Pyruvate and recombinant human basic fibroblast growth factor rescue the pluripotency and metabolism of bovine embryonic stem cells on low-density feeder layers
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ABSTRACT:
PROVIDER: PRJNA881957 | ENA |
REPOSITORIES: ENA
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Project description:Feeder cells still play an important role in the in vitro culture of bovine embryonic stem cells(bESCs). A suitable microenvironment or niche is essential for the self-renewal and pluripotency of stem cells cultured in vitro. Feeder cells participate in the construction of stem cell niches by secreting growth factors and extracellular matrix proteins. In this study, we used metabolomics and transcriptomics to investigate the effects of feeder layer deficiency on bESCs. bESCs co-cultured with low-density feeder cells experienced a decrease in pluripotent gene expression, cell differentiation, and central carbon metabolic activity. When cell-permeable pyruvate (Pyr) and recombinant human basic fibroblast growth factor (rhbFGF) were added to the culture system, the pluripotency of bESCs on low-density feeder layers was rescued, and acetyl-coenzyme A(AcCoA) synthesis and fatty acid de novo synthesis increased. rhbFGF enhances the effects of Pyr and activates the overall metabolic level of bESCs grown on low-density feeder layers.
2022-09-22 | GSE213631 | GEO
Project description:The pluripotency maintenance of pluripotent stem cells (PSCs) cultured in vitro requires the suitable microenvironment, which is commonly provided by the feeder layer. However, the preparation of feeder layer is time consuming and labor exhaustive. More importantly, the feeder cells treated with mitomycin or gamma-ray irradiation brings heterologous contamination to stem cells. The feeder-free PSC cultures are associated with high costs because of the requirement for additional supplements and special media. In this study, we characterize the pluripotency and metabolic status of bovine ESCs-F7 (classic bESCs lines, abbreviated as F7), which were cultured on methanol fixed mouse embryonic fibroblasts (MT-MEFs) or mitomycin C treated MEFs (1M-MEFs). MT-MEFs could be reused several times and were highly resistant to digestive enzymes. The relative expression levels of pluripotent markers were different between F7 cultured on MT-MEFs (marked as MT-F7) and those cultured on the 1M-MEFs (1M-F7). The long-term cultured MT-F7 cells formed embryoid bodies in vitro, showing the ability to differentiate into endodermal, ectodermal, and mesodermal germ layers like 1M-F7. RNA-sequencing analysis showed that the replacement of the feeder layer from 1M-MEFs to MT-MEFs lead to a novel steady transition of the F7, which included alteration of the expression patterns of genes that regulate pluripotency and metabolism. Further, the long-term cultured bovine expanded pluripotent stem cells (bEPSCs) on MT-MEFs (MT-bEPSCs) formed classical colonies, maintained pluripotency, and demonstrated elevated level of metabolic activity. In conclusion, this study demonstrated that methanol-fixed MEFs were efficient feeder layer that maintain the unique pluripotency and the distinctive metabolic characteristics of the bPSCs cultured in vitro.
2022-05-01 | GSE199731 | GEO
Project description:Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells
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Project description:The objective of this study was to reprogram peripheral blood-derived late-endothelial progenitor cells (EPCs) to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs were retrovirally-transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs.
2012-12-16 | GSE42947 | GEO
Project description:BASIC: Biology, Affect, Stress, Imaging and Cognition in pregnancy
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Project description:The objective of this study was to reprogram peripheral blood-derived late-endothelial progenitor cells (EPCs) to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs were retrovirally-transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs. Six samples were analyzed. The gene expression profile of four iPS clones were compared to the H9 human embryonic stem cell line and the parent endothelial progenitor cell line.
2012-12-16 | E-GEOD-42947 | biostudies-arrayexpress
Project description:Gene expression profiling of human iPSCs [201B7] under on-feeder, feeder-free [StemFit], and FGF inhibitor [100 nM PD173074] -treated cultures, and differentiating cells.
2022-03-09 | GSE195689 | GEO
Project description:suppression of Pyruvate carboxylase
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Project description:A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the human pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Here, we made use of the distinct MifR-dependent phenotypes to elucidate mechanisms associated with microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that cells located within microcolonies experience stressful, oxygen limited, and energy starving conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate partly restored microcolony formation in ∆mifR biofilms. Moreover, addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation. Consistent with the finding of denitrification genes not demonstrating distinct expression patterns in biofilms forming or lacking microcolonies, addition of nitrate did not alter microcolony formation. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation to the oxygen limitation and energy starvation of the P. aeruginosa biofilm environment.
2012-05-01 | GSE35286 | GEO
Project description:Most porcine embryonic stem cells (pESC) derived from in vitro blastocysts Still require complex media compositions and feeder layers which make difficulty to adapt their broad and routine use. We derived pESC using simplified culture media (FIW) consisting of small molecules FGF2, IWR-1, and WH-4-023 in serum-free medium. We established several pESC-FIW lines capable of single cell passaging with short cell doubling time (about 12 hours). These cells expressed pluripotency markers, OCT4, SOX2, and NANOG, as well as cell surface markers, SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed stable proliferation rate and normal karyotype even after long-term culture over 50 passages. Established pESC showed formative characteristics based on the negative expression for the naive marker KLF4 and primed marker T, whereas strong expression for the formative marker OTX2 with high levels of related genes. Transcriptome analysis showed that pESC-FIW had similar cellular identities to reported pESC maintained in complex media formulations and had characteristics of gastrulating epiblast cells. In addition, pESC-FIW could be maintained for multiple passages in a feeder-free condition on fibronectin coated plate using mTeSR™, a commercial medium for feeder-free culture. These results indicate that the canonical WNT (IWR-1) inhibition and the SRC inhibition (WH-4-023) are sufficient to establish pESC capable of single cell passaging and feeder-free expansion under the serum-free condition. Easy to maintain-pESC can be a tool for gene editing application useful for agriculture and biomedicine, and lineage commitment studies.
2024-08-07 | GSE240454 | GEO