Genome-wide analysis of gene expression in human embryonic stem cells (hESCs) and their differentiated cells
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ABSTRACT: In order to investigate the gene expression changes in human embryonic stem cells (hESCs) during differentiation, we performed a microarray analysis from RNAs isolated from undifferentiated hESCs and their differentiated cells incubated for 1 week or 2 weeks in ESC medium. Human SNUhES3 ESCs (Seoul National University Hospital, Seoul, Korea) were cultured on mitotically-arrested STO feeder cells (ATCC, Manassas, USA) in DMEM/F12 supplemented with 20% knockout serum replacement (KSR). The embryoid bodies were incubated for 1 or 2 weeks in the above ESC medium without bFGF. Cells were then lysed and RNA was isolated.
Project description:To better understand the extent to which induced pluripotent stem cells (iPSCs) faithfully recapitulate the characteristics of embryonic stem cells (ESCs) under (undiff)erentiated condition, KSR condition and FBS condition and how both compare to somatic tissues under these conditions, we employed whole-genome transcriptome analysis on all twenty one hESC lines available on the pre-2008 NIH Human Pluripotent Stem Cell Registry, eight human iPSCs derived at NIH by retroviral transduction of human fibroblasts and twenty human somatic tissues. One standard culture protocol was used in conjunction with rigorous quality control. Expanded description of methods used and are available at: http://stemcelldb.nih.gov. 230 samples: 44 human ESC (UNDIFF), 16 human iPSC (UNDIFF) 44 human ESC (KSR), 15 human iPSC (KSR), 44 human ESC (FBS), 15 human iPSC (FBS), 1 human Brain, 1 human Placenta, 1 human Skeletal Muscle, 1 human Heart, 1 human Liver, 1 human Lung, 1 human Kidney, 1 human Esophagus, 1 human Ovary, 1 human Testes, 1 human Spleen, 1 human Adipose, 1 human Thyroid, 1 human Thymus, 1 human Small Intestine, 1 human Prostate, 1 human Cervix, 1 human Colon, 1 human Bladder, 1 human Trachea) and 32 processing controls (UniRef).
Project description:Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers and epigenetic marks. To measure dissociation rates of H3.3, we utilized a TET-repressible ESC line, ES[MC1R(20)], with the expression cassette integrated at the ROSA26 locus. We transfected MC1R ESCs with HA/FLAG-tagged H3.3 controlled by tetracycline response elements. For ‘TET-OFF’ experiments, ESCs were cultured over several passages (weeks) on feeder cells in the absence of DOX and were subsequently passaged onto feeder-free plates prior to the inhibition of HA-H3.3 expression. To repress HA/FLAG-H3.3 expression we treated cells with 2 ?g/ml doxycycline hyclate before crosslinking with formaldehyde at various time points. Measurement of H3.3-HA enrichment over time-course was performed using two replicates of ChIP-seq experiments. As a control, input DNA sequencing was performed for every time point
Project description:Snai1 is a master factor of epithelial to mesenchymal transitioin (EMT), however, its role in embryonic stem cell (ESC) differentiation and lineage commitment remains undefined. We used microarrays to compare the global programme of gene expression between control and Snai1 knockout ESCs-derived EB and teratoma. For EBs, control and Snai1 knockout ESCs were cultured as embryoid bodies in spotaneous differentiation media, RNA of 5 days EBs were collected for Affymetrix microarrays. For teratomas, control and Snai1 knockout ESCs were injected into nude mice to form teratomas. RNA of 6 weeks were collected for Affymetrix microarrays.
