RESUMEN
Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Doxiciclina/farmacología , Técnicas Genéticas , Factores de Transcripción/genética , Animales , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Femenino , Fibroblastos/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Provirus/genética , Provirus/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/efectos de los fármacosRESUMEN
Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.