Project description:Transcription profiling by high throughput sequencing of mouse embryonic fibroblasts, terminally differentiated neurons and embryonic stem cells
Project description:The inclusion of oocyte factors together with Yamanaka’s previously identified reprogramming factors (OCT4, SOX2, KLF4 with or without cMYC; OSK(M)) may facilitate the reprogramming process that leads to induced pluripotent stem cells (iPSCs). We previously applied label-free LC-MS/MS analysis to search for such facilitators of reprogramming (reprogrammome), resulting in a catalog of 28 candidates that are (i) able to robustly access the cell nucleus, and (ii) shared between mature mouse oocytes and pluripotent embryonic stem (ES) cells. In the present study we hypothesized that our 28 reprogrammome candidates would also be (iii) abundant in mature mouse oocytes, (iv) depleted after oocyte-to-embryo transition, and (v) able to potentiate or replace the OSKM factors during iPSC reprogramming. Using LC-MS/MS and isotopic labeling methods we found that the abundance profiles of the 28 proteins was below that of known oocyte-specific and housekeeping proteins. Out of the 28 proteins only arginine methyltransferase 7 (PRMT7) presented a substantially changing profile during mouse embryogenesis and impacted on the conversion of mouse fibroblasts into iPSCs. PRMT7 indeed could very efficiently replace SOX2 in a factor-substitution assay yielding iPSCs. These findings show that proteomics can be used to prioritize the functional analysis of reprogrammome candidates.
Project description:We performed bulk mRNA sequencing of either control or Zfp266 KO mouse embryonic fibroblasts (MEFs), intermediate reprogramming populations, iPSCs and ESCs. We reveal enhanced and early upregulation of pluripotent and ESC associated genes in Zfp266 KO reprogramming populations.
Project description:Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by Oct4, Sox2, Klf4, plus c-Myc. Recently, Sox2 plus Oct4 were shown to reprogram fibroblasts and Oct4 alone to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here we report that Bmi1 leads to dedifferentiation of mouse fibroblasts into NSC-like cells and, in combination with Oct4, replaces Sox2, Klf4 and c-Myc during reprogramming fibroblasts to iPS cells. Furthermore, activation of sonic hedgehog signalling (by Shh, purmorphamine, or oxysterol) replaces the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One-and two-factor iPS cells are similar to mouse embryonic and adult fibroblasts into iPS cells in global gene expression profile, epigenetic status, in vitro and in bibo differentiation into all three ferm layers, as well as teratoma formation and germline transmission in vivo. These data support that fibroblasts can be reprogrammed into iPS cells by Oct4 alone. Total RNAs were isolated from indicated cells and labeled with Cy3. Hybridization was performed once for each sample.
Project description:PTEN imparts tumor suppression in mice by cell autonomous and non-autonomous mechanisms. Whether these two tumor suppressor mechanisms are mediated through similar or distinct signaling pathways is not known. Here we generated and analyzed knockin mice that express a series of human cancer-derived mutant alleles of PTEN that differentially alter the Akt axis in either stromal or tumor cell compartments of mammary glands. We find that cell non-autonomous tumor suppression by Pten in stromal fibroblasts strictly requires activation of P-Akt signaling, whereas cell autonomous tumor suppression in epithelial tumor cells is independent of overt canonical pathway activation. These findings expose distinct Akt-dependent and independent tumor suppressor functions of PTEN in stromal fibroblasts and tumor cells, respectively, that can be used to guide clinical care of breast cancer patients Wild type, Pten null and PtenF341V primary mouse embryonic fibroblasts isolated from 13.5 day old embryos (E13.5) were cultured, RNA was extracted and Affymetrix gene expression arrays were performed.
Project description:Previously we found that human pluripotent stem cells (hPSCs) utilize glucose differently depending on the presence of the feeder cells, which are mouse embryonic fibroblasts, or MEFs. More specifically, feeder-free cultured hPSCs are more reliant on glycolysis for proliferation. Therefore, we hypothesized that secreted factors by MEFs might be responsible for reprogramming the metabolism of hPSCs. To test this hypothesis, we separated the components in the MEF-conditioned medium by using size-based fractionation columns, and tested whether each fraction alters the reliance of feeder-free hPSCs on glucose. We concluded that it was the protein fraction of the MEF-conditioned medium potentially responsible for reprogramming glycolytic metabolism in hPSCs. To further understand which specific protein(s) could alter the metabolism of hPSCs, we here conduct mass spectrometry based proteomics experiment.