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Effect of rBC2LCN on human embryonic stem cells

ABSTRACT: We show a recombinant lectin probe, rBC2LCN, is a new tool for fluorescence-based imaging and flow cytometry analysis of human pluripotent stem cells, as an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized using fluoresceinated rBC2LCN. Fluoresceinated rBC2LCN also separated live pluripotent stem cells from mixed cell population by flow cytometry.

ORGANISM(S): Homo sapiens

PROVIDER: GSE42976 | GEO | 2015/12/09

SECONDARY ACCESSION(S): PRJNA184054

REPOSITORIES: GEO

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Effect of rBC2LCN on human embryonic stem cells

Project description:We show a recombinant lectin probe, rBC2LCN, is a new tool for fluorescence-based imaging and flow cytometry analysis of human pluripotent stem cells, as an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized using fluoresceinated rBC2LCN. Fluoresceinated rBC2LCN also separated live pluripotent stem cells from mixed cell population by flow cytometry. Induced gene expression in human ES cells was measured at 3 days treatment of rBC2LCN lectin. WA01 (H1) cells were treated with 1, 10 and 100 ug/ml of rBC2LCN or 1 of FITC-rBC2LCN for three days. Two independent experiments were performed at each treatment experiment. Samples of human ES cells (KhES-1 and 3), human iPS cells (201B7 and 253G1) and HDF in each conventional culture condition were used for outgroup examples.

2015-12-09 | E-GEOD-42976 | biostudies-arrayexpress

Alternative routes to induced pluripotent stem cells revealed by reprogramming of neural lineage

Project description:During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as Nanog occurs after the mesenchymal to epithelial transition (MET). Here we report that both adult stem cells (neural stem cells- NSCs) and differentiated cells (astrocytes) of the neural lineage can activate Nanog in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the Nanog+Ecadherin+ populations expressed stabilization markers and had upregulated several cell cycle genes; and were transgene independent. Inhibition of Dot1L activity, which has previously been shown to increase MET, enhanced both the numbers of Nanog+ and Nanog+E-cadherin+ colonies, suggesting a MET independent pathway for activation of Nanog in NSCs. Expressing Sox2 in MEFs prior to reprogramming does not alter the ratio of Nanog colonies that express E-cadherin obtained. Taken together these results provide a novel pathway for reprogramming taken by cells of the neural lineage. Neural Stem Cells (NSCs) were induced to reprogram by the induction of Oct4, Sox2, c-Myc and Klf4. Reprogramming colonies that expressed either Nanog alone (N+E-) or Nanog and E-cadherin (N+E+) were sorted by flow cytometry and used as input for RNA-sequencing. There are 3 replicates each for the N+E- and N+E+ samples.

2016-02-18 | E-GEOD-76130 | biostudies-arrayexpress

PD-L1 regulates inflammatory programs of macrophages from human pluripotent stem cells.

Project description:CRISPR knock-out combined with flow cytometry and scRNA-seq show that PD-L1 ablation compromises macrophage development from human pluripotent stem cells

2022-11-29 | GSE218722 | GEO

Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states

Project description:Using a statistical framework to analyze single-cell transcriptomics data, we infer the gene expression dynamics of early mouse embryonic stem (mES) cell differentiation, uncovering discrete transitions across nine cell states. We validate the predicted transitions across discrete states using flow cytometry. Moreover, using live-cell microscopy, we show that individual cells undergo abrupt transitions from a naïve to primed pluripotent state. Using the inferred discrete cell states to build a probabilistic model for the underlying gene regulatory network, we further predict and experimentally verify that these states have unique response to perturbations, thus defining them functionally.

