Dedifferentiation of committed epithelial cells into stem cells in vivo

Abstract

Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states, notably cancer.

Figure 1. Secretory cells proliferate after basal cell ablation

a, Schematic representation of the ablation of CK5-expressing basal cells of the trachea. Secretory, ciliated and basal stem cells are shown in pink, blue and grey colors respectively. b, Schematic of the timeline of i-dox or i-PBS administration and tissue harvest. c, Immunostaining for basal (p63 (green) and CK5 (cyan)) and secretory cells (SCGB1A1 (green)) in combination with Ki67 (red) on either i-PBS (upper panels) or i-Dox (lower panels) treated mice (n=6). White arrows, Ki67+ cells. d, Quantification of the percentage of p63+ and SCGB1A1+ cells per total DAPI+ cells in i-PBS or i-Dox n=3. e, Percentage of p63+Ki67+ and SCGB1A1+Ki67+ cells relative to total Ki67+ cells in i-PBS and i-Dox (n=3) treated CK5-DTA mice. i-Dox, inhaled doxycycline; i-PBS, inhaled PBS. Nuclei, DAPI (blue). *- p<0.05 and ** - p<0.01; n=3 (3 mice per condition). Error bars, average±s.e.m. P-values, two tailed and paired t-test. Scale bar, 20µm.

Figure 2. Luminal secretory cells dedifferentiate into basal stem cells after stem cell ablation

a, Schematic representation of tamoxifen (Tam) and i-dox administration to Scgb1a1-YFP/CK5-DTA mice followed by tissue harvest (H). b, Immunostaining for CK5 (red) (left panels) and NGFR (red) (right panels) in combination with YFP (green) in i-PBS (upper panels) or i-Dox (lower panels) treated mice (n=3). White arrows indicate double positive cells. c, Flowcytometric analysis of dedifferentiated cells. EpCAM+GSIβ4+CD24- cells were analyzed for expression of SSEA1 and YFP in either i-Dox and i-PBS treated mice. i-Dox, inhaled doxycycline; i-PBS, inhaled PBS. Nuclei - DAPI (blue). n=3 (at least 2 mice each per condition). Scale bar, 20µm.

Figure 3. Secretory cells dedifferentiate in the absence of basal cells in an ex vivo sphere forming assay

a, Schematic representation of lineage labeling, sorting and ex vivo sphere forming assay. Schematic representation of different type of spheres anticipated from basal and secretory cell mixing assay. b, Quantification of the number of spheres that are either basal cell derived (grey bars) and secretory cell (green bars) derived. x-axis, the ratios (1:0, 0:1, 1:1, 1:9 and 9:1) of basal to secretory cells seeded. y-axis, number of spheres formed relative to the number of cells seeded. c, Immunostaining for p63 (red) (upper panels) or CK5 (red) (lower panels) in combination with YFP (green). Nuclei - DAPI (blue). n=3 (2 replicates per condition). Error bars, average±s.e.m. Scale bar 20µm.

Figure 4. Dedifferentiation potential of secretory cells is inversely proportional to their maturity

a, Schematic representation of dissociation and sorting of 3 subsets of secretory cells based on expression of SSEA and GFP. b, Sorting of secretory cell subsets followed by sphere forming assay. Representative images of the predominant type of cell aggregates (spheres or cell clusters) from secretory cell subsets. c, Immunostaining for CK5 (green) and p63 (red) on cell aggregates. d-e, Quantification of the number of spheres (d) or cell clusters (e) from secretory cell subsets. Nuclei - DAPI (blue). n=3 (2 replicates per condition). Error bars, average±s.e.m. Scale bar 20µm.

Figure 5. Dedifferentiated cells are functional stem cells both in vivo and ex vivo

a, Schematic representation of the regeneration of epithelium from dedifferentiated cells after SO2 or influenza induced injury. b, Timeline for the induction of dedifferentiation prior to infectious or toxic injury followed by tissue harvest. c, Co-labeling of YFP (green) with CK5 (red; left panels), SCGB1A1 (red; middle panels) and FoxJ1 (red; right panels) after SO₂ (upper panels) or influenza induced injury (lower panels). d. Ex vivo expansion, and differentiation of sorted dedifferentiated cells. e, Immunostaining for CK5(red), p63 (magenta) and YFP (green) on colonies from sorted dedifferentiated cells. f, Co-labeling of YFP (green) with p63 or Acetylated-Tubulin or SCGB1A1 (red). i-Dox, inhaled doxycycline; Tam, tamoxifen; H, harvest. Nuclei - DAPI (blue). n=3 (2 replicates/mice per condition). Scale bar 20µm.