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1.
Journal of Southern Medical University ; (12): 929-936, 2022.
Artículo en Chino | WPRIM | ID: wpr-941023

RESUMEN

OBJECTIVE@#To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms.@*METHODS@#EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.@*RESULTS@#The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05).@*CONCLUSION@#Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.


Asunto(s)
Humanos , Equinomicina/metabolismo , Cuerpos Embrioides/metabolismo , Hipoxia/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , beta Catenina/metabolismo
2.
Biol. Res ; 48: 1-9, 2015. ilus, graf
Artículo en Inglés | LILACS | ID: biblio-950823

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. 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/metabolismo
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