Project description:Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of the transcription factors OCT4, SOX2, KLF4, c-MYC as well as NANOG and LIN28. Here we report generation of induced pluripotent stem cells from human umbilical cord blood derived unrestricted somatic stem cells (USSC) using retroviral expression of the transcription factors OCT4, SOX2, KLF4 and C-MYC and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells. The reprogrammed cells (HUiPS) were analysed morphologically, by qRT-PCR, global miRNA and epigenetic profiling and gene expression microarrays, as well as in their in vitro and in vivo differentiation potential by embryoid body formation and teratoma assay. The cord blood iPS cells are highly similar to human embryonic stem cells morphologically, at their molecular signature as well as in their in vitro and in vivo differentiation potential. Human cord blood derived unrestricted somatic stem cells offer an attractive source of cells for the generation of induced pluripotent stem cells. Our findings open novel perspectives to generate HLA matched pluripotent stem cell banks based on existing cord blood banks. Besides its obvious relevance of such a second generation cord blood iPS bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible. For transcriptome profiling, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit - Ambion) following the manufacturer's instructions (IVT: 10h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto HumanRef-8 v3 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials / instrumentation supplied / suggested by the manufacturer. Six sample types were analyzed, each one of them in duplicate. USSC: human umbilical cord blood unrestricted somatic stem cells (duplicates) HUiPS: human iPS cells from human umbilical cord blood USSC, hand-picked cols (duplicates) H9 hESC: H9 human ESCs grown on low-density CF1 MEFs (duplicates) H1 hESC: H1 human ESCs grown on low-density CF1 MEFs (duplicates) 1F hNiPS: One factor (Oct4) human iPS cells from hNSCs, hand-picked cols (duplicates) 2F hNiPS: Two factors (Oct4, Klf4) human iPS cells from hNSCs, hand-picked cols (duplicates)

Project description:Chavez2009 - a core regulatory network of OCT4 in human embryonic stem cells A core OCT4-regulated network has been identified as a test case, to analyase stem cell characteristics and cellular differentiation. This model is described in the article: In silico identification of a core regulatory network of OCT4 in human embryonic stem cells using an integrated approach. Chavez L, Bais AS, Vingron M, Lehrach H, Adjaye J, Herwig R BMC Genomics, 2009, 10:314 Abstract: BACKGROUND: The transcription factor OCT4 is highly expressed in pluripotent embryonic stem cells which are derived from the inner cell mass of mammalian blastocysts. Pluripotency and self renewal are controlled by a transcription regulatory network governed by the transcription factors OCT4, SOX2 and NANOG. Recent studies on reprogramming somatic cells to induced pluripotent stem cells highlight OCT4 as a key regulator of pluripotency. RESULTS: We have carried out an integrated analysis of high-throughput data (ChIP-on-chip and RNAi experiments along with promoter sequence analysis of putative target genes) and identified a core OCT4 regulatory network in human embryonic stem cells consisting of 33 target genes. Enrichment analysis with these target genes revealed that this integrative analysis increases the functional information content by factors of 1.3 - 4.7 compared to the individual studies. In order to identify potential regulatory co-factors of OCT4, we performed a de novo motif analysis. In addition to known validated OCT4 motifs we obtained binding sites similar to motifs recognized by further regulators of pluripotency and development; e.g. the heterodimer of the transcription factors C-MYC and MAX, a prerequisite for C-MYC transcriptional activity that leads to cell growth and proliferation. CONCLUSION: Our analysis shows how heterogeneous functional information can be integrated in order to reconstruct gene regulatory networks. As a test case we identified a core OCT4-regulated network that is important for the analysis of stem cell characteristics and cellular differentiation. Functional information is largely enriched using different experimental results. The de novo motif discovery identified well-known regulators closely connected to the OCT4 network as well as potential new regulators of pluripotency and differentiation. These results provide the basis for further targeted functional studies. This model is hosted on BioModels Database and identified by: MODEL1305010000 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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