Project description:Reprogramming occurs after nuclear transfer into zygotes whose genomes have been removed in mitosis, but not after nuclear transfer into zygotes enucleated in interphase. Our results suggest that there is a previously unappreciated barrier to successful human nuclear transfer, and that future studies should focus on the requirements for somatic genome activation. 1-3 embryos were used for analysis. RNA amplification was done using two or three rounds of T7-mediated RNA amplification using the Illumina Total Prep RNA Amplification kit. Somatic cells 1-000 and 1-011 required only one round of RNA amplification because starting amounts of RNA were 100-500ng, while embryonic samples were amplified from single cells or embryos.
Project description:Somatic cells can be reprogrammed to pluripotency using different methods. In comparison to pluripotent cells obtained through somatic nuclear transfer, induced pluripotent stem cells (iPSCs) exhibit a higher number of epigenetic errors. Furthermore, most of these abnormalities have been described to be intrinsic to the iPSC technology. Here we investigate whether the aberrant epigenetic patterns detected in iPSCs are specific to transcription factor-mediated reprogramming. We used germline stem cells (GSCs), which are the only adult cell type that can be converted into pluripotent cells (gPSCs) under specific culture conditions, and compared GSC-derived iPSCs and gPSCs at the transcriptomic, epigenetic and functional level. Our results show that both reprogramming methods generate indistinguishable states of pluripotency. GSC-derived iPSCs and gPSCs retained similar levels of donor cell-type memory and exhibited comparable numbers of reprogramming errors. Therefore, our study demonstrates that the epigenetic memory detected in iPSCs is not intrinsic to transcription-factor mediated reprogramming. Total RNA from 12 different in vitro mouse cell lines, 2 technical replicates per sample: germline stem cells (GSCs, 2 independent cell lines), GSC-derived induced pluripotent stem cells (iPSCs, 4 independent cell lines), germline-derived pluripotent stem cells (gPSCs, 4 independent cell lines), embryonic stem cells (ESCs), fibroblast-derived induced pluripotent stem cells (Fib-iPSCs)
Project description:Although nuclear transfer allows the reprogramming of somatic cells to totipotency, little is known concerning the kinetics by which it takes place or the minimum requirements for its success. Here, we demonstrate that reprogramming can be achieved within a few hours and a single cell-cycle as long as two key constraints on reprogramming are satisfied. First, the recipient cell chromosomes must be removed during mitosis. Second, the nuclear envelope of the donor cell must be broken down and its chromosomes condensed, allowing an embryonic nucleus to be constructed around the incoming chromosomes. If these requirements are not met, then reprogramming fails and embryonic development arrests. These results point to a central role for processes intimately linked to cell division in mediating efficient transitions between transcriptional programs. tail tip skin fibroblasts were transferred into mitotic mouse zygotes, blastomeres or oocytes.
Project description:Here we performed genome-wide RNA-seq and Reduced Representation Bisulfite Sequencing (RRBS-seq) in isogenic human induced pluripotent stem cells (iPSCs) and somatic cell nuclear transfer-derived embryonic stem cells (nt-ESCs), genetically matched in vitro fertilization-derived ESCs (IVF-ESCs), and their respective differentiated cells (cardiomyocytes and endothelial cells). We generated the transcriptome and DNA methylome map in human pluripotent stem cells and their differentiated cells with single-nucleotide resolution. We compared the genetic (genetic makeup) and epigenetic (reprogramming approach) influence on the gene expression and DNA methylation profiles and found that genetic composition is the major contributor of the transcriptional and epigenetic variances observed in the undifferentiated and differentiated cells originated from different reprogramming mechanisms.
Project description:Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.