Long-Term In Vitro Expansion of Epithelial Stem Cells Enabled by Pharmacological Inhibition of PAK1-ROCK-Myosin II and TGF-β Signaling

Cell Rep. 2018 Oct 16;25(3):598-610.e5. doi: 10.1016/j.celrep.2018.09.072.

Abstract

Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.

Keywords: PAK1/ROCK/Myosin II; TGF-β; cell culture method; cell therapy; epithelial stem and progenitor cells; feeder-free; regenerative medicine.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation
  • Cell Proliferation
  • Cells, Cultured
  • Epithelial Cells / cytology*
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism
  • Feeder Cells / cytology
  • Feeder Cells / drug effects
  • Feeder Cells / metabolism
  • Gene Expression Regulation / drug effects
  • Humans
  • In Vitro Techniques
  • Keratinocytes / cytology
  • Keratinocytes / drug effects
  • Keratinocytes / metabolism
  • Myosin Type II / antagonists & inhibitors*
  • Myosin Type II / genetics
  • Myosin Type II / metabolism
  • Neoplasms / drug therapy
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Signal Transduction
  • Small Molecule Libraries / pharmacology*
  • Stem Cells / cytology*
  • Stem Cells / drug effects
  • Stem Cells / metabolism
  • Transforming Growth Factor beta / antagonists & inhibitors*
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / metabolism
  • Xenograft Model Antitumor Assays
  • p21-Activated Kinases / antagonists & inhibitors*
  • p21-Activated Kinases / genetics
  • p21-Activated Kinases / metabolism
  • rho-Associated Kinases / antagonists & inhibitors*
  • rho-Associated Kinases / genetics
  • rho-Associated Kinases / metabolism

Substances

  • Small Molecule Libraries
  • Transforming Growth Factor beta
  • PAK1 protein, human
  • p21-Activated Kinases
  • rho-Associated Kinases
  • Myosin Type II