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Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media.

ABSTRACT: We assessed the pluripotency of human induced pluripotent stem cells (iPSCs) maintained on an automated platform using StemFlex and TeSR-E8 media. Analysis of transcriptome of single cells revealed similar expression of core pluripotency genes, as well as genes associated with naive and primed states of pluripotency. Analysis of individual cells from four samples consisting of two different iPSC lines each grown in the two culture media revealed a shared subpopulation structure with three main subpopulations different in pluripotency states. By implementing a machine learning approach, we estimated that most cells within each subpopulation are very similar between all four samples. The single-cell RNA sequencing analysis of iPSC lines grown in both media reports the molecular signature in StemFlex medium and how it compares to that observed in the TeSR-E8 medium.

SUBMITTER: Daniszewski M

PROVIDER: S-EPMC6135898 | biostudies-other | 2018 Sep

REPOSITORIES: biostudies-other

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Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media.

Daniszewski Maciej M Nguyen Quan Q Chy Hun S HS Singh Vikrant V Crombie Duncan E DE Kulkarni Tejal T Liang Helena H HH Sivakumaran Priyadharshini P Lidgerwood Grace E GE Hernández Damián D Conquest Alison A Rooney Louise A LA Chevalier Sophie S Andersen Stacey B SB Senabouth Anne A Vickers James C JC Mackey David A DA Craig Jamie E JE Laslett Andrew L AL Hewitt Alex W AW Powell Joseph E JE Pébay Alice A

iScience 20180823

We assessed the pluripotency of human induced pluripotent stem cells (iPSCs) maintained on an automated platform using StemFlex and TeSR-E8 media. Analysis of transcriptome of single cells revealed similar expression of core pluripotency genes, as well as genes associated with naive and primed states of pluripotency. Analysis of individual cells from four samples consisting of two different iPSC lines each grown in the two culture media revealed a shared subpopulation structure with three main s ...[more]

PMID: 30267684

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Project description:The epicardium plays an important role in coronary vessel formation and Tgfbr3-/- mice exhibit failed coronary vessel development associated with decreased epicardial cell invasion. Immortalized Tgfbr3-/- epicardial cells display the same defects. Tgfbr3+/+ and Tgfbr3-/- cells incubated for 72 hours with VEH or ligands known to promote invasion via TGFβR3 (TGFβ1, TGFβ2, BMP2), for 72 hours were harvested for RNA-seq analysis. We selected for genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells when incubated with VEH (604), TGFβ1 (515), TGFβ2 (553), or BMP2 (632). Gene Ontology (GO) analysis of these genes identified dysregulated biological processes consistent with the defects observed in Tgfbr3-/- cells, including those associated with extracellular matrix interaction. GO and Gene Regulatory Network (GRN) analysis identified distinct expression profiles between TGFβ1-TGFβ2 and VEH-BMP2 incubated cells, consistent with the differential response of epicardial cells to these ligands in vitro. Despite the differences observed between Tgfbr3+/+ and Tgfbr3-/- cells after TGFβ and BMP ligand addition, GRNs constructed from these gene lists identified NF-ĸB as a key nodal point for all ligands examined. Tgfbr3-/- cells exhibited decreased expression of genes known to be activated by NF-ĸB signaling. NF-ĸB activity was stimulated in Tgfbr3+/+ epicardial cells after TGFβ2 or BMP2 incubation, while Tgfbr3-/- cells failed to activate NF-ĸB in response to these ligands. Tgfbr3+/+ epicardial cells incubated with an inhibitor of NF-ĸB signaling no longer invaded into a collagen gel in response to TGFβ2 or BMP2. These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. Our approach successfully identified a signaling pathway important in epicardial cell behavior downstream of TGFβR3. Overall, the genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFβR3 signaling.

Project description:BackgroundSkeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia), 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy), and 16 wk (market age) from two genetic lines: a randombred control line (RBC2) maintained without selection pressure, and a line (F) selected from the RBC2 line for increased 16 wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage.ResultsA total of 3474 genes were differentially expressed (false discovery rate; FDR < 0.001) by overall effect of development, while 16 genes were differentially expressed (FDR < 0.10) by overall effect of genetic line. Ingenuity Pathways Analysis was used to group annotated genes into networks, functions, and canonical pathways. The expression of 28 genes involved in extracellular matrix regulation, cell death/apoptosis, and calcium signaling/muscle function, as well as genes with miscellaneous function was confirmed by qPCR.ConclusionsThe current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of their protein products.

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Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media.

Publications

Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media.

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