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

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.

Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells

“…As expected from previous studies (8,13) There were morphological and functional differences between the earlypassage conventional cells and the laterpassage CRC-expanded cells. Later-passage CRC ALI cultures, especially in the nonproprietary UNC ALI media, were thinner, with fewer ciliated cells, whereas later-passage cells in USG-containing VALI media had abundant goblet cells.…”

“…Ectocervical keratinocyte CRCs expressed markers of somatic stem cells rather than embryonic or induced pluripotent stem cells, and both keratinocytes and airway epithelial CRCs, in the absence of Y, retained their original organ-specific identity in air-liquid interface (ALI) cultures (8). Enhanced proliferation and lentivirus transduction of human and mouse airway epithelial cells cultured in the presence of Y were observed (9), the CRC technique was useful to study primary ciliary dyskinesia nasal curettage cells (10), and the method has been used to expand nasal respiratory epithelial cells manipulated with CRISPR/Cas9 (11).…”

Lung organoids: current uses and future promise

“…Various methods have been developed for isolating and growing these basal cells from different regions of the normal human respiratory system, including nasal epithelium, ʻlarge airways', which include the trachea, primary bronchi and intralobar bronchi down to about the third or fourth generation, and from bronchial brushings (Hackett et al, 2011;Randell et al, 2011). The most efficient methods for expanding and cloning TRP63 + KRT5 + human basal cells involves culturing them either on irradiated mouse 3T3-J2 fibroblasts in the presence of the Rho kinase inhibitor Y-27632 (Butler et al, 2016;Kumar et al, 2011;Suprynowicz et al, 2012), or with a Rho kinase inhibitor together with inhibitors of Smaddependent signaling through the BMP and TGFβ pathways and an activator of Wnt signaling (Mou et al, 2016). The progenitor properties and differentiation capacity of these basal cells can then be followed in organoid cultures.…”

Airway Progenitor Clone Formation Is Enhanced by Y-27632–Dependent Changes in the Transcriptome

“…Relatedly, it is critical to understand the mechanism that underlies the enhanced expansion and passage of CRC cells. CRC-mediated cell amplification has been previously associated with several pathways, including those involved in cell immortalization (6)(7)(8)21), induced pluripotency (22), and reprogramming to a more stem-like state (8).…”

“…These problems were recently addressed in both bronchial and alveolar epithelial cells using a new culture method called conditional reprogramming culture (CRC) (6)(7)(8)(9). The near-limitless number of cells generated from a single specimen using this method has made extensive characterization of bronchial airway epithelial cell function possible in small numbers of donor subjects (7,8,10). However, the analysis of bronchial airway epithelium from a broad cross-section of subjects with clinically defined airway diseases is needed to help distinguish airway disease endotypes (11)(12)(13).…”