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Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies

ABSTRACT: We report the miRNA profiling in MEF cells, ES cells and three Pluripotent Stem Cells obtained by three different reprogramming approaches from MEF cells based on Solexa sequencing. iPS cells are reprogrammed by four factors (OSKM) from MEF cells. NT-ESCs were established by reprogramming MEF cells into ESCs using nuclear transfer. NT-iPSCs were established to reflect the combination of nuclear transfer and iPS technologies. iPSCs, NT-ESCs, and NT-iPSCs were exactly derived from the same MEF cells. The results indicate NT-ESCs give expression to the unique miRNAs other than both ESCs and iPSCs while pluripotent cells acquire or retain the pluripotent specific miRNAs compared with MEF. Furthermore, the comparison of different reprogramming cells suggests that several miRNAs have key roles in distinctly developmental potential reprogrammine cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE52950 | GEO | 2014/07/05

SECONDARY ACCESSION(S): PRJNA230521

REPOSITORIES: GEO

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Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies

Project description:We report the miRNA profiling in MEF cells, ES cells and three Pluripotent Stem Cells obtained by three different reprogramming approaches from MEF cells based on Solexa sequencing. iPS cells are reprogrammed by four factors (OSKM) from MEF cells. NT-ESCs were established by reprogramming MEF cells into ESCs using nuclear transfer. NT-iPSCs were established to reflect the combination of nuclear transfer and iPS technologies. iPSCs, NT-ESCs, and NT-iPSCs were exactly derived from the same MEF cells. The results indicate NT-ESCs give expression to the unique miRNAs other than both ESCs and iPSCs while pluripotent cells acquire or retain the pluripotent specific miRNAs compared with MEF. Furthermore, the comparison of different reprogramming cells suggests that several miRNAs have key roles in distinctly developmental potential reprogrammine cells. Small RNA profiles of MEF, ES, iPS, NT-ES and NT-iPS cells were generated by Solexa sequencing. MEF and ES cells were performed in triplicate. iPS, NT-ES and NT-iPS cells were sequenced in duplicate.

2014-07-05 | E-GEOD-52950 | biostudies-arrayexpress

mRNA expression data from iPSCs, ntESCs and iPSC-nt-ESCs

Project description:We generated three kinds of genetically identical mouse reprogrammed cells: induced pluripotent stem cells (iPSCs), nuclear transfer embryonic stem cells (ntESCs) and iPSC-nt-ESCs that are established after successively reprogramming of iPSCs by nuclear transfer (NT). NtESCs show better developmental potential than iPSCs, whereas iPSC-nt-ESCs display worse developmental potential than iPSCs. We used microarrays to distinguish the gene expression differences among three pluriptoent stem cells and identified that imprinted genes had a similar expression pattern in iPSCs and iPSC-nt-ESCs. We sought to obtain genetic identical pluripotent stem cell lines in order to minimize genetic variations among different reprogrammed cells. To that end, we established a genetically homogenous secondary reprogramming system, in which mouse embryonic fibroblasts (MEFs) carrying doxycycline (dox)-inducible lentiviruses expressing Oct4, Sox2, Klf4 and c-Myc were isolated and used as donors for different reprogramming experiments. We generated iPSCs after plating MEFs in the presence of dox in ES culture conditions, and then derived ntESCs after transplantation of the nucleus from the same MEFs into enucleated oocyte. Furthermore, we successively reprogrammed iPSCs by means of NT and established a set of nt-iPSC lines. Pluripotent stem cells generated from different reprogramming strategies were for RNA extraction and hybridization on Affymetrix microarrays.

2011-05-31 | E-GEOD-24705 | biostudies-arrayexpress

mRNA expression data from iPSCs, ntESCs and iPSC-nt-ESCs

2011-05-31 | GSE24705 | GEO

Coding mutations and loss-of-imprinting in human pluripotent cells derived by nuclear transfer and defined factors [RNA-Seq]

Project description:Human pluripotent stem cells can be derived from somatic cells by forced expression of defined factors, and more recently by nuclear-transfer into human oocytes, revitalizing a debate on whether one reprogramming approach might be advantageous over the other. Here we compared the genetic and epigenetic stability of human nuclear-transfer embryonic stem cell (NT-ESC) lines and isogenic induced pluripotent stem cell (iPSC) lines, derived from the same somatic cell cultures of fetal, neonatal and adult origin. Both cell types shared similar genome-wide gene expression and DNA methylation profiles. Importantly, NT-ESCs and iPSCs have comparable numbers of de novo coding mutations but significantly higher than parthenogenetic ESCs. Similar to iPSCs NT-ESCs displayed clone- and gene-specific aberrations in DNA methylation and allele-specific expression of imprinted genes, similarly to iPSCs. The occurrence of these genetic and epigenetic defects in both NT-ESCs and iPSCs suggests that they are inherent to reprogramming, regardless of the underlying technique. RNA sequencing analysis was performed on a total of 12 human cell lines, including: an isogenic set of 3 nuclear-transfer embryonic stem cell (NT-ESC) lines, 2 RNA-reprogrammed induced pluripotent stem cell (iPSC) lines and their parental neonatal fibroblast cell line; an isogenic set of 1 NT-ESC line, 3 iPSC lines and their parental adult fibroblast cell line (derived from a type 1 diabetic subject); as well as 1 control embryonic stem cell (ESC) line.

