Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Generation of induced pluripotent stem cells in rabbits: potential experimental models for human regenerative medicine

ABSTRACT: Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2 and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of bFGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4 and NANOG, while the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state, showing high similarity to their embryonic stem (ES) cell counterparts we generated recently. However, their global gene expression analysis revealed a slight, but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and ES cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans. Comparison of rabbit embryonic stem cells, rabbit somatic cells, and rabbit induced pluripotent stem cells at several timing (passage number 6, 7, 22, 23) of cultures. Eight samples were used for assessment.

ORGANISM(S): Oryctolagus cuniculus

SUBMITTER: Arata Honda

PROVIDER: E-GEOD-23190 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type1 transmembrane protein, is the predominant glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds predominantly to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched Oglycan comprising an H type3 structure as the most probable rBC2LCN glycan ligand (Kd, 4.0 x 10-5 M) prepared from human 201B7 iPS cells, suggesting that H type3 is a novel potential pluripotency marker. We conclude that podocalyxin is the predominant glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. Gene expressions in human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs). Human iPS cells (MRC5-iPS, AM-iPS, UtE-iPS, PAE-iPS) were induced from four different somatic cells by infection of the retroviral vectors pMXs encoding OCT3/4, SOX2, KLF4 and c-MYC, simultaneously. Human iPS cells (TIG/MKOS#19, #56, #106) were generated through reprogramming by Sendai virus infection-mediated expression of OCT4, SOX2, KLF4, and c-MYC.

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: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

Dataset Information

Generation of induced pluripotent stem cells in rabbits: potential experimental models for human regenerative medicine

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure