Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells
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ABSTRACT:
PROVIDER: PRJNA821268 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18530722_1.fastq.gz | Fastqsanger.gz | |||
| SRR18530722_2.fastq.gz | Fastqsanger.gz | |||
| SRR18530723_1.fastq.gz | Fastqsanger.gz | |||
| SRR18530723_2.fastq.gz | Fastqsanger.gz | |||
| SRR18530724_1.fastq.gz | Fastqsanger.gz |
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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:The pluripotency maintenance of pluripotent stem cells (PSCs) requires the suitable microenvironment, which commonly provided by feeder layers. However, the preparation of feeder layers is time consuming and labor exhaustive, and the feeder cells treated with mitomycin C or γ-ray irradiation bring heterologous contamination. In this study, mouse embryonic fibroblasts (MEFs) were treated by methanol to generate chemical fixed feeder cells, and bovine embryonic stem cells F7 (bESC-F7) cultured on this feeder layer. Then the pluripotency and metabolism of bESC-F7 cultured on methanol-fixed MEFs (MT-MEFs) named MT-F7 was compared with mitomycin C treated MEFs (MC-MEFs). The results showed that bESC-F7 formed alkaline phosphatase positive colonies on MT-MEFs, the relative expression of pluripotent markers of these cells was different from the bESCs cultured on the MC-MEFs (MC-F7). The long-term cultured MT-F7 formed embryoid bodies, showed the ability to differentiate into three germ layers similar to MC-F7. The analyses of RNA-seq data showed that MT-MEFs lead bESCs to novel steady expression patterns of genes regulating pluripotency and metabolism. Furthermore, the bovine expanded pluripotent stem cells (bEPSCs) cultured on MT-MEFs formed classical colonies, maintained pluripotency, and elevated metabolism. In conclusion, MT-MEFs were efficient feeder layer that maintain the distinctive pluripotency and metabolism of PSCs.
| S-EPMC9163156 | biostudies-literature
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.
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Project description:We report how methanol fixation influences transcriptome profile in single cell RNA-seq. We generatad Smart-seq2 data from two cell lines, and both live and fixed cells from each cell line were processed and analyzed to illustrate fixaiton effect.
2021-05-18 | GSE150993 | GEO
Project description:Introduction: The application of single-cell RNA sequencing has greatly improved our under-standing of various cellular and molecular mechanisms involved in physiological and pathophysi-ological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. Materials and Methods: In this study, we selected two AML samples that had been fixed in methanol for 12–18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. Results: We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. Conclusions: The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effec-tively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.
2023-08-17 | GSE237239 | GEO
Project description:Engineered Methylobacterium methanol batch exponential
2014-12-22 | GSE34662 | GEO
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:We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis
2014-06-10 | GSE58303 | GEO
Project description:Background: Methanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes. Results: The methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by genetic adaption in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1. Conclusion: The methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity.
2015-08-01 | GSE71590 | GEO