RESUMO
We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match â¼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Povo Asiático/genética , Eletroporação , Perfilação da Expressão Gênica , Frequência do Gene , Vetores Genéticos , Antígenos HLA/genética , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Plasmídeos/genética , Doadores de TecidosRESUMO
Telomerase is critically important for the maintenance of a constant telomere length, which in turn, is related to the concepts of longevity and oncogenesis. In addition, it has been well documented that telomerase activity is expressed in immune cells in a highly regulated manner. We have studied systemic anaphylaxis in mouse telomerase reverse transcriptase knockout (mTERT(-/-)) mice to understand the significance of telomerase activity and telomere stability in mast cells, which induce a type I allergic response. Compared with wild-type mice, mTERT(-/-) mice displayed largely attenuated, IgE-mediated, passive anaphylactic responses, which were observed even in the early generations of mTERT(-/-) mice, and had decreased numbers of mast cells in vivo and impaired development of bone marrow-derived mast cells (BMMCs) induced by IL-3 or stem cell factor in vitro. Moreover, in mTERT(-/-) mice, BMMCs exhibited a large morphology and low proliferation rate, while they possessed a comparable degranulation capacity and cell surface expression level of c-kit and FcepsilonRI. These findings imply that telomerase activity has a definitive impact on the type I allergic response by altering the character of effecter mast cells.
Assuntos
Anafilaxia/genética , Anafilaxia/imunologia , Deleção de Genes , Mastócitos/imunologia , Telomerase/genética , Anafilaxia/patologia , Animais , Feminino , Histamina/sangue , Histocitoquímica , Masculino , Mastócitos/metabolismo , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Telomerase/análiseRESUMO
In the production of large-volume parenterals in Japan, equipment and devices such as tanks, pipework, and filters used in production processes are exhaustively cleaned and sterilized, and the cleanliness of water for injection, drug materials, packaging materials, and manufacturing areas is well controlled. In this environment, the bioburden is relatively low, and less heat resistant compared with microorganisms frequently used as biological indicators such as Geobacillus stearothermophilus (ATCC 7953) and Bacillus subtilis 5230 (ATCC 35021). Consequently, the majority of large-volume parenteral solutions in Japan are manufactured under low-heat sterilization conditions of F0 <2 min, so that loss of clarity of solutions and formation of degradation products of constituents are minimized. Bacillus oleronius (ATCC 700005) is listed as a biological indicator in "Guidance on the Manufacture of Sterile Pharmaceutical Products Produced by Terminal Sterilization" (guidance in Japan, issued in 2012). In this study, we investigated whether B. oleronius is an appropriate biological indicator of the efficacy of low-heat, moist-heat sterilization of large-volume parenterals. Specifically, we investigated the spore-forming ability of this microorganism in various cultivation media and measured the D-values and z-values as parameters of heat resistance. The D-values and z-values changed depending on the constituents of large-volume parenteral products. Also, the spores from B. oleronius showed a moist-heat resistance that was similar to or greater than many of the spore-forming organisms isolated from Japanese parenteral manufacturing processes. Taken together, these results indicate that B. oleronius is suitable as a biological indicator for sterility assurance of large-volume parenteral solutions subjected to low-heat, moist-heat terminal sterilization.
Assuntos
Bacillus/isolamento & purificação , Indústria Farmacêutica/normas , Soluções de Nutrição Parenteral/normas , Esterilização/normas , Bacillus/fisiologia , Meios de Cultura/análise , Meios de Cultura/normas , Indústria Farmacêutica/métodos , Humanos , Indicadores e Reagentes/análise , Indicadores e Reagentes/normas , Soluções de Nutrição Parenteral/análise , Soluções Farmacêuticas/análise , Soluções Farmacêuticas/normas , Esterilização/métodosRESUMO
Telomerase, the reverse transcriptase that maintains telomere DNA, is usually undetectable in most adult tissues but is positive in embryonic tissues and in cancers. In addition, freshly islolated or in vitro-activated lymphocytes were shown to express high levels of telomerase activity, although its expression in myeloid cells including dendritic cells (DCs) is largely unknown. Here, we investigated telomerase activity during the differentiation and maturation process of DCs. In vitro culture of bone marrow (BM) cells with granulocyte macrophage-colony stimulating factor and interleukin-4 induced a dramatic increase of telomerase activity accompanied with their differentiation into DCs. Furthermore, stimulation with microbial components such as lipopolysaccharide (LPS), which triggers maturation of DCs, augmented the activity. In vivo responses of telomerase activity were also observed in splenic DCs by injection of LPS intraperitoneally. It is interesting that in old mice, telomerase activity of splenic DCs was significantly higher than young mice but rather decreased after LPS stimulation. By measuring expression of cell-surface activation markers, splenic DCs of old mice responded poorly to LPS stimulation. Such poor responses to LPS were also observed in BM-derived DCs. These different features of DCs between young and old mice may contribute to a pathogenesis to microbial infections.
