Stem/progenitor cell dynamics during salivary gland development and regeneration demonstrated by the double pulse-chase paradigm

Abstract

Introduction: Active and quiescent stem cells coexist in hair follicles and intestinal crypts; however, their localization and differentiation potential in the salivary dynamics are unknown. This study aimed to clarify the cellular dynamics that occur during salivary gland development and regeneration in the duct ligation model with a focus on the role of label retaining cells (LRCs), presumably quiescent stem cells, in these processes.

Methods: Doxycycline-inducible TetOP-histone 2B (H2B)-green fluorescent protein (GFP) transgenic mice [GFP expression was induced during embryonic day 15 (E15)-postnatal day 7 (P7)] followed by EdU (5-ethynyl-2'-deoxyuridine) administration at P10-14 to chase the LRCs during development. In addition, LRCs were labeled with GFP immediately before salivary gland duct ligation and EdU was administered after the ligation was released to chase the LRCs with GFP and EdU during tissue repair.

Results and conclusions: During development, GFP (+) EdU (-) LRCs were abundant in striated duct cells (SDCs) and GFP (+) EdU (+) LRCs were primarily localized to the intercalated duct cells (IDCs) at P21. Labeling of the GFP (+) LRCs faded as well as the EdU (+) LRCs at P70. During tissue repair, GFP (+) EdU (+) LRCs were colocalized in the IDCs and myoepithelium cells (MECs), whereas the GFP (+) EdU (+) acinar cells (ACs) appeared over time. These results suggest that salivary gland quiescent stem/progenitor cells are present in the IDCs during development and that quiescent stem/progenitor cells in the IDCs and MECs differentiate into ACs during tissue repair.

Keywords: Adult stem cell; Growth and development; Regeneration; Salivary glands; Transgenic mice.

Graphical abstract

Fig. 1

GFP expression at P0, P10, and P30 (a–c) demonstrated by confocal laser microscopy and statistical analysis of GFP (+) cells in striated duct cells (SDCs), intercalated duct cells (IDCs), and acinar cells (ACs) (d–f) when GFP was labeled with E15 and the experimental design (n). For labeling at E15, GFP was diluted by P10 because of the rapid cell proliferation during early development. GFP (+) label retaining cells (LRCs) are significantly located to the ducts at P0, P10, and P30 (a–c). The brightness of the SDCs is significantly higher compared with that of the IDCs at P0 (d–f). Immunohistochemical staining for Ki67 and αSMA (g–l) and statistical analysis of Ki67 expression (m). Ki67 is highly expressed throughout the salivary gland at P0, whereas it decreases in expression and is localized to the IDCs by P30 (g–i). Statistical analysis showing that the high proliferative activity at P0 is significantly reduced at P5, P10, P15, and P30 (m). αSMA expression begins to appear in the ACs at P0 and in the ACs and IDCs at P30 (j–l). All samples are female mice. Scale bars = (a–c, g–l) 50 μm.

Fig. 2

GFP and EdU expressions at week 3 (a, d, j, m), week 4 (b, e, k, n), and week 10 (c, f, l, o) as demonstrated by confocal laser microscopy (a–f) and immunohistochemistry for SOX2 and Ki67 (j–o) and the experimental design (q). Figures d–f are magnified views of a–c, respectively. (a–f) GFP (+) EdU (+) cells are primarily localized to the IDCs and myoepithelium cells (MECs). (g–i) Comparing the chromaticity of SDCs with IDCs, the SDCs take up GFP and IDCs take up EdU at week 3 with significant differences, whereas no significant difference is observed at weeks 4 and 10. (j–l) SOX2 immunoreactivity, a possible stem cell marker, is localized to the duct cells and MECs. (m–o) Ki67 staining show proliferation in the ACs, ducts, and MECs at week 3 and localized to the IDCs and MECs through week 10. (p) Although the percentage of Ki67 expression does not significantly differ at weeks 3, 4, and 10, the localization of proliferative activity changes as described above. Arrows indicate MECs; double arrows indicate IDCs. The samples at 3 and 4 weeks are male mice; the samples at 10 weeks are female mice. Scale bars = (a–c, j–o) 50 μm, (d–f) 20 μm.

Fig. 3

H&E staining (a–f), Ki67- (g–l, t), and AQP5-immunoreactions (m–r, u) before ligation of the main excretory duct, 3 days and 1 week after ligation, and 1, 2, and 4 weeks after de-ligation and the experimental design (s). (a–f) The atrophy of ACs and the loss of granular ducts are recognizable immediately after ligation. Subsequently, the ACs and glandular ducts regenerate 2–4 weeks after the ligation is released. (g–l, t) The percent Ki67 expression increased after ligation, with a bimodal peak at 3 days and 1 week after ligation, returning to pre-ligation levels 2 weeks after de-ligation. Proliferation is observed in the duct cells and MECs 3 days after ligation and in the ACs and MECs 1 week after ligation. (m–r, u) The AC marker AQP5 is significantly decreased immediately after ligation and return to pre-ligation levels 4 weeks after de-ligation. All samples are male mice.

