RESUMEN
Glycosphingolipids (GSLs) are glycoconjugates that function as mediators of cell adhesion and modulators of signal transduction. Some well-defined markers of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are glycoconjugates, such as SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. However, Comprehensive GSL profiles of hiPSCs have not yet been elucidated. The global images of GSLs from the parental cells, hiPSCs, and differentiated cells revealed that there are parental cell-independent specific glycolipids, including Globo H (fucosyl-Gb5Cer) and H type1 antigen (fucosyl-Lc4Cer) that are novel markers for undifferentiated hiPSCs. Interestingly, undifferentiated hiPSCs expressed H type 1 antigen, specific for blood type O, regardless of the cells' genotypes. Thus, in this study, we defined the dynamics of GSL remodeling during reprogramming from parental cell sets to iPSC sets and thence to iPSC-neural cells.
Asunto(s)
Diferenciación Celular , Glucolípidos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Sistema del Grupo Sanguíneo ABO/genética , Biomarcadores , Línea Celular , Cromatografía Liquida , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Citometría de Flujo , Variación Genética , Genotipo , Glicoesfingolípidos/metabolismo , Humanos , Inmunohistoquímica , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Polisacáridos/biosíntesis , Espectrometría de Masas en TándemRESUMEN
CD98hc is a type II transmembrane protein that covalently links to one of several L-type amino acid transporters. CD98hc was first identified as a lymphocyte activation marker. In this study, we examined the role that CD98hc plays in the functions of macrophages using tissue specific knock-out miceCD98hc (CD98hc(flox/-)LysM-cre mice). When isolated peritoneal macrophages were incubated for 48 h, the macrophages obtained from the knock-out mice showed round-shaped morphologies, while almost all of the cells obtained from the control mice were spindle-shaped. The macrophage functions such as the antigen-presenting, phagocytic, and fusion activities, have been reported to decrease in CD98hc-deficient peritoneal macrophages. In addition, when the CD98hc deficient macrophages were stimulated with either IFN-γ/LPS or IL-4, the production of NO(2) or arginase-I decreased in comparison to that observed in the control macrophages. These findings show that the CD98hc molecules play an important role in the activation and functions of macrophages.
Asunto(s)
Cadena Pesada de la Proteína-1 Reguladora de Fusión/inmunología , Macrófagos Peritoneales/inmunología , Animales , Células Cultivadas , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Ratones , Ratones Noqueados , FagocitosisRESUMEN
From June 1996 to January 2001, 91 patients with B-cell non-Hodgkin lymphoma or B-cell acute lymphoblastic leukemia up to 18 years of age were enrolled in Tokyo Children's Cancer Study Group (TCCSG) NHL B9604 protocol study. Five-day intensive chemotherapy courses including high-dose methotrexate and high-dose cyclophosphamide were used for localized disease (Groups A and B). High-dose cytarabine was added for advanced disease (Groups C and D). Fifteen patients experienced an adverse event. There were three induction failures, eight relapses (three local, four bone marrow (BM), one BM + local), two toxic deaths and two second malignant neoplasm. Event-free survival at 6 years in Group D and in all patients was 82.4% +/- 9.2% and 81.9% +/- 4.4%, respectively. The TCCSG NHL B9604 protocol achieved an excellent treatment outcome especially in patients with the most advanced disease (Group D: high BM blast cell burden and/or central nervous system involvement).
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Linfoma de Burkitt/mortalidad , Niño , Preescolar , Ciclofosfamida/administración & dosificación , Citarabina/administración & dosificación , Humanos , Lactante , Metotrexato/administración & dosificación , Neoplasias Primarias Secundarias , Recurrencia , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.
Asunto(s)
Clonación de Organismos/métodos , Cabras , Técnicas de Transferencia Nuclear , Oocitos , Adenohipófisis/citología , Anomalías Múltiples/veterinaria , Animales , Temperatura Corporal , Peso Corporal , Células Cultivadas , Clonación de Organismos/efectos adversos , Medios de Cultivo , ADN Mitocondrial , Transferencia de Embrión , Femenino , Genotipo , Complejo Mayor de Histocompatibilidad , Masculino , Ovario/citologíaRESUMEN
To generate severe combined immunodeficient (SCID) livestocks for xenotransplantation, we have attempted to generate a SCID phenotype without gene knockout. Based on the reported mouse RAG1 mutants, we constructed the corresponding rabbit RAG1 mutants by mutagenesis of three residues within the catalytic domain: D602A, D710A, and E964A. As expected, these mutants each exhibited no catalytic activity on artificial substrates and inhibited recombination by the wild type RAG1. Moreover, replacement of the N-terminus of RAG1 with enhanced green fluorescent protein (EGFP) greatly increased protein stability, and the triple mutant RAG1 showed a twofold increase in its ability to inhibit wild type activity in vitro. We generated mice transgenic for the latter mutant to assess its effect on V(D)J recombination in vivo. Serum IgM levels in four out of seven transgenic mice were reduced to approximately 30-50% of control levels in four out of seven transgenic mice. Our results suggest that immunodeficient animals for regenerative medicine could be generated without gene knockout.