Citation

  • Authors: Horiguchi K. et al.
  • Year: 2021
  • Journal: Histochem Cell Biol s00418-021-02009-5
  • Applications: in vitro / siRNA / INTERFERin
  • Cell type: Rat primary anterior pituitary cells

Method

- CD9-positive cells isolated from the IL of adult Wistar rats were plated in a 96-well chamber with a laminin-coated surface. Cells were then cultured for 72 h in 200 μL Medium 199 with 10% FBS at 37 °C in a humidifed atmosphere of 5% CO2 and 95% air. - For transfection of siRNAs, the culture medium was replaced with 200 μL per well of Medium 199 with 10% FBS supplemented with transfection reagent (INTERFERin at 1:100 v/v; PolyPlus Transfection) and siRNAs against Tspan1 mRNA. - The bottom of each well was scratched with a 10 μL pipette tip for the wound healing assay. A non-silencing siRNA without homology to any known mammalian gene was used as a negative control. - After 72 h cultivation with siRNA reagents, cells were observed using the cellSens Dimension system and processed for qPCR.

Abstract

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL), and secretes important hormones for growth, sexual development, metabolism, and reproduction. In the marginal cell layer (MCL) facing Rathke's cleft between the IL and AL, cluster of differentiation (CD) 9-, CD81-, S100β-, and SOX2-quadruple positive (CD9/CD81/S100β/SOX2-positive) cells in the adult IL are settled as tissue-resident stem/progenitor cells supplying hormone-producing cells to the AL. However, it is unclear how CD9/CD81/S100β/SOX2-positive cells in the IL-side MCL migrate into the AL across Rathke's cleft. In the present study, we performed chimeric pituitary tissue culture using S100β/GFP-transgenic rats and Wistar rats, and traced the footprint of S100β/GFP-expressing cells. We detected IL-side S100β/GFP-expressing cells in the AL tissue, demonstrating that these cells migrate from the IL to the AL. However, the cells failed to migrate in the opposite direction. Consistently, scanning electron microscopic analysis revealed well-developed cytoplasmic protrusions in the IL-side MCL, but not in the AL-side MCL, suggesting that IL-side CD9/CD81/S100β/SOX2-positive cells had higher migratory activity. We also searched for a specific marker for IL-side CD9/CD81/S100β/SOX2-positive cells and identified tetraspanin 1 (TSPAN1) from microarray analysis. Downregulation of Tspan1 by specific siRNA impaired cell migration and significantly reduced expression of snail family transcriptional repressor 2 (Slug), a marker of epithelial-mesenchymal transition (EMT). Therefore, CD9/CD81/S100β/SOX2-positive cells in the IL-side MCL can be stem/progenitor cells that provide stem/progenitor cells to the AL-side MCL via SLUG-mediated EMT and cell migration.

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