Project description:The derivation of functional, transplantable HSCs from an pluripotent stem cells in vitro holds great promise for clinical therapies, but is unachieved. In order to achieve full functionality of HSCs, it is vital to determine the extent to which PSCs can currently be differentiated to the HSC program in vitro and identify the remaining dysregulated genetic pathways. Microarrays were used to compare the transcritomes of ESC-derived immunophenotypic HSPCs to endogenous HSPCs from various stages of development to determine the programs important for human HSC development and function, and which programs were lacking in ESC-derived hematopoietic cells. CD34+CD38-CD43+CD90+ HSPCs were sorted from human placenta and embryoid bodies, and CD34+CD38-CD45+CD90+ HSPCs sorted from fetal liver and embryoid bodies co-cultured on OP9-M2 stroma, the RNA was extracted, library created and hybridized to the Affymetrix microarray
Project description:Gata5 efficiently promotes cardiomyocyte fate from murine ESCs.By removing serum from the culture conditions, GATA4 and GATA6 are each also able to efficiently promote cardiogenesis in ESC derivatives, with some distinctions. Thus, GATA factors can function in ESC derivatives upstream of other cardiac transcription factors to direct specification of progenitors with cardiac potential. We used microarray to compared the global gene expression of Gata5-induced cardiac cells with other growth factor directed ESC-derived cardiac cells iGata5 ESCs were induced with doxycycline at day 4 and harvested at day16 for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Snai1 is a master factor of epithelial to mesenchymal transitioin (EMT), however, its role in embryonic stem cell (ESC) differentiation and lineage commitment remains undefined. We used microarrays to compare the global programme of gene expression between control and Snai1 knockout Flk1+ and Flk1- cells sorted from 4 day EBs. Control and Snai1 knockout ESCs were cultured as embryoid bodies in spotaneous differentiation media, 4 days EBs were dissociated and sorted by anti-Flk1 antibody to separated Flk1+ and Flk1- cells, total RNA were collected for Affymetrix microarrays
Project description:During development, two cell-types born from closely related progenitor pools often express the identical transcriptional regulators despite their completely distinct characteristics. This phenomenon highlights the necessity of the mechanism that operates to segregate the identities of the two cell-types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). Here we demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate V2a and MN pathways. Together, our study uncovers a widely applicable gene regulatory principle for segregating related cell fates. ChIP DNA samples from Chx10-ESC-derived MNs were prepared for sequencing according to the Illumina protocol, and sequenced on the Illumina HiSeq 2000. The peak calling was conducted with MACS software (Zhang et al., 2008). MEME-ChIP Suite (Bailey et al., 2009; Machanick and Bailey, 2011) and TOMTOM algorithm (Gupta et al., 2007) was used for motif analysis.
Project description:During development, two cell-types born from closely related progenitor pools often express the identical transcriptional regulators despite their completely distinct characteristics. This phenomenon highlights the necessity of the mechanism that operates to segregate the identities of the two cell-types throughout differentiation after initial fate commitment. To understand this mechanism, we investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). Here we demonstrate that the paired homeodomain factor Chx10 functions as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate V2a and MN pathways. Together, our study uncovers a widely applicable gene regulatory principle for segregating related cell fates. RNA samples from Chx10-ESC-derived MNs were prepared for sequencing according to the Illumina protocol, and sequenced on the Illumina HiSeq 2000. We will then compare the transcriptome changes between -Dox (no Chx10) and +Dox (Chx10) in order to identify genes rregulated by Chx10.
Project description:In order to investigate the gene expression changes in human embryonic stem cells (hESCs) during differentiation, we performed a microarray analysis from RNAs isolated from undifferentiated hESCs and their differentiated cells incubated for 1 week or 2 weeks in ESC medium.
Project description:Myogenic differentiation relies on Pax7 function. We used embryonic stem cells lacking functional Pax7 to follow its role in derivation of skeletal myoblasts. Microarray analysis allowed us to compare transcriptomes of undifferentiated and differentiating embryonic stem cells of two genotypes, i.e. Pax7+/+ and Pax7-/- at day 7 and 21 of culture. Undifferentiated mouse embryonic stem cells, embryiod bodies at day 7, and embryoid body outgrowths at day 21 of differentiation were collected for RNA extraction and hybridization on Affymetrix microarrays.