title: A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis.
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.
authors: 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.
authors: Adam D
authors: AD
authors: Gracz
authors: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors: Ian A
authors: IA
authors: Williamson
authors: Department of Cell Biology &Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors: Kyle C
authors: KC
authors: Roche
authors: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors: Michael J
authors: MJ
authors: Johnston
authors: Department of Pathology and Laboratory Medicine, University of Kansas, Kansas City, Kansas 66160, USA.
authors: Fengchao
authors: F
authors: Wang
authors: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill North Carolina 27599, USA.
authors: Yuli
authors: Y
authors: Wang
authors: UNC/NC State Biomedical Engineering, Chapel Hill North Carolina 27599, USA.
authors: Peter J
authors: PJ
authors: Attayek
authors: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill North Carolina 27599, USA.
authors: Joseph
authors: J
authors: Balowski
authors: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors: Xiao Fu
authors: XF
authors: Liu
authors: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors: Ryan J
authors: RJ
authors: Laurenza
authors: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors: Liam T
authors: LT
authors: Gaynor
authors: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill North Carolina 27599, USA.
authors: Christopher E
authors: CE
authors: Sims
authors: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors: Joseph A
authors: JA
authors: Galanko
authors: 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.
authors: Linheng
authors: L
authors: Li
authors: 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.
authors: Nancy L
authors: NL
authors: Allbritton
authors: 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.
authors: Scott T
authors: ST
authors: Magness
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D018920:Coculture Techniques; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D019879:Paneth Cells / Q000166:cytology / Q000378:metabolism*; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis; D055153:Stem Cell Niche / Q000235:genetics; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D046888:Tissue Array Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins