Generation of parthenogenetic iPS cells from parthenogenetic neural stem cell
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Marcos Araúzo-Bravo
PROVIDER: E-GEOD-15561 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Generation of parthenogenetic induced pluripotent stem cells from parthenogenetic neural stem cells.
Do Jeong Tae JT Joo Jin Young JY Han Dong Wook DW Araúzo-Bravo Marcos J MJ Kim Min Jung MJ Greber Boris B Zaehres Holm H Sobek-Klocke Ingeborg I Chung Hyung Min HM Schöler Hans R HR
Stem cells (Dayton, Ohio) 20091201 12
Somatic cells can achieve a pluripotent cell state in a process called pluripotential reprogramming. Multipotent stem cells can differentiate into cells of only one lineage, but pluripotent stem cells can give rise to cells of all three germ layers of an organism. In this study, we generated induced pluripotent stem (iPS) cells from bimaternal (uniparental) parthenogenetic neural stem cells (pNSCs) by transduction with either four (4F: Oct4, Klf4, Sox2, and c-Myc) or two (2F: Oct4 and Klf4) tran ...[more]
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Project description:In pluripotential reprogramming, a pluripotent state is established within somatic cells. In this study, we have generated induced pluripotent stem (iPS) cells from bi-maternal (uniparental) parthenogenetic neural stem cells (pNSCs) by transduction with four (Oct4, Klf4, Sox2, and c-Myc) or two (Oct4 and Klf4) transcription factors. The parthenogenetic iPS (piPS) cells directly reprogrammed from pNSCs were able to generate germline-competent himeras, and hierarchical clustering analysis showed that piPS cells were clustered more closer to parthenogenetic ES cells than normal female ES cells. Interestingly, piPS cells showed loss of parthenogenetic-specific imprinting patterns of donor cells. Microarray data also showed that the maternally imprinted genes, which were not expressed in pNSCs, were upregulated in piPS cells, indicating that pluripotential reprogramming lead to induce loss of imprinting as well as re-establishment of various features of pluripotent cells in parthenogenetic somatic cells.
2010-01-23 | GSE15561 | GEO
Project description:Pluripotency can be induced in murine and human fibroblast by transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc). Previously we reported that two factors (Oct4 and Klf4) are sufficient for reprogramming adult mouse neural stem cells (NSCs) to a pluripotent state. However, although NSCs endogenously express the factors Sox2, c-Myc, and Klf4, our previous report does not elucidate why exogenous expression of either Klf4 or c-Myc is still required for reprogramming. Here we report that exogenous expression of Oct4 is sufficient to generate one-factor induced pluripotent stem (1F iPS) cells without any oncogenic factors, such as c-Myc and Klf4, from mouse adult NSCs, which endogenously express Sox2, c-Myc, and Klf4, and also intermediate reprogramming markers alkaline phosphatase (AP), stage-specific embryonic antigen-1 (SSEA-1). These results extend our previous report proposing that somatic cells can be reprogrammed to a pluripotent state with a reducing number of reprogramming factors when the complementing factors are endogenously expressed in the somatic cells. Experiment Overall Design: 10 hybridizations in total. Experiment Overall Design: NSC-derived iPS cells by one-factor (Oct4) in triplicate: Experiment Overall Design: - NSC_1F_iPS_1 Experiment Overall Design: - NSC_1F_iPS_2 Experiment Overall Design: - NSC_1F_iPS_3 Experiment Overall Design: One-factor (Oct4) iPS cell-derived NSC in triplicate: Experiment Overall Design: - 1F_iPS_NSC_1 Experiment Overall Design: - 1F_iPS_NSC_2 Experiment Overall Design: - 1F_iPS_NSC_3 Experiment Overall Design: Neural stem cell (NSC) derived from brain of OG2/Rosa26 mice: Experiment Overall Design: - NSC_1 Experiment Overall Design: - NSC_2 Experiment Overall Design: - NSC_3 Experiment Overall Design: - NSC_4
2009-02-08 | E-GEOD-12499 | biostudies-arrayexpress
Project description:Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility to generate patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Considering that ectopic expression of c-Myc causes tumourigenicity in offspring and retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here, we show that adult mouse neural stem cells (NSCs) express higher endogenous levels of Sox2 and c-Myc than ES cells and that exogenous Oct4 together with either Klf4 or c-Myc are sufficient to generate iPS cells from NSCs. These two-factor (2F) iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germline, and form chimeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors. Experiment Overall Design: 8 hybridizations in total. Experiment Overall Design: NSC derived iPS cells by 2 factors (Oct4 and Klf4) in triplicate: Experiment Overall Design: - iPS cell_2F_1 Experiment Overall Design: - iPS cell_2F_2 Experiment Overall Design: - iPS cell_2F_3 Experiment Overall Design: Embryonic Stem cells (ESC) in triplicate: Experiment Overall Design: - ESC_1 Experiment Overall Design: - ESC_2 Experiment Overall Design: - ESC_3 Experiment Overall Design: NSC cultures in duplicates: Experiment Overall Design: - NSC_2 Experiment Overall Design: - NSC_3 Experiment Overall Design: NSC derived iPS cells by 4 factors (Oct4, Sox2, c-Myc and Klf4) in triplicate: Experiment Overall Design: - iPS cell_4F_1 Experiment Overall Design: - iPS cell_4F_2 Experiment Overall Design: - iPS cell_4F_3
2008-10-20 | E-GEOD-10806 | biostudies-arrayexpress
Project description:Brief expression of pluripotency-associated factors such as OCT4, KLF4, SOX2 and c-MYC (OKSM), in combination with differentiation-inducing signals, was reported to trigger transdifferentiation of fibroblasts into alternative cell types. Here, we show that OKSM expression gives rise to both induced pluripotent stem cells (iPSCs) and iNSCs under conditions that were previously shown to induce only NSC transdifferentiation. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing via an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originate from cells transiently expressing Oct4, whereas ablation of Oct4-positive cells prevented iNSC formation. Lastly, use of an alternative transdifferentiation cocktail that lacks OCT4 and was reportedly unable to support induced pluripotency, yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation using OKSM and related reprogramming factors requires passage through a transient iPSC state. 5 samples were anlyzed in total, 2 induced pluripotent stem cells (iPSCs), 1 neural stem cells (NSCs) and 2 induced NSCs (iNSCs)
2015-04-30 | E-GEOD-65951 | biostudies-arrayexpress
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.
2010-09-18 | E-GEOD-24208 | biostudies-arrayexpress
Project description:The differentiation capability of induced pluripotent stem cells (iPSCs) towards certain cell types for disease modelling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from either fetal neural stem cells (fNSCs), dermal fibroblasts or CD34+ hematopoietic stem cells. Neural progenitor cells (NSCs) and neurons could be generated at similar efficiencies from all iPSCs. Whole genome transcriptome analysis and an expression analysis of neural genes in iPSC-derived NSCs revealed a separation of neuroectoderm- from mesoderm-derived cells. Furthermore, we found genes, which were similarly expressed between fNSCs and neuroectoderm- but not mesoderm-derived NSCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival and integration of neuroectoderm-derived cells in vivo. These results indicated distinct origin- dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The RNA samples from in vivo transplanted and FACS sorted cells were amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre, USA) from 100 pg to 50 microg. 43 samples were analyzed Fib, Human fibroblast F134, 2 replicates CD34+, Human Cord Blood CD34+ Hematopoyetic Stem Cell (HSC), 1 replicate fNSC, Human fetal Neural Stem Cells (NSC), 1 replicate fNSC-1FiPSCa-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates fNSC-1FiPSCb-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates fNSC-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCc-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone c, in vitro differentiated to NSC, 1 replicate H1-ESC-NSC, Human H1 embryonic stem cell (ESC), in vitro differentiated to NSC, 2 replicates HUES6-ESC-NSC, Human HUES6 embryonic stem cell (ESC), in vitro differentiated to NSC, 1 replicate fNSC-1FiPSCa, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate fNSC-1FiPSCb, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, 1 replicate fNSC-2FiPSCa, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-2FiPSCa, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, 1 replicate Fib-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, 1 replicate Fib-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, 1 replicate H1-ESC, Human H1 embryonic stem cell (ESC), 3 replicates HUESC6-ESC, Human HUESC6 embryonic stem cell (ESC) , 2 replicates Fib-4FiPSCa-NSC-In Vivo, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vivo differentiated to NSC, 2 replicates fNSC-1FiPSCa-NSC-In Vivo, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vivo differentiated to NSC, 4 replicates
2014-09-12 | E-GEOD-55107 | biostudies-arrayexpress
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.
