Figure 1. Identification of neonatal PSCs. (A) Neonatal pulmonary cells were labeled
with Sca-1 and SSEA-1 antibodies and analyzed using flow cytometry. The data are representative of five independent experiments. (B) Representative flow cytometry histograms of SSEA-1 and Sca-1-stained cell population in the lung single-cell suspension. (C) Cell number of SSEA-1+- and Sca-1+- enriched pulmonary cell extracted from whole lung/mouse (n = 3-5 mice per group). Data are means ± SD and represent three independent experiments. ** P <0.01.Figure 2. Whole-mount view of SSEA-1
+pulmonary cells in lung tissues. Neonatal
and adult mice derived lung sections were stained with anti-SSEA-1 (green) and the nuclei were counterstained with DAPI (blue). Bar: 500 µm. Data are representative of at least five independent experiments.Figure 3. Expression of SSEA-1
+pulmonary cells in lung tissues. Neonatal and adult
mice derived lung sections were stained with anti-SSEA-1 (green) and CCSP (red); the nuclei were counterstained with DAPI (blue). The bronchiole (Br) and terminal bronchiolar (TB) airway were determined by CCSP (red) staining. Neonatal (left) and adult (right) SSEA-1+ pulmonary cells were located within TB adjacent to the bronchioalveolar duct junction (BADJ). Bars: 50 µm. The arrows point to the BADJ.Data are representative of at least five independent experiments.
Figure 4. Characterization of neonatal SSEA-1
+pulmonary cells. (A) Flow
cytometric analysis of surface marker expression in neonatal SSEA-1+ pulmonary cells.The gray areas represent matched isotype controls. Data are representative of three independent experiments. (B) FACS analysis of intracellular SPC and surface T1α expression in SSEA-1+ PSCs. SSEA-1+ PSCs expressed SPC (a type II pneumocyte marker) but were negative for T1α (a type I pneumocyte marker). Data are representative of two independent experiments. (C) Enriched SSEA-1+ PSCs were purified from neonatal mice, and the CCSP expression levels were determined using RT-QPCR with specific primers. The mRNA levels were normalized using the housekeeping gene GAPDH. Bars indicate mean ± SD of three independent
prepared, and the CCSP protein levels were determined by immunoblots stained with a specific antibody against CCSP. β -actin was used as an internal control. Data are representative of two independent experiments.
Figure 5. Neonatal SSEA-1
+pulmonary cells are negative for pneumocyte-related
markers. Newborn mice-derived pulmonary single-cell suspensions and paraffin-fixed
lung tissues were stained with SSEA-1 (green), alveolar cell markers (red) AQP5 (a type I pneumocyte marker), and proSPC (a type II pneumocyte marker). The nuclei were counterstained with DAPI (blue). Bars, 20 µm.Figure 6. Neonatal SSEA-1
+pulmonary cells are self-renewal and clonogenicity. (A)
Representative photographs of sphere colonies cultured in Matrigel with Transwell plates. (B) Representative photographs of primary and secondary sphere colonies. Bars, 100 µm. (C) Efficiency of secondary spheres formation. The results are expressed as means ± SD (n = 3).Figure 7. Neonatal SSEA-1
+pulmonary cells are multipotent. (A) SSEA-1
+ PSCs cultured on Matrigel-coated plate for 15-20 days were stained with pro SPC (green; left) and AQP5 (green; right) antibody to identify the type I pneumocyte; the nuclei were counterstained with DAPI (blue). Bars, 20 µm. (B) SSEA-1+ PSCs seeded in an ALI culture for 15 days were labeled for ZO-1 (green), γ -tubulin (red), and DAPI (blue).Bars, 20 µm. (C) Scanning electron microscopic images of in vitro-cultured SSEA-1+ PSCs at ALI for 15 days, showing ciliated and nonciliated cells. Bar, 10 µm.
Figure 8. Neonatal SSEA-1
+PSCs suppress TSLP and eotaxin production. Lung
epithelial cells (2×104) derived from adult mice were co-cultured with neonatal SSEA-1+ PSCs (1×104) in the presence of (A) LPS (2 µg/ml) or (B) IL-4 (100 ng/ml) for 24 hours. TSLP and eotaxin contents in culture supernatants were determined using ELISA.Values are the means ± SD. The data are representative of three independent experiments. * P < 0.05, ** P < 0.01.
Figure 9. SSEA-1
+PSCs suppress TSLP and eotaxin production through cell-cell contact independent manner, respectively. Co-culture system of adult lung epithelial
cells (2×104; bottom) and neonatal SSEA-1+ PSCs (1×104; insert) were separated with a Tranwell and stimulated with (A) LPS (2 µg/ml) or (B) IL-4 (100 ng/ml) for 24 hours.TSLP and eotaxin contents in culture supernatants were determined using ELISA.
Values are the means ± SD. The data are representative of three independent experiments. * P < 0.05, ** P < 0.01.
Figure 10. Neonatal lung SSEA-1
+PSCs reduced TSLP and eotaxin production from adult lung epithelial cells through CCSP-dependent and -independent manner. Lung epithelial cells derived from adult mice were co-cultured with neonatal
SSEA-1+ PSCs and stimulated with (A) LPS or (B) IL-4 for 24 hours in the presence of control antibody or CCSP neutralizing antibody (5 µg/ml). TSLP and eotaxin contents in LPS- and IL-4-stimulated culture supernatants were determined using ELISA, respectively. Data are means ± SD and representative of three independent experiments.** P < 0.01.
