Project description:This SuperSeries is composed of the following subset Series: GSE23968: Large intergenic non-coding RNAs as novel modulators of reprogramming: ESCs, fibroblast, and fibroblast-derived iPSC (gene expression) GSE23970: Large intergenic non-coding RNAs as novel modulators of reprogramming: human embryonic stem cells, CD34+ cells, and CD34+ derived induced pluripotent stem cells (LincRNA expression) GSE23973: Large intergenic non-coding RNAs as novel modulators of reprogramming: siRNA (gene expression) GSE24181: Large intergenic non-coding RNAs as novel modulators of reprogramming: human embryonic stem cells, fibroblasts, and fibroblast-derived induced pluripotent stem cells (LincRNA expression) Refer to individual Series
Project description:Expression profiling of non coding RNAs using tilling arrays from ES, Fibroblasts and Fibroblasts derived iPSC LincRNA profiles of human embryonic stem cells, fibroblasts, and fibroblast-derived induced pluripotent stem cells Total RNA was extracted from human ESCs, fibroblast, and fibroblast-derived iPSC lines, then hybridized to Nimblegen tiling array GPL8791; Human lincRNA 364k Tiling Array V1
Project description:We recently showed that some human induced pluripotent stem cell (iPSC) clones were defective in neural differentiation and were marked with the activation of long term repeats (LTRs) of human endogenous retroviruses (HERVs). We herein demonstrated that these LTRs were transiently overexpressed during the generation of iPSCs and contributed to reprogramming. When the generation of iPSCs was completed, LTRs were re-suppressed to levels similar to those in human ES cells. However, differentiation-defective iPSC clones maintained high LTR expression levels, which indicated that these clones failed to complete reprogramming. lincRNA-RoR, a long intergenic non-coding RNA (lincRNA) that was previously shown to support the induction and maintenance of pluripotency, was detected among the LTR-driven transcripts. Short hairpin RNAs against the conserved sequence in LTRs or lincRNA-RoR markedly reduced the efficiency of iPSC generation. Reprogramming factors including OCT3/4, SOX2, and KLF4 bound to most LTRs. The expression of KLF4 was low in normal iPSC clones, but remained high in differentiation-defective clones. The forced expression of KLF4 in human embryonic stem cells led to the activation of LTRs and defects in neural differentiation. These results demonstrated that the transient overexpression of KLF4/LTR/lincRNA-RoR played crucial roles in reprogramming toward pluripotency in humans, whereas a failure in its re-silence resulted in differentiation defects. OCT3/4, SOX2 and KLF4 bindings in human dermal fibroblast and iPSC
Project description:We recently showed that some human induced pluripotent stem cell (iPSC) clones were defective in neural differentiation and were marked with the activation of long term repeats (LTRs) of human endogenous retroviruses (HERVs). We herein demonstrated that these LTRs were transiently overexpressed during the generation of iPSCs and contributed to reprogramming. When the generation of iPSCs was completed, LTRs were re-suppressed to levels similar to those in human ES cells. However, differentiation-defective iPSC clones maintained high LTR expression levels, which indicated that these clones failed to complete reprogramming. lincRNA-RoR, a long intergenic non-coding RNA (lincRNA) that was previously shown to support the induction and maintenance of pluripotency, was detected among the LTR-driven transcripts. Short hairpin RNAs against the conserved sequence in LTRs or lincRNA-RoR markedly reduced the efficiency of iPSC generation. Reprogramming factors including OCT3/4, SOX2, and KLF4 bound to most LTRs. The expression of KLF4 was low in normal iPSC clones, but remained high in differentiation-defective clones. The forced expression of KLF4 in human embryonic stem cells led to the activation of LTRs and defects in neural differentiation. These results demonstrated that the transient overexpression of KLF4/LTR/lincRNA-RoR played crucial roles in reprogramming toward pluripotency in humans, whereas a failure in its re-silence resulted in differentiation defects. Nine samples were prepared as intermediate state of cells between human dermal fibroblast and iPSC. One iPSC clone and 4 subclones derived from defective iPSC exhibit normal differentiation ability.
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.
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.