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Nat Cell Biol. 2015 Mar;17(3):340-9. doi: 10.1038/ncb3104. Epub 2015 Feb 9.

A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis.

Author information

1
1] Department of Cell Biology &Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA [2] Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
2
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
3
Department of Cell Biology &Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
4
Department of Pathology and Laboratory Medicine, University of Kansas, Kansas City, Kansas 66160, USA.
5
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill North Carolina 27599, USA.
6
UNC/NC State Biomedical Engineering, Chapel Hill North Carolina 27599, USA.
7
1] Department of Pathology and Laboratory Medicine, University of Kansas, Kansas City, Kansas 66160, USA [2] Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
8
1] Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill North Carolina 27599, USA [2] UNC/NC State Biomedical Engineering, Chapel Hill North Carolina 27599, USA.
9
1] Department of Cell Biology &Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA [2] Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA [3] UNC/NC State Biomedical Engineering, Chapel Hill North Carolina 27599, USA.

Abstract

Stem cells reside in 'niches', where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.

PMID:
25664616
PMCID:
PMC4405128
DOI:
10.1038/ncb3104
[Indexed for MEDLINE]
Free PMC Article
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