Figure 11. Transplantation of SSEA-1
+PSCs did not change serum OVA-specific
IgE in the asthmatic mice. (A) Distribution of SSEA-1
+ PSCs in adoptive transferred mice. The lung sections were stained with an anti-GFP- (green) and an anti-panCK (red) antibody, and they were counterstained with DAPI (blue). Bars, 50 µm. (B) Magnified view of the boxed area in A. Bar, 50 µm. (C) Transplanted PSCs maintained SSEA-1 expression after repeated OVA challenge in vivo. The lung sections were stained with an anti-GFP- (green) and an anti-SSEA-1 (red) antibody, and they were counterstained with DAPI (blue). Bar, 20 µm. (D) The mice received an i.v. injection of 1×105 cells of SSEA-1+ PSCs or SSEA-1- pulmonary cells isolated from neonatal GFP-mice after the second aerosol treatment. OVA-specific IgE was detected in serum 24 hours after the last inhalation exposure to OVA. Data are means ± SEM and representative of two independent experiments (n = 5 mice per group).Figure 12. Transplantation of SSEA-1
+PSCs before the OVA challenge reduced
the level of inflammatory mediators and the cellular infiltration of the lungs. 1×10
5 SSEA-1+ PSCs isolated from neonatal mice were i.v. injected into OVA-induced asthmatic mice prior to an OVA aerosol challenge. (A) The BAL fluid was taken 24 hours after the last aerosol OVA exposure. Differential counts of the cells in the BAL fluid were obtained by FACS analysis, and (B) The levels of IL-4, IL-5, TSLP, and eotaxin in the BAL fluid was determined by ELISA. n = 3-5 mice per group. Bars represent means ± SD. * P < 0.05, ** P <0.01. The data from one representative experiment of two are shown.Figure 13. Transplantation of SSEA-1
+PSCs improves AHR and airway
eosinophilia in the asthmatic mice. (A) Flowchart of the method used to produce the
OVA-induced asthmatic murine model. (B) Airway function was measured by invasive body plethysmography. The results are expressed as means ± SEM of the lung resistance (RL) in the ratio of RL after PBS nebulization. Data are represent two independent experiments (n = 4-7 mice per group). ** P < 0.01. (C) Cell number in the recovered BAL fluid was counted after staining by trypan blue. (D) Eosinophil counts in the BAL fluid were obtained by FACS analysis. Data are means ± SEM and representative of two independent experiments (n = 6-8 mice per group). ** P < 0.01.Figure 14. Transplantation of SSEA-1
+PSCs reduces airway inflammatory
cytokine/chemokine production in the asthmatic mice. BAL fluid was taken 24 hours
after the last aerosol OVA exposure. The level of eotaxin, TSLP, IL-4, IL-5, and IL-13 in BAL fluid was determined using specific ELISA kits. Data are means ± SEM and representative of two independent experiments (n = 4-6 mice per group). ** P < 0.01.Figure. 15. Transplantation of SSEA-1
+PSCs reduces airway inflammation. H&E
staining of lung sections in untreated, SSEA-1+ PSCs-, SSEA-1- pulmonary cells-treated, and healthy group. Bars, 200 µm. Arrows denote infiltrated leukocytes; br, bronchus; v, vessel. Data are representative of two independent experiments.Figure 16. Transplantation of SSEA-1
+PSCs decreased the infiltration of
inflammatory cells into lung in the asthmatic mice. Enlarge view of Figure 15. Bars,
200 µm. Arrows denote infiltrated leukocytes; br, bronchus; v, vessel.Figure 17. Transplantation of SSEA-1
+PSCs increase Foxp3
+Treg population.
Total cell number in (A) thoracic and (B) cervical LN. Cell number and percentage of Foxp3-GFP+ CD4+ Treg in (C) thoracic and (D) cervical LN determined by FACS analysis (n = 5-9 mice/group). Values are presented as the means ± SD. The data are representative of two independent experiments. * P < 0.05, ** P <0.01.
Figure 18. Transplantation of SSEA-1
+PSCs preserve CCSP production. (A) The
expression of CCSP in lung sections from untreated-, SSEA-1+ PSCs-treated-, and healthy mice was analyzed using immunostaining with an anti-CCSP antibody (green);and the nuclei were counterstained with DAPI (blue). The data are representative of two independent experiments. Bars, 50 µm. (B) The transcript levels of CCSP in lung tissues were analyzed using RT-QPCR with specific primers. The mRNA levels were normalized with GAPDH as a housekeeping gene. * P < 0.05, ** P <0.01.
Figure 19. Transplantation of SSEA-1
+PSCs inhibits unregulated cell proliferation in asthma. (A) Schematic representation of an OVA-induced asthma model with BrdU
administration. (B) Representative BrdU immunostaining of proliferating cells (red);and the nuclei were counterstained with DAPI (blue). The data are representative of two independent experiments. Bars, 100 µm. (C) Graphic representation of decreased BrdU incorporation in the lungs of SSEA-1+ PSCs-recipient mice. Two to three consecutive cryosections from each mouse lung were examined, and the average number of BrdU-positive nuclei per 1,000 nuclei was used as the value for the mouse. The data are expressed as the means ± SD, n = 3 mice/group. ** P < 0.01.
Figure 20. BrdU-incorporated cell population in asthma. Immunofluorescence
staining of T1α, CD3 or TTF-1 (green), BrdU (red) and DAPI (blue) in asthmatic lung tissue. Bars, 20 µm.VII. REFERENCES
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