RESUMEN
BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allograft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4(+) T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA (-/-) hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA (-/-) hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA (-/-) hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA (-/-) hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA (-/-) hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4(+) T cells, which are the main effector cells of cellular immunity in allograft.
Asunto(s)
Diferenciación Celular/genética , Desoxirribonucleasas/metabolismo , Eliminación de Gen , Células Madre Embrionarias Humanas/metabolismo , Proteínas Nucleares/genética , Transactivadores/genética , Animales , Células Presentadoras de Antígenos/metabolismo , Antígenos CD/metabolismo , Antígeno B7-2/metabolismo , Autorrenovación de las Células , Células Dendríticas/metabolismo , Desoxirribonucleasas/clasificación , Cuerpos Embrioides/metabolismo , Fibroblastos/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Inmunoglobulinas/metabolismo , Inmunohistoquímica , Interferón gamma/metabolismo , Cariotipo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones SCID , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Teratoma , Células Tumorales Cultivadas , Antígeno CD83RESUMEN
BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allo-graft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4+ T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA(-/-)hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA(-/-)hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA(-/-)hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA(-/-)hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA(-/-)hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4+ T cells, which are the main effector cells of cellular immunity in allograft.
Asunto(s)
Humanos , Animales , Ratones , Proteínas Nucleares/genética , Transactivadores/genética , Diferenciación Celular/genética , Eliminación de Gen , Desoxirribonucleasas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Teratoma , Células Dendríticas/metabolismo , Inmunoglobulinas/metabolismo , Inmunohistoquímica , Glicoproteínas de Membrana/metabolismo , Células Tumorales Cultivadas , Antígenos de Histocompatibilidad Clase II/genética , Antígenos CD/metabolismo , Interferón gamma/metabolismo , Ratones SCID , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Desoxirribonucleasas/clasificación , Antígeno B7-2/metabolismo , Cuerpos Embrioides/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Cariotipo , Fibroblastos/metabolismo , Autorrenovación de las Células , Células Presentadoras de Antígenos/metabolismoRESUMEN
Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease (ZFN) technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1, 3-galactosyl-transferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer (SCNT). Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.
Asunto(s)
Alelos , Endonucleasas/metabolismo , Galactosiltransferasas/genética , Técnicas de Transferencia Nuclear , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Datos de Secuencia Molecular , PorcinosRESUMEN
OBJECTIVE: To evaluate the pathogenicity of SSM-CVB3 in a macaque model. METHODS: The clinical symptoms of macaques were recorded; hematological, biochemical and histopathological evaluations were completed; viral titers and neutralization titers (NT-titers) in sera were tested; and the mRNA levels of SSM-CVB3, coxsackievirus and adenovirus receptor (CAR) and decay accelerating factor (DAF) were determined. RESULTS: After SSM-CVB3 infection, the macaques showed a lack of activity, a poor appetite, a higher body temperature, and severe diarrhea. The macaques also developed hematuria and albuminuria at 4 to 10 days post-inoculation. Virus titers (5.1-6.5 LogTCID(50)/mL) were higher at 6 to 10 days post-inoculation, and NT-titers (6.5-7.3 Log2) reached plateaus at 8 to 14 days post-inoculation. The infected macaques developed serious anemia with decreased RBC and WBC, but the percentages of LYM were increased. The levels of CK, CK-MB, AST and ALT in the sera were 84-169 U/L, 87.6-271.1 U/L, 43-87 U/L and 43-82 U/L, respectively, and all of those were higher than normal. Histological analysis showed obvious cardiac, hepatic and renal damages in the infected macaques and the mRNA contents of SSM-CVB3, CAR and DAF in the heart, liver and kidneys of infected macaques were higher (P<0.05). CONCLUSION: This was the first report on experimental SSM-CVB3 infections in macaques with serious hepatic and renal damage, except for myocarditis. The information obtained from this study suggests that the SSM-CVB3 strain and this macaque model could be used for studying CVB3-induced cardiac, hepatic or renal diseases.
