Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells

TLDR

The study examined whether Y‑27632 and feeder cells induce a stem‑like phenotype in epithelial cells and explored the diagnostic and therapeutic potential of generating adult stem‑like cells in vitro without genetic manipulation. CRCs are induced rapidly within two days by reprogramming the entire cell population via Y‑27632 and feeder cells, rather than selecting a minor subpopulation. Conditionally reprogrammed cells exhibit an indefinite, karyotype‑stable, nontumorigenic proliferative state, display adult stem‑cell markers (α6, β1 integrins, ΔNp63α, CD44, TERT), reduced Notch signaling, increased nuclear β‑catenin, lack embryonic/iPS transcription factors, are reversible upon removal of Y‑27632 and feeders, and can differentiate into tissue‑specific epithelia in air‑liquid interface cultures.

Abstract

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air–liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.