An RNAi screen probing signaling control of human embryonic stem cells establishes the cell cycle-regulated restriction of the exit from pluripotency (CCNB1 overexpression)
ABSTRACT: Analysis of effect of Cyclin B1 overexpression on differentiating hESCs at the gene expression level. The hypothesis tested in the present study was that Cyclin B1 overexpression prevents the exit from pluripotency in hESCs. Results provide important information on the effect of Cyclin B1 on hESCs, such as changes in expression of TGFb-related genes and pluripotency markers, as well as mesodermic and neuroectodermic genes. Total RNA obtained from CCNB1 overexpressing hESCs incubated in medium without bFGF and TGF-beta for 0, 48 and 96 hours.
Project description:Analysis of effect of S-phase arrest and replication checkpoint activation on differentiating hESCs at the gene expression level. The hypothesis tested in the present study was that replication checkpoint activation prevents the exit from pluripotency in hESCs. Results provide important information of the response of hESCs to replication arrest, such as upregulation of genes involved in the TGF-beta signaling pathway, and subsequent maintenance of pluripotency marker expression. Total RNA obtained from hESCs incubated in medium without bFGF and TGF-beta supplemented with either DMSO, Aphidicolin or Aphidicolin+AZD7762 for 0, 48 and 96 hours.
Project description:Human induced pluripotent stem (iPS) cells are capable of differentiating into derivatives of the three embryonic germ layers both in vitro and in vivo. To date the the molecular differences between teratoma-forming cells and non-teratoma-forming cells has not been analysed. A cell line, B1, bears typical ES cell-like morphology, expression of pluripotency-associated genes, and in vitro pluripotency capacity, but fails to form teratomas after subcutaneously injected into immune-deficient mice based on histological analysis. Besides histological analysis, we characterized the tumors derived from line B1, and teratomas derived from bona fida iPS and ES (line H1) cells respectively, using microarray-based gene expression analysis. The expression levels of pluripotency-associated markers in B1 cells were comparable to that in iPS and ES cells, while the complexity of tissue expression commitment was decreased upon spontaneous differentiation of B1 cells as compared to iPS and ES cells. Total RNA obtained from HFF1 (human foreskin fibroblast) cells, line B1, iPS-A4, iPS-B4 and ES (line H1) cells, and their derived tumors in immune-deficient mice.
Project description:The unique cellular state of embryonic stem cells is maintained by pluripotency-associated transcription factors such as OCT4. The transcriptional regulatory circuitries between human and mouse pluripotent stem cells exhibit notable differences. Transposable elements have altered the pluripotency network by contributing new cis-regulatory elements to mammalian genomes. In our study, we found that the expression of HERVH is enriched in human embryonic stem cells (hESCs) and depletion of HERVH in hESCs induced differentiation. To confirm the differentiation phenotype and identify potential downstream target genes of HERVH, we conducted the microarray analysis on hESCs treated with control shRNA against Luciferase and shRNA against HERVH. Total RNA was extracted from cells treated with control shRNA or HERVH shRNA for 5 days. 3 replicates each.
Project description:Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling. Total RNA obtained from mid gestation human amniotic fluid cells (AFSCs/2nd trimester AFSCs), early gestation human amniotic fluid cells (eAFSCs/1st trimester AFSCs) and human embryonic stem cells (hESCs) as described in the corresponding Materials and Methods sections.
Project description:Recently, (in vitro) pluripotent EpiSCs were derived from the post-implantation egg cylinder stage epiblasts of mouse and rat. These EpiSCs resemble and correspond very closely to the conventional human embryonic stem cells (hESCs) in the colony morphology and culture/signaling requirements for maintaining pluripotency, but exhibit a range of significant phenotypic and signaling response differences from the conventional mouse ES cells (mESCs). These observations strongly support the notion that EpiSCs and hESCs are intrinsically similar, and raise an attractive hypothesis: as mESCs and EpiSCs/hESCs represent two distinct pluripotency states: the mESC-like state representing the ICM of pre-implantation blastcyst and the EpiSC-like state representing the post-implantation epiblasts, whether the epiblast state (including conventional hESCs) can be converted back to the ICM state. Despite studies providing evidence that epiblast-like cells exist and transition back and forth within colony of conventional mESCs; mESCs and EpiSCs share substantial set of pluripotency transcriptional factors, including Oct4, Sox2 and Nanog; and mESCs are more stable in culture, in the present study we found that EpiSCs differentiated rapidly under mESC culture conditions and no “spontaneously” converted mESC could be readily identified and isolated over serial passages at the population or clonal level. Remarkably, we found that blockage of the TGFβ pathway or inhibition of the H3K4 demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes of EpiSCs towards mESC phenotypes with activation of some ICM-specific gene expression. However, full conversion of EpiSCs to a mESC-like state with competence to chimeric contribution can only be readily generated with a combination of inhibitors of LSD1 and ALK. These observations underscore a powerful and direct