Unknown,Transcriptomics,Genomics,Proteomics

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Derivation of rat induced pluripotent stem cells that share similarities with embryonic stem cells using a transposon system

ABSTRACT: Induced pluripotent stem (IPS) cells have attracted enormous attention due to their vast potential in regenerative medicine, pharmaceutical screening and basic research. The majority of prior established rat IPS cells were generated from somatic cells by retroviral and lentiviral transduction with expression of Oct4, Sox2, Klf4 and c-Myc and using chemical inhibitors of key differentiation pathways. A major difficulty in the application of this technology is the efficient delivery of reprogramming factors and the long-term maintenance of properties of stem cells. Here, we employed the PiggyBac (PB) transposon carrying four 2A peptide-linked reprogramming factors for generating rat IPS cells. These stable rat IPS cells are similar to embryonic stem (ES) cells in morphology, proliferation, teratoma formation, expression characteristic pluripotency markers, developmental potential, and germline transmission. Transcriptional profiling of the IPS cells revealed both pathways in common with ES cells from rat and unique signaling pathway to our cells, including Wnt, TGF and Notch. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying germline pluripotency and pave the way for exploration of cell-based therapies using the rat. To compare the gene expression profiling between rat IPS cells and ES cells to show if the rat IPS cells had been reprogrammed into pluripotent status like rat ES cells at the gene expression level.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Sheng Yang

PROVIDER: E-GEOD-35841 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen, a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified key pathways and regulators of prostatic differentiation including Wnt5a, BPM7, PTEN, and NKx3.1 using E-PZ-iPS-lke cells. Our results suggest that iPS-like cells can be derived from prostatic epithelial cells. These cells are pluripotent and capable of prostatic differentiation, and therefore provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. Bisulphite converted DNA from the 10 samples were hybridised to the Illumina Infinium HumanMethylation450K Beadchip v1.2

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.

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Derivation of rat induced pluripotent stem cells that share similarities with embryonic stem cells using a transposon system

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