Project description:Human embryonic stem (hES) cells have the capacities to propagate for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self-renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. The hES-T3 cells with normal female karyotype cultured on either mouse embryonic fibroblasts (MEF) in hES medium (containing 4 ng/ml bFGF) (T3MF) or feeder-free Matrigel in MEF-conditioned medium (supplemented with additional 4 ng/ml bFGF) (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self-renewal and pluripotency of hES cells can be maintained by continuing culture on these two conditions. However, the expression profiles, especially microRNAs, of the hES-T3 cells cultured on Matrigel in hES medium supplemented with 4 ng/ml bFGF and 5 ng/ml activin A (T3BA) were found to be different from those of T3MF and T3CM cells. In T3BA cells, four hES cell-specific microRNAs miR-372, miR-302d, miR-367 and miR-200c, as well as three other microRNAs miR-199a, miR-19a and miR-217, were found to be up-regulated, whereas five miRNAs miR-19b, miR-221, miR-222, let-7b and let-7c were down-regulated by activin A. Thirteen abundantly differentially expressed mRNAs, including NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, CD24 and COX6A1 genes, targeted by seven over-expressed miRNAs were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3BA and T3CM cells. The NR4A2, ERBB4 and CXCR4 target genes were further found to be regulated by EGF and/or TNF. The 50 abundantly differentially expressed genes targeted by five under-expressed miRNAs were also identified. The abundantly expressed mRNAs in T3BA and T3CM cells were also analyzed for the network and signaling pathways, and roles of activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the self-renewal and pluripotency of hES cells. In this investigation, both miRNA and mRNA expression profiles from human embryonic stem cells grown on MEF feeder (T3MF), feeder-free Matrigel in MEF-conditioned medium (T3CM) and in hES medium (containing 4 ng/ml bFGF) supplemented with 5 ng/ml activin A (T3BA) were quantitatively determined. Several target genes of T3BA and T3CM cells-specific miRNAs were identified. ***This submission represents the mRNA expression component of the study only***

Project description:Human embryonic stem (hES) cells have the capacities to propagate for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self-renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. The hES-T3 cells with normal female karyotype cultured on either mouse embryonic fibroblasts (MEF) in hES medium (containing 4 ng/ml bFGF) (T3MF) or feeder-free Matrigel in MEF-conditioned medium (supplemented with additional 4 ng/ml bFGF) (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self-renewal and pluripotency of hES cells can be maintained by continuing culture on these two conditions. However, the expression profiles, especially microRNAs, of the hES-T3 cells cultured on Matrigel in hES medium supplemented with 4 ng/ml bFGF and 5 ng/ml activin A (T3BA) were found to be different from those of T3MF and T3CM cells. In T3BA cells, four hES cell-specific microRNAs miR-372, miR-302d, miR-367 and miR-200c, as well as three other microRNAs miR-199a, miR-19a and miR-217, were found to be up-regulated, whereas five miRNAs miR-19b, miR-221, miR-222, let-7b and let-7c were down-regulated by activin A. Thirteen abundantly differentially expressed mRNAs, including NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, CD24 and COX6A1 genes, targeted by seven over-expressed miRNAs were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3BA and T3CM cells. The NR4A2, ERBB4 and CXCR4 target genes were further found to be regulated by EGF and/or TNF. The 50 abundantly differentially expressed genes targeted by five under-expressed miRNAs were also identified. The abundantly expressed mRNAs in T3BA and T3CM cells were also analyzed for the network and signaling pathways, and roles of activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the self-renewal and pluripotency of hES cells.

Project description:Epiblast stem cells (EpiSC) are pluripotent stem cells derived from mouse postimplantation embryos between E5.5 to E7.5, a time window in which gastrulation commences. Therefore, EpiSC represent a valuable tool for studying mammalian postimplantation development in vitro. Beyond their pluripotent features, EpiSC can also be reprogrammed into a mouse embryonic stem cell-like (mESC) state. Published reports showed that EpiSC reversion requires transcription factor overexpression, while others suggest that applying stringent mESC culture conditions alone was sufficient to revert EpiSC to mESC-like cells. In an effort to clarify these discrepancies, we systematically compared a panel of independent EpiSC lines. Indeed, the different lines shared a number of defining EpiSC characteristics such as teratoma formation ability, regardless of the day of isolation from the embryo. However, despite being grown in identical, self-renewal-promoting conditions, some lines displayed strong expression of genes associated with mesendodermal differentiation. Strikingly, these same lines were not revertable to a mESC-like state by applying stringent mESC culture conditions. Moreover, expression of mesendodermal marker genes correlated in a negative manner with the ability to efficiently undergo neural induction. Overall, our data suggest that different EpiSC lines may self-renew in distinct developmental ground states, which has important consequences for functional readouts such as revert ability to a mESC-like state and directed differentiation. For EpiSC lines derivation E5.5 embryos were plated onto irradiated mouse embryonic feeder cells (MEF cells) seeded at low density. Outgrowths were picked and expanded manually. Basal medium consisted of Knock-Out-DMEM, 20% Knock-Out Serum Replacement, Non Essential Amino Acids, L-Glutamine and beta-Mercaptoethanol (all Gibco). For cultivation on feeders 5ng/ml bFGF (Peprotech) was added to the medium to yield EpiSC medium. For preparation of mouse embryonic fibroblast-conditioned medium, EpiSC medium incubated on inactivated MEFs, followed by addition of another 5ng/ml bFGF (CM+). For feeder free culture, EpiSCs were seeded onto FCS-coated dishes. EpiSC were routinely passaged using 1mg/ml Collagenase IV (Invitrogen). Mouse ESC were cultured in standard conditions - Knock-Out-DMEM, 15% Knock-Out Serum Replacement (both Gibco), 5% fetal calf serum (Biowest), Non Essential Amino Acids, L-Glutamine and beta-Mercaptoethanol (all Gibco), supplemented with either homemade or commercially available LIF (ESGRO) at 1000U/ml. Some experiments were carried out in chemically defined N2B27 medium 11 in the presence of 10ng/ml bFGF. For reversion experiments, EpiSC were harvested using Accutase (Invitrogen) and seeded as single cells at approximately 16.000 cells / well of a 6-well plate (Sarstedt) in CM+. After 24h medium was changed to PCL medium (basal medium, LIF (1000U/ml), PD0325901 (Axon Medchem 1µM), and CHIR99021 (Axon Medchem 3µM). After 48h treatment, reversion medium was further supplemented with 10ÂµM SB431542 (Sigma) to eliminate remaining EpiSC.

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Expression data from porcine embryonic stem cells

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