcripción/genéticaRESUMEN
Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinal disorders involving the progressive dysfunction of photoreceptors and the retinal pigment epithelium, for which there is currently no treatment. The rd6 mouse is a natural model of autosomal recessive retinal degeneration. Given the known contributions of oxidative stress caused by reactive oxygen species (ROS) and selective inhibition of potent ROS peroxynitrite and OH·by H2 gas we have previously demonstrated, we hypothesized that ingestion of H2 water may delay the progression of photoreceptor death in rd6 mice. H2 mice showed significantly higher retinal thickness as compared to controls on optical coherence tomography. Histopathological and morphometric analyses revealed higher thickness of the outer nuclear layer for H2 mice than controls, as well as higher counts of opsin red/green-positive cells. RNA sequencing (RNA-seq) analysis of differentially expressed genes in the H2 group versus control group revealed 1996 genes with significantly different expressions. Gene and pathway ontology analysis showed substantial upregulation of genes responsible for phototransduction in H2 mice. Our results show that drinking water high in H2 (1.2-1.6 ppm) had neuroprotective effects and inhibited photoreceptor death in mice, and suggest the potential of H2 for the treatment of RP.
Asunto(s)
Agua Potable , Degeneración Retiniana , Retinitis Pigmentosa , Animales , Modelos Animales de Enfermedad , Hidrógeno/metabolismo , Hidrógeno/farmacología , Ratones , Células Fotorreceptoras de Vertebrados/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Degeneración Retiniana/patología , Retinitis Pigmentosa/patologíaRESUMEN
Replication competent oncolytic herpes simplex virus (HSV) vectors have been used extensively to treat solid tumors with promising results. However, highly defective HSV vectors will be needed for applications that require sustained therapeutic gene expression in the absence of vector-related toxicity or inflammation. These vectors require complementing cell lines for their manufacture, creating significant challenges to achieve high yields of infectious virus particles. We recently described an improved upstream process for the production of a non-cytotoxic HSV vector for gene therapy applications. Here, we sought to optimize the downstream conditions for purification and long-term storage of the same vector, JΔNI5. We compared different methods to remove cellular impurities and concentrate the vector by monitoring both physical and biological titers, resulting in the establishment of optimal conditions for vector production. To optimize the long-term storage parameters for non-cytotoxic HSV vectors, we evaluated vector stability at low temperature and sensitivity to freeze-thaw cycles. We report that suboptimal purification and storage methods resulted in loss of vector viability. Our results describe effective and reproducible protocols for purification and storage of HSV vectors for pre-clinical studies.
RESUMEN
Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4/Klf4, c-MYC/c-Myc and RONIN/Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent 'on demand' generation of hiPSCs for personal regenerative and pharmaceutical applications.