Project description:Nanog null neural stem (NS) cells were reprogrammed to naive pluripotency in 2i/LIF conditions with chick (c) and zebrafish (z) Nanog orthologs. Global gene expression was compared to iPS cells derived with mouse (m) Nanog. Murine iPS cells derived with zebrafish nanog, chick nanog, and mouse nanog orthologs (2 replicates each).
Project description:This SuperSeries is composed of the following subset Series: GSE32464: Global gene expression analysis in murine iPS cells derived with mouse and human Nanog orthologs GSE32650: Global gene expression analysis in murine iPS cells derived with mouse, chick and zebrafish Nanog orthologs Refer to individual Series
Project description:Nanog null neural stem (NS) cells were reprogrammed to naive pluripotency in 2i/LIF conditions with chick (c) and zebrafish (z) Nanog orthologs. Global gene expression was compared to iPS cells derived with mouse (m) Nanog.
Project description:Nanog null neural stem (NS) cells were reprogrammed to naive pluripotency in 2i/LIF conditions with mouse (m) Nanog and human (h) Nanog. Global gene expression in resulting iPS cells was compared to embryonic stem (ES) cells and nanog null NS cells. Murine iPS cells derived with mouse nanog iPS and human nanog iPS and then compared to embryonic stem cells and nanog null neural stem cells (3 replicates each).
Project description:Nanog null neural stem (NS) cells were reprogrammed to naive pluripotency in 2i/LIF conditions with mouse (m) Nanog and human (h) Nanog. Global gene expression in resulting iPS cells was compared to embryonic stem (ES) cells and nanog null NS cells.
Project description:In this study we have compared functional and molecular properties of highly purified murine induced pluripotent stem (iPS) cell- and embryonic stem (ES) cell-derived cardiomyocytes (CM). In order to obtain large amounts of purified CM, we have generated a transgenic murine iPS cell line, which expresses puromycin resistance protein N-acetyltransferase and EGFP under the control of the cardiomyocyte-specific α-myosin heavy chain promoter (alphaMHC-Puro-IRES-GFP, aPiG). We demonstrate that murine aPIG-iPS and aPIG-ES cells differentiate into spontaneously beating CM at comparable efficiencies. When selected with puromycin both cell types yielded more than 97% pure population of CMs. Both aPIG-iPS and aPIG-ES cell-derived CM express typical cardiac transcripts and structural proteins and possess similar sarcomeric organization. Action potential recordings revealed that iPS- and ES cell-derived CM respond to beta-adrenergic and muscarinic receptor modulation, express functional voltage-gated sodium, calcium and potassium channels and possess comparable current densities. Comparison of global gene expression profiles of CM generated from iPS and ES cells revealed that both cell types cluster close to each other but are highly distant to undifferentiated ES or iPS cells as well as unpurified iPS and ES cell-derived embryoid bodies (EB). Both iPS and ES cell-derived CMs express genes and functional categories typical for CM. They are enriched in genes involved in transcription and genes coding for structural proteins involved in cardiac muscle contraction and relaxation. They also express genes involved in heart and muscle developmental processes, ion export and ion binding processes and various metabolic processes for ATP synthesis. These CMs downregulate genes involved in immune response, cell cycle and cell division, thus demonstrating the CMs population is mitotically inactive. Most surface signaling pathways are also downregulated. Thus, a transgenic aPiG-iPS cell line can provide a robust supply of highly purified and functional CMs for future in vitro and in vivo studies. Seven different experimental groups were included into analysis: undifferentiated murine ES cells (1) and undifferentiated murine iPS cells (2), murine ES cell-derived embyroid bodies (3) and murine iPS cell-derived embryoid bodies at day 16 of differentiation (4), murine ES cell-derived cardiomyocytes (5) and murine iPS cell-derived cardiomyocytes (6) at day 16 of differentiation (they were generated by puromycin selection for 7 days prior to RNA isolation). Adult mouse tail tip fibroblasts (7) were used as a control for iPS cells. Total RNA samples were prepared from three independent biological replicates in groups 1-6. In group 7, single RNA probes were analyzed as three technical replicates.
Project description:It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical reprogramming factors Oct4, Sox2, Klf4 and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog-/- fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPS cells from Nanog-/- fibroblasts that effectively contributed to chimeric mice. Thus while Nanog may be an important mediator of reprogramming it is not required for establishing pluripotency in the mouse, even under standard conditions. In order to further evaluate the equivalency of Nanog null iPSC to nanog null ESCs, we have performed RNAseq on two independent nanog null iPSC lines, as well as Nanog Null ESC, WT ESC and iPSCs as well as MEFs. As a negativve control for reprogramming we have analyzed a partially reprogrammed iPSC line.
Project description:Whole genome sequencing was performed on several murine iPS cell clones (and their parental cells) from each of three independent reprogramming experiments. Hundreds of single nucleotide variants (SNVs) were detected in each clone, with an average of 11 in coding regions. Affymetrix Mouse Exon 1.0ST arrays were used to compare expression patterns in MPSVII iPS lines, and embryo-derived MPSVII ES cells. Unsupervised hierarchal clustering analysis showed that the iPS clones and ES cell lines clustered randomly, suggesting that their global patterns of gene expression are highly similar. Taken together, our data suggest that most of the genetic variation in iPS cell clones is not caused by reprogramming, but is rather a consequence of cloning individual cells, “capturing” random mutations that preexisted in the single cells that were reprogrammed. These mutations can sometimes contribute to reprogramming “fitness”, thus providing a selective advantage for rare cells when they overexpress reprogramming factors. Mouse embryonic fibroblasts (MEFs) derived from a murine disease model (Mucopolysaccaridosis type VII- MPSVII) were used. Affymetrix Mouse Exon 1.0ST arrays were used to compare expression patterns in MPSVII iPS lines, and embryo-derived MPSVII ES cells. Expression patterns in four separate iPS clones were compared to MPSVII ES cells. Control hybridization was performed with B6 Blu ES cells and MEFs.