Transcriptomic analysis in intestinal stem cells | Scott T. Magness, PhD
Microphysiological systems (MPSs) are small-scale cell culture devices that simulate biological niches and represent a wide array of emerging technologies used in industry and academia for basic science and drug screening applications. While MPSs are powerful research tools, the microscale volumes intrinsic to the MPS renders transcriptomic analyses challenging or impossible as the minimal cellular material provides insufficient quantities of RNA for conventional RNA sequencing workflows. Using intestinal stem cells (ISCs) isolated from human tissue donors, we developed a number of MPSs to study the impact of hypoxia on ISCs, fatty acid handling and metabolism in absorptive enterocyte (AE) monolayers, and gastric metaplasia in single organoids. We leveraged the Fluidigm® Juno™ and Advanta™ systems to generate Illumina® libraries on a small number of cells cultured on the MPS devices. Very high quality sequencing data were obtained from picograms to nanograms of RNA. Transcriptomic data generated from the three different MPSs revealed that:
1) unique gene expression patterns are found in human ISCs during short-, medium-, and long-term induction of hypoxia,
2) ISC-derived AEs mature in culture over time similar to what is observed in vivo, and
3) clonal metaplastic organoids display heterogeneous transcriptomic signatures. These findings demonstrate the utility of the Juno and Advanta systems for enabling transcriptomic studies on extremely small sample sizes and RNA quantities.
→ABOUT THE SPEAKER:
Scott T. Magness, PhD
University of North Carolina - Chapel Hill,
NC State University Joint Departments of Biomedical Engineering
UNC Departments of Medicine, Cell Biology & Physiology
For Research Use Only. Not for use in diagnostic procedures.
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