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Quantitative Analysis of Intestinal Stem Cell Dynamics Using Microfabricated Cell Culture Arrays

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Somatic Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1842))

Abstract

Regeneration of intestinal epithelium is fueled by a heterogeneous population of rapidly proliferating stem cells (ISCs) found in the base of the small intestine and colonic crypts. ISCs populations can be enriched by fluorescence-activated cell sorting (FACS) based on expression of combinatorial cell surface markers, and fluorescent transgenes. Conventional ISC culture is performed by embedding single ISCs or whole crypt units in a matrix and culturing in conditions that stimulate or repress key pathways to recapitulate ISC niche signaling. Cultured ISCs form organoid, which are spherical, epithelial monolayers that are self-renewing, self-patterning, and demonstrate the full complement of intestinal epithelial cell lineages. However, this conventional “bulk” approach to studying ISC biology is often semiquantitative, low throughput, and masks clonal effects and ISC phenotypic heterogeneity. Our group has recently reported the construction, long-term biocompatibility, and use of microfabricated cell raft arrays (CRA) for high-throughput analysis of single ISCs and organoids. CRAs are composed of thousands of indexed and independently retrievable microwells, which in combination with time-lapse microscopy and/or gene-expression analyses are a powerful tool for studying clonal ISC dynamics and micro-niches. In this protocol, we describe how CRAs are used as an adaptable experimental platform to study the effect of exogenous factors on clonal stem cell behavior.

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Acknowledgments

We would like to thank Eric Bankaitis, Ph.D. and Bailey Zwarcyz, MS for critical reading of the manuscript. Additionally, we would like to acknowledge Adam Gracz, Ph.D. for his work in developing the crypt and villus-enrichment methodology used here. We acknowledge all authors who contributed to the publication of this protocol which is based on “A high-throughput platform for stem cell-niche cocultures and downstream expression analysis” published in Nature Cell Biology 2016, 17(3): 340-349. The original work described in this chapter was funded by the National Institutes of Health, R01 DK091427 (Magness), R03 EB013803 (Y. Wang/Magness), R01 EB012549 (Allbritton), Small Business Innovation Research R43 GM106421 (Y. Wang/Magness), U01 DK085507-01 (Li), University Cancer Research Fund of the University of North Carolina (Magness/Allbritton), and the Center for Gastrointestinal Biology and Disease P30 DK034987 (Magness, Y. Wang, Galanko).

Author information

Authors and Affiliations

  1. Department of Medicine, Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Leigh A. Samsa, Ian A. Williamson & Scott T. Magness

  2. NC State/UNC Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Ian A. Williamson & Scott T. Magness

  3. Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Scott T. Magness

Authors
  1. Leigh A. Samsa
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  2. Ian A. Williamson
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  3. Scott T. Magness
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Corresponding author

Correspondence to Scott T. Magness .

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Editors and Affiliations

  1. Basic Research Laboratory, Stem Cell Regulation and Animal Aging Section, National Cancer Institute, Frederick, MD, USA

    Shree Ram Singh

  2. Department of Medicine-Hematology/Oncology, Rutgers New Jersey Medical School, Newark, NJ, USA

    Pranela Rameshwar

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Samsa, L.A., Williamson, I.A., Magness, S.T. (2018). Quantitative Analysis of Intestinal Stem Cell Dynamics Using Microfabricated Cell Culture Arrays. In: Singh, S., Rameshwar, P. (eds) Somatic Stem Cells. Methods in Molecular Biology, vol 1842. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8697-2_10

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  • DOI: https://doi.org/10.1007/978-1-4939-8697-2_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8696-5

  • Online ISBN: 978-1-4939-8697-2

  • eBook Packages: Springer Protocols

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