Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of hiPSCs adapted to homemade weekend-free media

ABSTRACT: Homemade weekend free media are a promising way for the labs to reduce the manpower and high costs associated with the culture of human induced pluripotent stem cells (hiPSCs) in feeder-free conditions. However, as these media would have a different concentration of various growth factors, cells may perceive a change in the signalling environments and change their transcription profile. To investigate the extent of this change, we decided to perform RNA sequencing and study the change of gene expression as compared to cells grown in commercial, daily changed E8.

INSTRUMENT(S): NextSeq 1000

ORGANISM(S): Homo sapiens

SUBMITTER: Elisa Balmas

PROVIDER: E-MTAB-14237 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The mammary gland is a unique apocrine gland made up of a branching network of ducts that end in alveoli. It is an ideal system to study the molecular mechanisms associated with cell proliferation, differentiation, and oncogenesis. MFG-E8, also known as Lactadherin, is a vital glycoprotein related to the milk fat globule membrane and initially identified to get secreted in bovine milk. Our previous report suggests that a high level of MFG-E8 is indicative of high milk yield in dairy animals. Here, we showed that MFGE-E8 controls the cell growth and morphology of epithelial cells through a network of regulatory transcription factors. To understand the comprehensive action, we downregulated its expression in MECs by MFG-E8 specific shRNA. We generated a knockdown proteome profile of differentially expressed proteins through a quantitative iTRAQ experiment on a high-resolution mass spectrometer (Q-TOF). The downregulation of MFG-E8 resulted in reduced phagocytosis and cell migration ability, whereas it also leads to more lifespan to knockdown vis-a-vis healthy cells, which is confirmed through BrdU, MTT, Neutral red, and Caspase 3/7. The bioinformatics analysis revealed that MFG-E8 knockdown perturbs a large number of intracellular signaling, eventually leading to cessation in cell growth. Based on the Weighted Gene Coexpression Network Analysis and directed network, we found that MFG-E8 is activated by CX3CL1, TP63, and CSF2 and leads to the activation of SOCS3 and CCL2 for the regulation of cell proliferation. We further proved that the depletion of MFG-E8 resulted in activated cytoskeletal remodeling by MFG-E8 knockdown, which results in the activation of three independent pathways ZP4/JAK-STAT5, DOCK1/STAT3, and PIP3/AKT/mTOR. Overall, this study suggests that MFG-E8 expression in mammary epithelial cells is an indication of intracellular deterioration in cell health. To date, to the best of our knowledge, this is the first study that explores the downstream targets of MFG-E8 involved in the regulation of mammary epithelial cell health.

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RNA-seq of hiPSCs adapted to homemade weekend-free media

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