Fig. 4

Confocal laser microscopic images (a–f, a’–f’), SOX2- (g–i, g’–i’), and αSMA-immunoreactions (j–l, j’–l’) of GFP- and EdU-labeled submandibular glands on the ligated side and control side at 1 week (a, d, g, j, a’, d’, g’, j’), 2 weeks (b, e, h, k, b’, e’, h’, k’) and 9 weeks (c, f, i, l, c’, f’, i’, l’) after de-ligation and the experimental design (m). (a, d) In ligated side, AC atrophy is observed and no ACs retaining labels for GFP and EdU are recognizable. (b, e) Over time, some ACs retain both GFP and EdU labels. (g–l) SOX2 is consistently localized in the duct cells and MECs, and αSMA is maintained even during tissue injury. In control side, (a’–f’) GFP (+) LRCs are localized to the SDCs, IDCs, ACs, and MECs, whereas EdU is primarily taken up in the SDCs and MECs. (g’–i’) SOX2 is also expressed in the duct cells and MECs. Figures d–f, d’–f’ are higher magnified views of a–c, a’–c’, respectively. Arrows indicate MECs; double arrows indicate IDCs. The samples at 1 and 2 weeks after de-ligation are male mice, and the samples at 10 weeks after de-ligation are female mice. Scale bars = (a–c, g–l, a’–c’, g’–l’) 50 μm, (d–f, d’–f’) 20 μm.

Fig. 5

Laser microscopic images of GFP- and EdU-labeled submandibular glands at 3 days (a, d, g, j), 1 week (b, e, h, k), and 2 weeks (c, f, i, l) after de-ligation on the ligated side (a–f) and control side (g–l) and the experimental design (m). (a–f) GFP (+) LRCs are localized in the MECs, IDCs, and ACs, whereas EdU is incorporated into the MECs and IDCs. Co-localization of GFP and EdU is also observed in the MECs and the IDCs. (g–l) GFP (+) LRCs are localized in the IDCs and MECs, although the labeling is considerably diluted. In contrast, EdU is primarily taken up by the IDCs and MECs. No cells with both GFP and EdU labels are observed. Figures d–f, j–l are magnified views of a–c, g–i, respectively. Arrows indicate MECs; double arrows indicate IDCs. All samples are female mcie. Scale bars = (a–c, g–i) 50 μm, (d–f, j–l) 20 μm.

Fig. 6

Schematic diagrams showing the experimental designs (a, e) and the chronological changes of the LRCs during development (b–d) and regeneration (f–h). (a–d) During development, the SDCs divide at a slower rate at P0, followed by the sequential active cell proliferation of the IDCs and the ACs. The presence of asymmetric dividing cells in the IDCs during P10-14 suggests that the IDCs are a source of cells during development. (e–h) During regeneration, GFP (+) LRCs and EdU (+) cells are found in the SDCs, IDCs, and MECs, being co-localized in the IDCs and MECs; however, some ACs retain the GFP and EdU label based on the healing progress. This suggests that the LRCs in the IDCs and MCs differentiate into ACs.

Supplementary Fig. 1

Number of animals used and the schedule for each experiment.

Supplementary Fig. 2

GFP and vimentin expressions at week 3 (a, d), week 4 (b, e), and week 10 (c, f) as demonstrated by confocal laser microscopy (a–f) and the experimental design (g). Figures d–f are magnified views of a–c, respectively. (a–f) the localization of GFP-positive cells (green) was clearly different from that of vimentin-positive cells (red). The samples at 3 and 4 weeks are male mice, and the samples at 10 weeks are female mice. Scale bars = (a–c, j–o) 50 μm, (d–f) 20 μm.

Supplementary Fig. 3

GFP and CK14/19 expressions at week 3 (a, d, g, j), week 4 (b, e, h, k), and week 10 (c, f, i,l) as demonstrated by confocal laser microscopy (a–l) and the experimental design (m). Figures d–f, j–l are magnified views of a–c, g–i, respectively. (a–l) The cells with the GFP-positive nucleus and CK-positive cytoplasm are observed. The samples at 3 and 4 weeks are male mice, and the samples at 10 weeks are female mice. Scale bars = (a–c, g–i) 50 μm, (d–f, j–l) 20 μm.

Supplementary Fig. 4

GFP and CK14/19 expressions on the ligated side at 1 week (a, d, g, j), 2 weeks (b, e, h, k), and 9 weeks (c, f, i,l) after de-ligation as demonstrated by confocal laser microscopy (a–l) and the experimental design (m). Figures d–f, j–l are magnified views of a–c, g–i, respectively. (a–l) The cells with the GFP-positive nucleus and CK-positive cytoplasm are recognizable. The samples at 1 and 2 weeks after de-ligation are male mice, and the samples at 9 weeks after de-ligation are female mice. Scale bars = (a–c, g–i) 50 μm, (d–f, j–l) 20 μm.

Supplementary Fig. 5

SOX2-immunoreactions (a–c, e–j) at week 3 (a), week 4 (b), and week 10 (c), and GFP- and EdU-labeled submandibular glands on the ligated side and control side at 1 week (e, h), 2 weeks (f, i) and 9 weeks (g, j) after de-ligation and the experimental design (d, k). (a–c, e–j) SOX2 expression is observed in the developing and regenerating submandibular gland, although its expression was weaker compared with that in the sublingual gland. The samples at 3 and 4 weeks are male mice, and the samples at 10 weeks are female mice. The samples at 1 and 2 weeks after de-ligation are male mice, and the samples at 10 weeks after de-ligation are female mice. Scale bars = (a–c, e–j) 50 μm.