Regulation of trachebronchial tissue-specific stem cell pool size

Abstract

Tissue-specific stem cell (TSC) number is tightly regulated in normal individuals but can change following severe injury. We previously showed that tracheobronchial epithelial TSC number increased after severe naphthalene (NA) injury and then returned to normal. This study focused on the fate of the supernumerary TSC and the signals that regulate TSC pool size. We used the Keratin 5-rTA/Histone 2B:green fluorescent protein (GFP) model to purify basal cells that proliferated infrequently (GFP(bright) ) or frequently (GFP(dim) ) after NA injury. Both populations contained TSC but TSCs were 8.5-fold more abundant in the GFP(bright) population. Interestingly, both populations also contained a unipotential basal progenitor (UPB), a mitotic basal cell subtype whose daughters were terminally differentiated basal cells. The ratio of TSC to UPB was 5:1 in the GFP(bright) population and 1:5 in the GFP(dim) population. These data suggested that TSC proliferation in vivo promoted TSC-to-UPB differentiation. To evaluate this question, we cloned TSC from the GFP(bright) and GFP(dim) populations and passaged the clones seven times. We found that TSC number decreased and UPB number increased at each passage. Reciprocal changes in TSC and UPB frequency were more dramatic in the GFP(dim) lineage. Gene expression analysis showed that β-catenin and Notch pathway genes were differentially expressed in freshly isolated TSC derived from GFP(bright) and GFP(dim) populations. We conclude that (a) TSC and UPB are members of a single lineage; (b) TSC proliferation in vivo or in vitro promotes TSC-to-UPB differentiation; and (c) an interaction between the β-catenin and Notch pathways regulates the TSC-to-UPB differentiation process.

Keywords: Multipotential stem cells; Notch; Terminal differentiation; Unipotential basal progenitor; Wnt/β-catenin.

Figure 1. The chromatin label-retention system is cell type-specific and doxycycline (dox) dependent

(A) Schematics depicting transgene structure. Keratin 5 (K5), tetracycline (TET), green fluorescent protein (GFP). (B–C) Images depicting expression of Histone 2B:GFP (H2B:GFP) when mice are fed standard chow (B) or dox chow (C). (D–E) Dual immunofluorescence analysis (DIF) of the tracheobronchial epithelium (TBE) from animals that were fed standard chow (D) or dox chow (E). K5-red, H2B:GFP-green, and DAPI (blue). (F) Experimental design. NA-naphthalene. DIF analysis of TBE from mice that were recovered to day 6 (G) or day 40 (H). Arrows-H2B:GFP+ nuclei (n=6 tracheas).

Figure 2. Localization and phenotype of Histone 2B:GFP positive (H2B:GFP+) cells

(A–C) Whole mount images of trachea recovered from doxycycline (dox) and naphthalene (NA)-treated mice on recovery day 40. (A, C) external and (B) luminal surface. Asterisks: cartilage rings. Dashed lines: border between cartilaginous and membranous regions. Arrows: white-GFP+; yellow-GFPlow nuclei. (D–I) Dual immunofluorescence analysis of H2B:GFP+ cell phenotype on recovery day 6 (D–F) or 40 (G–I). Arrows: white H2B:GFP+ cells that co-express Keratin (K) 5 (D, G) or CCSP (E, H); green: H2B:GFP+ cells that do not express CCSP (E, H) or ACT (F, I) (n=6 tracheas).

Figure 3. Isolation of viable Histone 2B:GFP^bright (GFP^bright) cells

(A–B) FLOW cytometry histograms depicting cell count vs. GFP fluorescence intensity for CD45-/CD31-/TER119-/DAPI- cells derived from bitransgenic (BiTg) mice fed standard chow (no Dox, blue line) or BiTg mice fed doxycycline (dox) chow and treated with naphthalene (NA). Cells from dox/NA treated mice were evaluated on recovery day 6 (short term recovery, red line) or day 40 (long term recovery, green line). (C–D) Flow cytometry bivariate plots depicting the Sca1 and CD49f surface phenotype of GFP^bright (C) and GFP^dim (D) cells. (n = 7 analyses). (E) Real time RT-PCR analysis of Keratin (K) 5 and aldehyde dehydrogenase 1a1 (Aldh1a1) mRNAs in GFP^bright (small check) and GFP^dim (large check) populations. Mean ± SEM (n=4). (F) Dual immunofluorescence analysis of Keratin (K) 5 and GFP expression in cytospin preparations of GFP^bright cells. (n= 4 analyses).

