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An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells.
Cotton, Michael J; Ariel, Pablo; Chen, Kaiwen; Walcott, Vanessa A; Dixit, Michelle; Breau, Keith A; Hinesley, Caroline M; Kedziora, Katarzyna M; Tang, Cynthia Y; Zheng, Anna; Magness, Scott T; Burclaff, Joseph.
Afiliación
  • Cotton MJ; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC, 27599, USA.
  • Ariel P; Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
  • Chen K; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC, 27599, USA.
  • Walcott VA; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
  • Dixit M; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC, 27599, USA.
  • Breau KA; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
  • Hinesley CM; Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
  • Kedziora KM; Department of Cell Biology, Center for Biologic Imaging (CBI), University of Pittsburgh, Pittsburgh, PA, 15260, USA.
  • Tang CY; School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
  • Zheng A; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
  • Magness ST; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC, 27599, USA. scott_magness@med.unc.edu.
  • Burclaff J; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. scott_magness@med.unc.edu.
Sci Rep ; 14(1): 15195, 2024 07 02.
Article en En | MEDLINE | ID: mdl-38956443
ABSTRACT
The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ciclo Celular / Células Epiteliales / Mucosa Intestinal Límite: Humans Idioma: En Revista: Sci Rep Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ciclo Celular / Células Epiteliales / Mucosa Intestinal Límite: Humans Idioma: En Revista: Sci Rep Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido