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.

Conflict of interest statement

Conflict of interest statement: Georgetown University has submitted a PCT patent application on this cell culture technology entitled “Immortalization of epithelial cells and methods of use” (PCT/US2011/060378).

Figures

Fig. 1.

Rapid induction of epithelial stem cell markers by ROCK inhibitor and feeders. Irradiated 3T3 J2 fibroblasts were plated on 10-cm tissue culture dishes in complete DMEM at 75% confluence. Two hours later, primary HECs (growing in KGM) were plated on 10-cm dishes with (F+Y+J2) or without (F+Y) J2 cells, in F-medium containing 10 µM Y-27632. Whole-cell lysates were prepared after 2–4 d of growth, and after 5 or 10 passages (P5, P10) for analysis by Western blotting. Control cells (KGM) were harvested from KGM. Lanes contain equal amounts of protein. Molecular mass markers (in kDa) are shown on the left.

Fig. 2.

ROCK inhibitor and feeders reprogram primary epithelial cells. Flow cytometric analysis of integrin α6 (A) and E-cadherin (B) on the surface of primary HECs in KGM, or growing on irradiated J2 cells in F-medium containing 10 µM Y-27632 (F+Y+J2) for 2 d, 4 d, or 11 passages. Unlabeled cells were not incubated with the primary antibodies.

Fig. 3.

Induction of nuclear β-catenin and telomerase during conditional reprogramming. (A) Nuclear and nonnuclear fractions were prepared from primary HECs growing in KGM, or in F-medium containing 10 µM Y-27632 in the presence (F+Y+J2) absence (F+Y) of irradiated J2 cells for 2 d, 3 d, 5 passages (P5) or 10 passages (P10). The fractions were solubilized for SDS/PAGE and Western blot analysis (equal cell equivalents per lane). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TfR, transferrin receptor. (B) Quantitative real-time RT-PCR measurement of hTERT mRNA levels in primary HECs growing in KGM, or on irradiated J2 cells in F-medium containing 10 µM Y-27632 for 2 d, 4 d or 11 passages (P11). The level of hTERT in KGM was set to 1.0. Some P11 reprogrammed HECs were harvested and replated in KGM for 3 d (P11→KGM) before RNA isolation and PCR analysis. Error bars indicate SEM (n = 3).

Fig. 4.

Rapid loss of the stem cell phenotype after removing reprogramming conditions. Western blots of whole cell lysates that were prepared from primary HECs growing in KGM, in F-medium with irradiated J2 cells and 10 µM Y-27632 for 11 passages (F+Y+J2), or for 3 d in KGM after 11 passages in F-medium with irradiated J2 cells and Y-27632 ((F+Y+J2)→KGM). Lanes contain equal amounts of protein. Molecular mass markers (in kDa) are shown on the right.

Fig. 5.

Tissue-specific developmental potential is maintained during conditional reprogramming. (A) H&E-stained histological sections of normal cervix, and of primary HECs that were differentiated in air–liquid interface culture after 43 passages on irradiated J2 cells in F-medium containing 10 µM Y-27632. Serial sections of the samples were deparaffinized, rehydrated, and stained with primary antibodies specific for cytokeratin 14 (K14), involucrin and filaggrin, followed by labeling with fluorescent secondary antibodies and Hoechst dye 33258 (DNA). (Scale bars: 100 µm.) (B) Histological sections of primary tracheal-bronchial cells that were cultured to late passage as above, followed by differentiation in vitro. Sections were stained with H&E or a combination of alcian blue and periodic acid-Schiff reaction (AB-PAS). Note the presence of ciliated cells (arrowheads) and mucus-producing cells (arrows). (Scale bar: 30 μm.) (C) Confocal microscopy of tracheal-bronchial CRCs from a second donor that were differentiated in air–liquid interface culture for 47 d, fixed and fluorescently labeled with phalloidin (F-actin), Hoechst dye 33342 (DNA), or antibodies demonstrating the presence of cilia (alpha-tubulin) and mucins 5AC and 5B (MUC5AC/MUC5B). An X–Z cross-section, extended focus X–Y view, and corresponding three-dimensional (3D) view are shown.

Fig. 6.

Conditional reprogramming does not up-regulate markers of iPSCs. Western blots of whole cell lysates prepared from primary HECs growing in KGM, or in F-medium containing 10 µM Y-27632 in the presence (F+Y+J2) or absence (F+Y) of irradiated J2 cells for 2 d, 3 d, or 4 d. As a positive control, an identical lysate was prepared from mouse ESCs (MESC). All antibodies were generated against human antigens (and demonstrated to cross-react with the mouse homologs), or were polyclonal antibodies raised against full-length mouse proteins (and demonstrated to cross-react with the human homologs). Lanes contain equal amounts of protein. Molecular mass markers (in kDa) are shown on the left.