Figure 4. The GFP^bright and GFP^dim populations contain functional tissue specific stem cells (TSC)

(A) Schematic of TSC-derived rim clone. (B) Confocal 3-dimencional projection of a rim clone generated by a GFP^bright cell that was cultured in doxycycline (dox)-free medium. Representative of 10 rim clones. (C) FLOW cytometry histogram depicting cell count vs. GFP fluorescence intensity for Passage (P) 1 rim clones that were derived from GFP^bright cells and cultured in dox-free medium. Representative of 3 analyses. (D) Flow cytometry bivariate plots depicting the Sca1 and CD49f surface phenotype of GFP^positive cells indicated in panel C. Representative of 3 analyses. (E) Merged phase contrast and fluorescence images of a P2 rim clone that was cultured in dox-free medium. Arrows: GFP+ cells. Arrowheads GFP− cells. Representative of 3 analyses. (F–G) Dual immunofluorescence analysis of GFP+ and Keratin 5 (K) 5. Green (GFP) channel only at 50X (F). Green (GFP) and red channels (K5) at 400x (G). (n = 3 analyses).

Figure 5. Tissue specific stem cells (TSC) that proliferate frequently in vivo have decreased mitotic potential in vitro

(A) Rim (white bars) and non-rim (hatched bars) clone forming cell frequency (CFCF) of freshly-isolated GFP^bright and GFP^dim cells (n=4) (B) Schematic of a non-rim clone. (C) Merged phase contrast and fluorescence images of a passage (P) 0 rim clone that was cultured in dox-free medium. Representative of 3 analyses. (D) Frequency of rim and non-rim clone formation as a function of passage. Green circles: GFP^bright rim clones. Green squares: GFP^bright non-rim clones; Red triangles: GFP^dim rim clones; Red inverted triangles: GFP^dim non-rim clones. Mean ± SEM (n=6). (E) Phase-contrast image of a P6 clone. Arrow: edge of the clone indicating the absence of a rim. (F) Western blot analysis of p53 protein level in rim clones from P3-P6. Lanes 1, 3, 5 and 7: clones derived from GFP^dim cells. Lanes 2, 4, 6 and 8: clones generated by GFP^bright cells. Lane 9, normal NIH3T3 cells. Lane 10, irradiated NIH3T3 cells, a positive control. Actin was used as the loading control.

Figure 6. Tissue specific stem cell (TSC) proliferation in vivo does not alter differentiation potential

(A) Schematic of the experimental design. (B–D) Dual immunofluorescence analysis of GFP (green) and Keratin (K) 5 (red) (B), GFP (green) and CCSP (red) (C), or GFP (green) and ACT (red) (D). Nuclei are counterstained with DAPI (blue). All images are enface views of the apical surface. Arrows: differentiated GFP+ cells. Representative of 3 analyses. (E) Differentiation potential of rim clone cells as a function of passage (P). Mean ± SEM (n=3). (F) Rate of differentiation to K5+, CCSP+, and ACT+ cells with increasing passage number. Data summarize those in Panel E. Mean ± SEM (n=3). (G) Frequency of filler cell differentiation to K5+, CCSP+, and ACT+ cells.

Figure 7. Wnt/β-catenin pathway genes are differentially expressed in GFP^bright and GFP^dim cells

(A–H) Quantitative RT-PCR analysis of Wnt/β-catenin pathway gene expression in freshly isolated GFP^bright (closed bar) and GFP^dim (open bar) cells. Mean ± SD (n = 3). (I) A summary of the gene expression data is shown in the Venn diagram. Genes that are significantly (p < 0.05) over expressed in the GFP^bright or in the GFP^dim cells are shown in the left and right respectively. Genes that are not significantly different between the two populations are shown in the overlapping area.