RESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Doxiciclina/farmacologia , Técnicas Genéticas , Fatores de Transcrição/genética , Animais , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Feminino , Fibroblastos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Provírus/genética , Provírus/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/efeitos dos fármacosRESUMO
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.
Assuntos
Linfócitos B/citologia , Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Animais , Núcleo Celular/genética , Células-Tronco Embrionárias/citologia , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Fatores de Transcrição/metabolismoRESUMO
Genome-wide DNA hypomethylation occurs in many human cancers, but whether this epigenetic change is a cause or consequence of tumorigenesis has been unclear. To explore this phenomenon, we generated mice carrying a hypomorphic DNA methyltransferase 1 (Dnmt1) allele, which reduces Dnmt1 expression to 10% of wild-type levels and results in substantial genome-wide hypomethylation in all tissues. The mutant mice were runted at birth, and at 4 to 8 months of age they developed aggressive T cell lymphomas that displayed a high frequency of chromosome 15 trisomy. These results indicate that DNA hypomethylation plays a causal role in tumor formation, possibly by promoting chromosomal instability.
