Dataset Information

ENRICHMENT IN C-KIT IMPROVES STEM CELL POTENTIAL OF FOETAL HUMAN AMNIOTIC FLUID CELLS: CHARACTERIZATION FROM SINGLE CELL ANALYSIS TO THE SECRETOME EFFECT [Bulk RNA-Seq]

ABSTRACT: Human amniotic fluid cells (hAFSCs) are a fascinating foetal cell-type that have important stem cell characteristics, since described as broadly multipotent immature cells with high self-renewal and no tumorigenic properties. However, amniotic fluid contains a heterogeneous population of amniocytes, from totally differentiated or progenitor cells to highly multipotent stem cells. There is no single approach to isolating the stem cell component, but the selection of a subpopulation of hAFSCs expressing c-Kit (or CD117) is widely employed, while a deep characterization of the two populations is still lacking. Here we performed single-cell and bulk RNAseq analysis to compare the gene expression profiles of adherent amniotic fluid cells and their subpopulation c-Kit+. Information deriving from this high throughput technology on the transcriptome was then confirmed for specific targets with protein expression experiments and functional analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE219024 | GEO | 2023/03/01

REPOSITORIES: GEO

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Project description:Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterised by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesise that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21. Mass spectra were analyzed using Mascot (version 2.2, Matrix Science), executing a spectral search against a concatenated International Protein Index (IPI) human protein database (version 3.54 containing 39,925 entries) and a decoy database. Parameters included: trypsin enzyme specificity, SILAC double measurements of Lys6 and Arg8, 1 missed cleavage, minimum peptide length of 7 amino acids, minimum of 1 unique peptide, top 6 MS/MS peaks per 100 Da, peptide mass tolerance of 20 ppm for precursor ion and MS/MS tolerance of 0.5 Da. Oxidation of methionine and N-terminal protein acetylation for variable modifications and cysteine caramidomethylation for fixed modification. All entries were filtered using a false positive rate of 1% both at the peptide and protein levels, and false positives were removed. Quantification via normalised H/L ratios was based on minimum of 3 peptide ratio counts. Protein group entries with a normalised ratio significance B score of =< 0.05 or significance A score of =< 0.05 were retained for further analysis.

Dataset Information

ENRICHMENT IN C-KIT IMPROVES STEM CELL POTENTIAL OF FOETAL HUMAN AMNIOTIC FLUID CELLS: CHARACTERIZATION FROM SINGLE CELL ANALYSIS TO THE SECRETOME EFFECT [Bulk RNA-Seq]

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