induction of reprogramming from the developmentally later-stage EpiSCs to a mESC-like stage by a synergy of signaling and direct epigenetic modulations. It also highlights a significant role for TGFβ pathway inhibition in promoting reprogramming to and sustaining true pluripotency, which further supports our recent studies in generating chimerism-competent rat pluripotent cells. Collectively, our studies provide a proof-of-concept demonstration that pluripotency-restricted EpiSCs can be readily converted to a mESC-like state in the absence of any genetic manipulation by precise pharmacological control of signaling pathways that distinguish the two pluripotency states and an epigenetic target simultaneously, and offer a convenient experimental system to further study the mechanism. Such method and concept may also provide an avenue for generating a new type of mESC-like human pluripotent cell. Global gene-expression analyses of the parnate/mAMFGi cells
Project description:Bone morphogenetic protein (BMP) signaling is known to support differentiation of human embryonic stem cells (hESCs) into mesoderm and extraembryonic lineages, whereas other signaling pathways can largely influence this lineage specification. Here, we set out to reinvestigate the influence of ACTIVIN/NODAL and fibroblast growth factor (FGF) pathways on the lineage choices made by hESCs during BMP4-driven differentiation. We show that BMP activation, coupled with inhibition of both ACTIVIN/NODAL and FGF signaling, induces differentiation of hESCs, specifically to βhCG hormone-secreting multinucleated syncytiotrophoblast and does not support induction of embryonic and extraembryonic lineages, extravillous trophoblast, and primitive endoderm. It has been previously reported that FGF2 can switch BMP4-induced hESC differentiation outcome to mesendoderm. Here, we show that FGF inhibition alone, or in combination with either ACTIVIN/NODAL inhibition or BMP activation, supports hESC differentiation to hCG-secreting syncytiotrophoblast. We show that the inhibition of the FGF pathway acts as a key in directing BMP4-mediated hESC differentiation to syncytiotrophoblast. Human embryonic Stem Cells (hESCs) were treated under defined conditions (N2B27) with BMP4 (B), SB431542 (SB) (ACTIVIN/NODAL inhibitor), SU5402 (SU) (FGFR1 inhibitor), FGF2 (F) either alone or in various combinations as mentioned, followed by isolation of total RNA.
Project description:Two independent protocols for deriving HLCs from hESCs and iPSCs were adopted and further characterization included immunocytochemistry, real-time RT-PCR, and in vitro functional assays. Comparative microarray-based gene expression profiling was conducted on these cells and compared to the transcriptomes of human fetal liver and adult liver progenitors. HLCs derived from hESCs and hiPSCs showed significant functional similarities, similar expression of genes important for liver physiology and common pathways. However, specific differences between the two cell types could be observed. Total RNA obtained from undifferentiated hESCs, iPSCs, HLCs (hepatocyte-like cells)-derived from hESCs and iPSCs, fetal forskin fibroblasts and fetal liver.
Project description:Gene expression analysis of control fibroblasts (NFH2), one AD-derived fibroblasts (NFH-46), NFH2-derived control-iPS cells (OiPS3, OiPS6), NFH46-derived AD-iPS cells (iPS5 and iPS 26B), hESCs (H1 and H9). Total mRNA obtained from AD fibroblasts (NFH-46) and control fibroblasts (NFH-2), from pluripotent stem cells AD-iPS cells (iPS5 and iPS 26B), control-iPS cells (OiPS3, OiPS6), and hESCs (H1 and H9).
Project description:A systems biology approach in which qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify 15 pluripotency regulating cytokines or cytokine related genes. IL-11 was validated as a novel factor capable of maintaing the undifferentiated state of human embryonic stem cells in the absence of exogenously added bFGF to the culture acting via a different mechanims than bFGF. Transcriptomic microarray data of eight overexpression and knock-down experiments were used for qualitative modeling based on boolean networks to predict novel factors - mainly cytokines - that maintain pluripotent human embryonic stem cells in the absence of bFGF in culture. The culture was maintained for at least 9 passages using and stained positive for alcaline phosphatase staining, OCT3/4, SOX2, NANOG, TRA1-60. Microarray based gene expression profiling showed that the IL-11 treatment occupies an intermediate state, between bFGF treatment and the negative control which is no cytokine treatment as judged by hierarchical clustering. Moreover, KEGG pathway analysis indicates common and additional distinct mechanisms of bFGF and IL-11 dependant pluripotency dependant mechanisms.
Project description:Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor β (TGF β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within one week under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021) and TGF β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. hESCs at about 20% confluence were treated with 3 μM CHIR99021, 2 μM SB431542, 0.1 μM Compound E (γ-Secretase Inhibitor XXI) in neural induction media containing Advanced DMEM/F12:Neurobasal (1:1), 1xN2, 1xB27, 1% Glutmax, 5 μg/ml BSA and 10 ng/ml hLIF, for 7 days. The culture was then split 1:3 for the next six passages using Accutase and cultured in neural induction media supplemented with 3 μM CHIR99021 and 2 μM SB431542 on X-ray inactivated MEF feeders or Matrigel-coated plates. After six passages, the cells were split 1:10 regularly. Global gene expression analysis of primitive neural stem cells