ABSTRACT
The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63(-/-) epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63(-/-) epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.
Subject(s)
Down-Regulation/genetics , Keratinocytes/metabolism , Multipotent Stem Cells/cytology , Phosphoproteins/genetics , Proteins/genetics , RNA-Binding Proteins/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Adult , Animals , Cell Differentiation , Cell Line , Cell Lineage , Cell Proliferation , Chimera , Embryo, Mammalian/cytology , Epidermal Cells , Gene Expression Profiling , Homeodomain Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Keratinocytes/cytology , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Multipotent Stem Cells/metabolism , Nanog Homeobox Protein , Octamer Transcription Factor-3/metabolism , Phosphoproteins/deficiency , Phosphoproteins/metabolism , Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , SOXB1 Transcription Factors/metabolism , Trans-Activators/deficiency , Trans-Activators/metabolism , Transcription Factors/deficiency , Transcription Factors/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/metabolismABSTRACT
The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30-75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.
