RESUMEN
Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.
Asunto(s)
Reprogramación Celular , Histonas , Células Madre Pluripotentes Inducidas , Factor 4 Similar a Kruppel , Reprogramación Celular/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Histonas/metabolismo , Diferenciación Celular/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Cromatina/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , TranscriptomaRESUMEN
Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.