RESUMEN
Differentiating somatic cells are progressively restricted to specialized functions during ontogeny, but they can be experimentally directed to form other cell types, including those with complete embryonic potential. Early nuclear reprogramming methods, such as somatic cell nuclear transfer (SCNT) and cell fusion, posed significant technical hurdles to precise dissection of the regulatory programmes governing cell identity. However, the discovery of reprogramming by ectopic expression of a defined set of transcription factors, known as direct reprogramming, provided a tractable platform to uncover molecular characteristics of cellular specification and differentiation, cell type stability and pluripotency. We discuss the control and maintenance of cellular identity during developmental transitions as they have been studied using direct reprogramming, with an emphasis on transcriptional and epigenetic regulation.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Expresión Génica Ectópica , Epigénesis Genética , Técnicas de Transferencia Nuclear , Células Madre Pluripotentes/metabolismo , Animales , Humanos , Células Madre Pluripotentes/citologíaRESUMEN
Input from various signaling pathways in conjunction with specific transcription factors (TFs), noncoding RNAs, and epigenetic modifiers governs the maintenance of cellular identity. Endogenous or exogenous TFs operate within certain boundaries, which are set, in part, by the cell type-specific epigenetic landscape. Ectopic expression of selected TFs can override the cellular identity and induce reprogramming to alternative fates. In this minireview, we summarize many of the classic examples and a large number of recent studies that have taken advantage of TF-mediated reprogramming to produce cell types of biomedical relevance.
Asunto(s)
Desdiferenciación Celular , Epigénesis Genética , Factores de Transcripción/metabolismo , Humanos , Factores de Transcripción/genéticaRESUMEN
Ectopic transcription factor expression enables reprogramming of somatic cells to pluripotency, albeit with generally low efficiency. Despite steady progress in the field, the exact molecular mechanisms that coordinate this remarkable transition still remain largely elusive. To better characterize the final steps of pluripotency induction, we optimized an experimental system where pluripotent stem cells are differentiated for set intervals before being reintroduced to pluripotency-supporting conditions. Using this approach, we identify a transient period of high-efficiency reprogramming where ectopic transcription factors, but not serum/LIF alone, rapidly revert cells to pluripotency with near 100% efficiency. After this period, cells reprogram with somatic-like kinetics and efficiencies. We identify a set of OCT4 bound cis-regulatory elements that are dynamically regulated during this transient phase and appear central to facilitating reprogramming. Interestingly, these regions remain hypomethylated during in vitro and in vivo differentiation, which may allow them to act as primary targets of ectopically induced factors during somatic cell reprogramming.
Asunto(s)
Diferenciación Celular/fisiología , Reprogramación Celular/fisiología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Fibroblastos , Regulación de la Expresión Génica , Genómica , Cinética , Ratones , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Células MadreRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The original version of this Article omitted the following from the Acknowledgements: "G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721)."This has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Genotipo , Células Madre Pluripotentes/fisiología , Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reparación del ADN , Edición Génica/métodos , Frecuencia de los Genes , Ingeniería Genética , Vectores Genéticos/genética , Recombinación Homóloga , Humanos , Patología Molecular , Donantes de TejidosRESUMEN
Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.
Asunto(s)
ADN Complementario/genética , Edición Génica/métodos , Trasplante de Células Madre Hematopoyéticas , Subunidad gamma Común de Receptores de Interleucina/genética , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/terapia , Animales , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular , Codón Iniciador/genética , Dependovirus , Exones/genética , Sangre Fetal/citología , Vectores Genéticos/genética , Voluntarios Sanos , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Ratones , Mutación , Parvovirinae/genética , Cultivo Primario de Células , Factores de Tiempo , Transducción Genética/métodos , Quimera por Trasplante/genética , Trasplante Heterólogo/métodos , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genéticaRESUMEN
A report of the 'Joint Keystone Symposium on Epigenomics and Chromatin Dynamics', Keystone, Colorado, 17-22 January 2012.