2010-09-18 | GSE24208 | GEO
Project description:Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) following the forced overexpression of a specific combination of transcription factors, of which Oct4 is essential. To better understand the mechanism underlying iPSC reprogramming, we investigated the immediate transcriptional response to ectopic Oct4 expression in somatic cells. To this end, we derived different somatic cell types from Oct4-inducible transgenic mice and induced Oct4 expression for different time periods. Using microarray analysis, we have identified early transcriptional Oct4 targets, including genes not previously known to interact with Oct4. Comparison of the sets of Oct4-regulated genes revealed pronounced differences among the individual cell types. Furthermore, a notable up-regulation of pluripotent markers could not be detected. In contrast, we observed a down-regulation of somatic markers specific to each cellular population. This finding suggests that Oct4 interferes with the somatic transcriptional program in a cell type-specific manner, thereby promoting an unstable epigenetic state that facilitates reprogramming rather than the direct initiation of a pluripotent gene expression network. Finally, we show that Mgarp, a gene expressed in pluripotent cells, is commonly up-regulated in all cell types after Oct4 induction. Unexpectedly, Mgarp expression decreases during the first steps of reprogramming due to a Klf4-dependent inhibition. Our data show that Oct4 counteracts Klf4 repressive activity and enhances Mgarp expression at the late stages of reprogramming. This temporal pattern of Mgarp expression is crucial for the efficient generation of iPSCs. In summary, our study provides new insights into the specific role that Oct4 plays during the early stages of reprogramming. Different somatic cell types were derived from transgenic mice containing both a tetracycline-inducible transactivator (rtTA-M2) and a tetracycline operon-controlled Oct4 expression cassette (TO) (Hochedlinger 2005, Cell 121:465-477) that were mated with GOF18-GFP mice in which the green fluorescent protein (GFP) is driven by 18 kb of the Oct4 regulatory region (Oct4-GFP) (Yoshimizu 1999, Dev Growth Differ 41:675-684), thus indicating endogenous Oct4 expression. In addition, a control neural stem cell (CtrlNSC) line was generated from transgenic mice that entail a tetracycline-inducible transactivator (irtTA-VP16-GBD) (Anastassiadis 2002, Gene 298:159-172) plus an OG2 Oct4-GFP reporter transgene (Yoshimizu 1999),but lack a TO cassette. A second control consisted of wild type (C57BL/6) mouse embryonic fibroblasts (CtrlMEF). Oct4-inducible mouse embryonic fibroblasts (TO-MEFs) and neural stem cell (TO-NSCs) were derived from 14.5 days post coitum (d.p.c.) embryos and cultured as previously described (Conti 2005, PLoS Biol 3:e283). Primary bone marrow cells (TO-BMCs) were isolated from 6-week old mice after flushing femora and tibiae and plated onto dishes with a combined coating of gelatin (PAA, Pasching, Austria), laminin (Santa Cruz Biotechnology, Santa Cruz, CA), and poly-L-lysine (Sigma, St. Louis, MO). Adherent cells were then cultivated in DMEM Low Glucose (Gibco, Carlsbad, CA) with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM/L glutamine (all PAA). The transgenic Oct4 expression was induced by either 6 µg/ml doxycycline (Sigma) or by 2 µg/ml doxycycline (Dox) plus 10–7 M dexamethasone (Sigma), depending on the respectively contained tetracycline transactivator. 5-azacytidine (10 nM, Sigma) and cycloheximide (30 µg/ml, Sigma) were applied as indicated. Generation, culture, and differentiation of ESCs and iPSCs were performed as previously described (Tiemann 2011, Cytometry A 79:426-435). Microarray Gene Expression Analysis Samples from Oct4-inducible cell types and controls were collected at different time points (0, 6, 12, and 24 hours after induction). Biotin-labeled cRNA was prepared with the linear TotalPrep RNA Amplification Kit (Ambion, Austin, TX) from 500 ng original RNA and hybridized onto MouseRef 8 v2.0 Expression BeadChips (Illumina, San Diego, CA). The chips were stained with streptavidin-Cy3 (GE Healthcare, Little Chalfont, UK) and scanned using the iScan reader (Illumina). Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix robust multiarray analysis model. Variance stabilization was performed by log2 scaling and the R-Bioconductor lumi package was used for normalization. 17 samples were analyzed. ESC: Mouse Embryonic Stem Cell (ESC) TO_MEF_0: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 0h TO_MEF-6: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 6h TO_MEF-12: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 12h TO_MEF-24: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 24h TO_BMC-0: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 0h TO_BMC-6: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 6h TO_BMC-12: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 12h TO_BMC-24: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 24h TO_NSC-0: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 0h TO_NSC-6: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 6h TO_NSC-12: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 12h TO_NSC-24: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 24h Ctrl_MEF_0: Control wild type Mouse Embryonic Fibroblast (MEF) untreated Ctrl_MEF_24: Control wild type Mouse Embryonic Fibroblast (MEF) 24h treated Ctrl_NSC_0: Control wild type Mouse Neural Stem Cell (NSC) untreated Ctrl_NSC_24: Control wild type Mouse Neural Stem Cell (NSC) 24h treated
2014-05-13 | E-GEOD-47061 | biostudies-arrayexpress
Project description:Glioblastoma-derived neural stem (GNS) cells were reprogrammed to induced pluripotent stem (iPS) cells by transgenic expression of OCT4 and KLF4. Gene expression profiling was performed in comparison to normal neural stem (NS) cells reprogrammed in parallel, as well as standard ES cells as an independent reference.
2013-03-15 | E-MTAB-1273 | biostudies-arrayexpress
Project description:Glioblastoma-derived neural stem (GNS) cells were reprogrammed to induced pluripotent stem (iPS) cells by transgenic expression of OCT4 and KLF4. Genome-wide DNA methylation status was profiled at 485,000 loci to assess epigenetic erasure and restoration due to reprogramming and redifferentiation to the neural stem (NS) cell state.
2013-03-15 | E-MTAB-1271 | biostudies-arrayexpress