Assuntos
Diferenciação Celular/fisiologia , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Telomerase/metabolismo , Animais , Células da Medula Óssea/citologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Injeções Intraperitoneais , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Baço/metabolismo , Regulação para CimaRESUMO
Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inativação do Cromossomo X/genética , Diferenciação Celular/genética , Linhagem Celular , Cromossomos Humanos X/genética , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Feminino , Regulação da Expressão Gênica , Genes Ligados ao Cromossomo X , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Análise de Sequência de DNARESUMO
We evaluated the teratoma-forming propensity of secondary neurospheres (SNS) generated from 36 mouse induced pluripotent stem (iPS) cell lines derived in 11 different ways. Teratoma-formation of SNS from embryonic fibroblast-derived iPS cells was similar to that of SNS from embryonic stem (ES) cells. In contrast, SNS from iPS cells derived from different adult tissues varied substantially in their teratoma-forming propensity, which correlated with the persistence of undifferentiated cells.
Assuntos
Células-Tronco Pluripotentes/citologia , Segurança , Animais , Linhagem Celular , Transformação Celular Neoplásica/patologia , Camundongos , Neurônios/citologia , Teratoma/patologiaRESUMO
The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.6% of 1-cell embryos from the C57BL mouse strain developed to the blastocyst stage, but 95.7% of embryos from the AKR mouse strain showed 2-cell block. Phosphate-free culture medium rescued the 2-cell block of AKR embryos and accelerated the first cell cycle of the embryos. Co-culture with BRL cells and a BRL-conditioned medium fractionated below 30 kDa also rescued the 2-cell block of AKR embryos. Examinations of in vitro development of reconstructed embryos and of embryos from F1 females between AKR and C57BL strains clearly demonstrated that the AKR cytoplast caused the 2-cell block. In the backcrossed female progeny between (AKR x C57BL) F1 males and AKR females, about three-quarters of the embryos were of the 2-cell blocking phenotype and about one-quarter were of the non-blocking phenotype. These results suggest that two genes are responsible for the 2-cell block of AKR embryos.
Assuntos
Ciclo Celular/fisiologia , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/metabolismo , Fosfatos/fisiologia , Animais , Ciclo Celular/genética , Células Cultivadas , Técnicas de Cocultura , Feminino , Masculino , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , FenótipoRESUMO
Ig-like transcripts (ILT/leukocyte Ig-like receptor/monocyte/macrophage Ig-like receptor or CD85) are encoded on human chromosome 19q13.4, designated the human leukocyte receptor complex, and are predominantly expressed on myeloid lineage cells. We investigated the transcriptional regulation of ILT1, ILT2, and ILT4 genes to elucidate control mechanisms operating on the specific expression of ILT receptors. Inhibitory ILT2 and ILT4 both have a similar genomic structure, in which the approximately 160-bp 5'-flanking regions function as core promoters with critically important PU.1 binding sites. However, an Sp1 family-binding GC-box is more influential in trans-activation of ILT2 than ILT4. Additionally, ILT4 transcription is tightly regulated by chromatin modifications accompanied by histone acetylation, which strictly controls expression within myeloid lineage cells. Activating ILT1 carries a core promoter corresponding to the intronic region of ILT2 and ILT4, where PU.1 and Runx1 binding sites are essential, but a downstream heat shock element also augments promoter activity. Thus, each ILT is regulated by a distinct transcriptional mechanism, although PU.1 acts as a common trans-acting factor. We also found that human CMV infection strongly trans-activates inhibitory ILT2 and ILT4 genes through the expression of immediate-early proteins.
Assuntos
Antígenos CD/genética , Família Multigênica/imunologia , Receptores Imunológicos/genética , Antígenos CD/química , Antígenos CD/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Citomegalovirus/imunologia , Éxons , Regulação da Expressão Gênica/imunologia , Genes Precoces/fisiologia , Humanos , Células Jurkat , Células K562 , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Glicoproteínas de Membrana , Dados de Sequência Molecular , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Sítio de Iniciação de Transcrição , Transcrição Gênica , Ativação Transcricional , Células U937 , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
The object of the present study was to investigate the validation of the sperm quality analyzer (SQA) and the hypo-osmotic swelling (HOS) test with standard sperm analysis methods in frozen-thawed ram and minke whale spermatozoa. In rams, highly significant correlations were observed in the percentage of motile spermatozoa (P<0.01) and sperm concentration (P<0.01) between the standard and SQA methods. But, the percentage of morphologically normal spermatozoa did not significantly correlate between the standard and SQA methods. The percentages of swollen spermatozoa at 15 minutes by the HOS test were significantly correlated with the motility by the standard (P<0.05) and by the SQA (P<0.05) methods. For minke whale spermatozoa, the SVI (sperm viability index) values by the standard method were significantly (P<0.001) correlated with the sperm motility index (SMI) values by SQA. The percentage of motile spermatozoa was also significantly correlated (P<0.01) with the motility measured by SQA. Using different hypo-osmotic solutions and incubation times, the HOS test with 25, 100 and 150 mOsM did not show significant variations. Motility observed by the standard method and the percentage of swollen spermatozoa were significantly correlated (P<0.05). These results indicate that the SQA and HOS test can be utilized to assess the post-thawing motility of ram and minke whale spermatozoa, and that the SQA and HOS test values are significantly correlated in ram spermatozoa. However, sperm concentration and morphologically normal spermatozoa are not assessed accurately by SQA in minke whales.