2014-11-06 | E-GEOD-61656 | biostudies-arrayexpress

Coding mutations and loss-of-imprinting in human pluripotent cells derived by nuclear transfer and defined factors [DNA methylation profiling]

Project description:Human pluripotent stem cells can be derived from somatic cells by forced expression of defined factors, and more recently by nuclear-transfer into human oocytes, revitalizing a debate on whether one reprogramming approach might be advantageous over the other. Here we compared the genetic and epigenetic stability of human nuclear-transfer embryonic stem cell (NT-ESC) lines and isogenic induced pluripotent stem cell (iPSC) lines, derived from the same somatic cell cultures of fetal, neonatal and adult origin. Both cell types shared similar genome-wide gene expression and DNA methylation profiles. Importantly, NT-ESCs and iPSCs have comparable numbers of de novo coding mutations but significantly higher than parthenogenetic ESCs. Similar to iPSCs NT-ESCs displayed clone- and gene-specific aberrations in DNA methylation and allele-specific expression of imprinted genes, similarly to iPSCs. The occurrence of these genetic and epigenetic defects in both NT-ESCs and iPSCs suggests that they are inherent to reprogramming, regardless of the underlying technique. Genome-wide DNA methylation profiling by Illumina Infinium HumanMethylation 450K Beadchip was performed on a total of 21 human cell lines, including: an isogenic set of 3 nuclear-transfer embryonic stem cell (NT-ESC) lines, 2 RNA-reprogrammed induced pluripotent stem cell (iPSC) lines and their parental neonatal fibroblast cell line; an isogenic set of 1 NT-ESC line, 6 iPSC lines and their parental adult fibroblast cell line (derived from a type 1 diabetic subject); as well as 7 control embryonic stem cell (ESC) lines.

2014-11-06 | E-GEOD-61461 | biostudies-arrayexpress

Coding mutations and loss-of-imprinting in human pluripotent cells derived by nuclear transfer and defined factors [RNA-Seq]

2014-11-06 | GSE61656 | GEO

Coding mutations and loss-of-imprinting in human pluripotent cells derived by nuclear transfer and defined factors [DNA methylation profiling]

2014-11-06 | GSE61461 | GEO

Epigenetic and transcriptional aberrations in human pluripotent stem cells reflect differences in reprogramming mechanisms [methylation array]

Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but existing cell types have imitations. Human embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the “gold standard”, but are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) can be produced from somatic cells by forced expression of pluripotency-associated factors, but are prone to genetic and epigenetic aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming, or secondary to the reprogramming method, we employed an alternative approach by somatic cell nuclear transfer (SCNT). SCNT-based reprogramming to NT-ESCs is mediated by factors present in oocyte’s cytoplasm, thus mimicking early embryogenesis. We generated genetically matched pluripotent stem cells and conducted genome-wide genetic, epigenetic and transcriptional analyses. We discovered that unlike iPSCs, NT-ESCs have a low burden of de novo copy number variations (CNVs), reflecting superior maintenance of genetic stability. Moreover, DNA methylation and transcriptome profiles of NT-ESCs corresponded closely to those of IVF-ESCs. In contrast, iPSCs harbored methylation abnormalities including residual CpG methylation typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT with the potential to satisfy the clinical requirements for cell replacement therapies. Bisulphite converted DNAs of two IVF-ESCs, two sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, and the parental fibroblast were hybridized to the Illumina Infinium HumanMethylation 450K Beadchip

2014-07-08 | E-GEOD-53060 | biostudies-arrayexpress

mRNA profiling of iPSCs and derivative NT-ESCs

Project description:Pluripotent cells can be derived from somatic cells by either overexpression of defined transcription factors (resulting in induced pluripotent stem cells (iPSCs)) or by nuclear transfer or cloning (resulting in NT-ESCs). To determine whether cloning further reprograms iPSCs, we used iPSCs as donor cells in nuclear transfer experiments. An iPSC clone derived from tail-tip fibroblasts using adenoviral vectors was used as donor cell in nuclear transfer experiments. RNA was isolated from both parental iPSC clone and derivative NT-ESCs lines and analyzed.

2010-05-27 | E-GEOD-20575 | biostudies-arrayexpress

Epigenetic and transcriptional aberrations in human pluripotent stem cells reflect differences in reprogramming mechanisms [RNA-Seq]

Project description:Human pluripotent stem cells hold great potential for regenerative medicine, but available cell types have important limitations. While embryonic stem cells derived from fertilized embryos (IVF-ESCs) are considered the "gold standard" of pluripotency, they are allogeneic to potential recipients. Autologous induced pluripotent stem cells (iPSCs) are prone to epigenetic and transcriptional aberrations. To determine whether accumulation of such aberrations is intrinsic to somatic cell reprogramming or secondary to the reprogramming method, we generated a genetically matched collection of human IVF-ESCs, iPSCs, and ESCs derived by somatic cell nuclear transfer (SCNT; NT-ESCs), and subjected them to genome-wide genetic, epigenetic and transcriptional analyses. SCNT-based reprogramming is mediated by the full complement of oocyte cytoplasmic factors, thus closely recapitulating early embryogenesis. NT-ESCs and iPSCs derived from the same somatic donor cells contained comparable numbers of de novo copy number variations (CNVs), suggesting that the two reprogramming methods may not differ significantly in mutagenic or selective pressure. On the other hand, the DNA methylation and transcriptome profiles of NT-ESCs corresponded very closely to those of IVF-ESCs, while iPSCs differed markedly from IVF-ESCs and harbored residual DNA methylation patterns typical of parental fibroblasts, suggesting incomplete reprogramming. We conclude that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal candidates for cell replacement therapies. Duplicate cDNA libraries of two IVF-ESCs, three sendai produced iPSC lines, two retro-virus produced iPSC lines, four NT-ESCs, and the parental fibroblast line were sequenced using Illumina HiSeq 2000. The sequence reads were mapped to hg19 reference genome and hits that passed quality filters were analyzed for differential expression.

2014-07-08 | E-GEOD-53094 | biostudies-arrayexpress

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