Human embryonic stem cells (hESCs) have the unique property of immortality, ability to infinitely self-renew and survive in vitro. In contrast to tumor-deribed cells, their immortality are free from any genomic abberations. Instead, they depend on the AKAP-Lbc/Rho signaling cascade. To understand the downstream way, we performed RNA-seq analyses between normal and AKAP-Lbc-depleted hESCs using the doxycyclin-inducible gene silensing strategy. Overall design: We use the genetically modified hESCs in which AKAP-13-targeting shRNA is induced by doxycyclin(dox) treatment. To minimize cell loss during treatment, anti-apoptotic factor Bcl-XL is overexpressed. We collected RNA from dox-treated and untreated cells in biological triplicate. We measured gene expression in these 2 sample groups using RNA-seq (illumina HiSeq) .
Rho-Signaling-Directed YAP/TAZ Activity Underlies the Long-Term Survival and Expansion of Human Embryonic Stem Cells.
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View SamplesHuman ES cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are usually generated and maintained on living feeder cells like mouse embryonic fibroblasts or on a cell-free substrate like Matrigel. For clinical applications, a quality-controlled, xenobiotic-free culture system is required to minimize risks from contaminating animal-derived pathogens and immunogens. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived substrate on which to maintain hiPSCs/hESCs. In this study, we examined whether PCM-DM could be used for the generation and long-term stable maintenance of hiPSCs. Decidua-derived mesenchymal cells (DMCs) were reprogrammed by the retroviral transduction of four factors (OCT4, SOX2, KLF4, c-MYC) and cultured on PCM-DM. The established hiPSC clones expressed alkaline phosphatase, hESC-specific genes and cell-surface markers, and differentiated into three germ layers in vitro and in vivo. At over 20 passages, the hiPSCs cultured on PCM-DM held the same cellular properties with genome integrity as those at early passages. Global gene expression analysis showed that the GDF3, FGF4, UTF1, and XIST expression levels varied during culture, and GATA6 was highly expressed under our culture conditions; however, these gene expressions did not affect the cells pluripotency. PCM-DM can be conveniently prepared from DMCs, which have a high proliferative potential. Our findings indicate that PCM-DM is a versatile and practical human-derived substrate that can be used for the feeder-cell-free generation and long-term stable maintenance of hiPSCs.
Feeder-free generation and long-term culture of human induced pluripotent stem cells using pericellular matrix of decidua derived mesenchymal cells.
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View SamplesThe expression of four transcription factors (OCT3/4, SOX2, KLF4, and c-MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. Expression of the c-MYC, also known as an oncogene, might induce carcinogenesis and thus, iPS cells produced with the use of c-MYC transduction cannot be used for human therapeutic applications. Furthermore, reprogramming efficiency was significantly reduced in the absence of c-MYC transduction. Here, we generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without c-MYC. Interestingly, clonally expanded MSCs, named 10F-15, could be used for iPS cell generation with 100-fold higher efficiency compared to that of other clonally expanded MSCs and human dermal fibroblasts. These iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES markers expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that MSCs isolated from human third molars are a valuable cell source for the generation of iPS cells.
Induction of pluripotent stem cells from human third molar mesenchymal stromal cells.
Specimen part, Cell line
View SamplesGeneChip-based screen for genes induced in the initial phase of neural differentiation from ES cells.
Intrinsic transition of embryonic stem-cell differentiation into neural progenitors.
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View SamplesThis SuperSeries is composed of the SubSeries listed below.
Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition.
Specimen part
View SamplesEndothelial-to-mesenchymal transition (EndMT) in which endothelial cells lose their characteristics and acquire mesenchymal property has recently been recognized as a driver of disease progression in wide range of pathologies. However, the regulatory mechanism of EndMT has not been fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induced EndMT. Hence, we analyzed functions of ERG and FLI1 using gene expression microarray and ChIP-seq to elucidate the regulatory mechanism of EndMT.
Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition.
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View SamplesPrevious study demonstrated that HDAC3 has a critical role in MM proliferation; however, the underlying mechanism has not yet been elucidated. We identify that HDAC3 inhibition targets DNMT1 through dual regulations. We demonstrate that knockdown of DNMT1 leads to apoptosis and significant growth inhibition in myeloma cells. HDAC3 inhibition by gene silencing or HDAC3 selective inhibitor BG45 downregulates an oncoprotein c-Myc through its acetylation. c-Myc directly regulates DNMT1 expression at its enhancer region. Furthermore, HDAC3 directly regulates the stability of DNMT1 protein through its acetylation. Pharmaceutical inhibition of HDAC3 and DNMT1 synergistically induce MM growth inhibition in in vitro and in vivo settings.
HDAC3 regulates DNMT1 expression in multiple myeloma: therapeutic implications.
Cell line
View SamplesRecent studies have implicated KDM3A, which catalyzes removal of H3K9 methylation, is associated with tumorigenesis. However, the biological role of KDM3A in multiple myeloma, has not been delineated. Here we identify KDM3A-KLF2-IRF4 axis dependence in multiple myeloma. We demonstrate that knockdown of KDM3A leads to apoptosis and significant growth inhibition in myeloma cells. Mechanistically, KDM3A directly regulates myeloma cell survival factor IRF4 expression through H3K9 demethylation at its promoter. We further show that KDM3A directly regulates KLF2 expression and that knockdown of KLF2 leads to growth inhibition in myeloma cells.
The KDM3A-KLF2-IRF4 axis maintains myeloma cell survival.
Specimen part, Cell line
View SamplesA comprehensive landscape of epigenomic events regulated by the Reelin signaling through activation of specific cohort of cis-regulatory enhancer elements (LRN-enhancers), which involves the proteolytical processing of the LRP8 receptor by the gamma-secretase activity and is required for learning and memory behavior Overall design: All RNA-Seq experiments were designed to evaluate the transcriptional program regulated by the Reelin-LRP8 signaling pathway in neuronal cells
LRP8-Reelin-Regulated Neuronal Enhancer Signature Underlying Learning and Memory Formation.
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View SamplesAlthough liganded nuclear receptors have been established to regulate RNA polymerase II (Pol II)-dependent transcription units, their role in regulating Pol III-transcribed DNA repeats remains largely unknown. Here we report that ~2-3% of the ~100,000-200,000 total human DR2 Alu repeats located in proximity to activated Pol II transcription units are activated by the retinoic acid receptor (RAR) in human embryonic stem cells to generate Pol III-dependent RNAs. These transcripts are processed, initially in a DICER-dependent fashion, into small RNAs (~28-65 nt) referred to as repeat-induced RNAs that cause the degradation of a subset of crucial stem-cell mRNAs, including Nanog mRNA, which modulate exit from the proliferative stem-cell state. This regulation requires AGO3-dependent accumulation of processed DR2 Alu transcripts and the subsequent recruitment of AGO3-associated decapping complexes to the target mRNA. In this way, the RAR-dependent and Pol III-dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II-dependent neuronal transcriptional program. Overall design: RNA-sequencing of polyA selected RNA molecules in NTera2/D1 cells and Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq).
DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation.
Specimen part, Treatment, Subject
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