2017-10-17 | GSE105054 | GEO

Metabolomics Workbench ST000003 - GNPS - Metabolomic analysis of mouse embryonic fibroblasts, embryonic stem cells, and induced pluripotent stem cells

Project description:mouse embryonic fibroblasts, embryonic stem cells, and induced pluripotent stem cells were compared via metabolomic analysis

2018-08-02 | MSV000082680 | GNPS

RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids and targeted DNA-seq of a colorectal biobank

Project description:This study has two components: (1) Human colon adenoma organoids (n=4 patients) were dissociated into single cells. Cells were incubated with a magnetic bead bound to an LGR5 antibody and run through a magnetic column. Magnet bound cells and flow through negative (FTN) cells were obtained. Magnet bound and FTN cells were incubated with an APC-check reagent (which binds to the magnetic bead on the LGR5 antibody) and DAPI, before being sorted by flow cytometry. 3 populations of live (DAPI-) cells were collected: FTN: Flow through negative. LGR5 negative by magnet and by flow cytometry SortedNeg: Magnet bound cells that were negative for LGR5 by flow cytometry SortedPos: Magnet bound cells that were positive for LGR5 by flow cytometry (2) Human colon organoids, as well as the tissue the organoid was derived from and adjcacent normal tissue (from n=19) were also profiled for known colorectal cancer associated mutations using the Qiagen Qiaseq Colorectal Cancer Panel, which provides targeted sequencing information for 71 genes.

2020-04-30 | E-MTAB-4698 | biostudies-arrayexpress

Global Gene Expression Profiles of Single-Cell Derived Fetal Rat Calvaria Cell Colonies with Adipocyte and Bone Markers

Project description:Single cell-derived colonies were obtained by sorting 21 days fetal Rat calvaria cells into microtitre wells using flow cytometry. Colonies were categorized according to a limited gene expression profiling for a specific bone (osteocalcin) and a specific adipocyte (adipsin) marker by real-time PCR. Colonies expressing neither, one or both bone and adipocyte markers are in the sample set. Microarray analysis will address the global gene expression profiles of CFU-O (bone marker only), CFU-F (neither bone nor adipocyte marker) and CFU-bipotential (both markers) derived from single cells. CFU-A (adipocyte marker only) samples are also available for analysis if warranted. Keywords: other

2005-12-09 | GSE3775 | GEO

Hybridisation of 11 8-cell neonatal foreskin keratinocyte colonies

Project description:Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms which enable this are ill defined. Using live imaging we saw two populations of cells those which generate expanding colonies and those which generate balanced colonies. We wanted to assess if there were any transcriptional differences between these two colony types.

2015-10-19 | E-MTAB-3128 | biostudies-arrayexpress

Epithelial-mesenchymal transition in cancer stem cells and during metastasis of a squamous cell carcinoma

Project description:Scientific background: Current understanding of carcinogenesis supposes a heterogeneous composition of malign tumors from cancer stem cells (CSCs) and stromal cells. CSCs most likely play an important role during treatment failure and metastasis. A possibly associated phenomenon is the phenotype of cancer cells undergoing an epithelial-mesenchymal transition (EMT). This study investigated the role of EMT in CSCs of a HPV-negative pharyngeal squamous cell carcinoma during metastasis. Methods: A cell line was established from a HPV-negative pharyngeal squamous cell carcinoma. Enrichment of cancer stem cells was performed by stem cell-enriching conditions in spheroid colonies. Gene expression profiling was performed (Human 8 × 60 K design array, Agilent Technologies) and the phenotype EMT was assessed by a gene set (MSigDB: M5930, Hallmark_epithelial_mesenchymal_transition) applying Gene set expression analysis (GSEA). A validation of the results was carried out by immunohistochemical staining and flow cytometry of CD44 und E-Cadherin. Results: Primary tumor and metastasis were highly positive for CD44. E-Cadherin was shown in primary tumor and the cell line derived from it. Flow cytometry showed a heterogeneous composition of the stem cell-enriched spheroid colonies with the appearance of a distinct population with the loss of E-Cadherin. GSEA showed significant enrichment of the EMT-phenotype in the primary tumor (FDR < 25 %, p < 5 %) compared to the other phenotypes. Conclusions: In this study, CSCs showed mesenchymal characteristics. EMT may be a dynamic state in HPV-negative pharyngeal squamous cell carcinoma that is active in CSCs during metastasis and at least partially turned back later.

2018-04-30 | GSE97510 | GEO

Are These Cardiomyocytes? Protocol Development Reveals Impact of Sample Preparation on the Accuracy of Identifying Cardiomyocytes by Flow Cytometry

Project description:Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation.

2019-08-12 | PXD014971 | panorama

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