RESULTS

3.1 Identification of potential stem/progenitor cells from neonatal mouse lungs

The development of the lungs occurs as a continuous process from embryogenesis to early adolescence in humans as well as mice⁹⁸. A stem cell pool present in the neonatal lung contributes to both the bronchiolar and alveolar lineages during lung development⁶⁹, whereas these stem cell populations are a rare and quiescent population in the adult lung^{70, 71}. Previous study showed that Oct-4-, SSEA-1-, Sca-1-, or CCSP-expressing pulmonary stem/progenitor cells undergo terminal differentiation to alveolar pneumocytes ⁹³. Therefore, we speculated that mouse pulmonary stem/progenitor cells might reside among the Sca-1⁺, SSEA-1⁺, Oct-4⁺ and CCSP⁺ cells. To test this hypothesis, single-cell suspensions from lung tissues of neonatal and adult mice were prepared. CCSP and Oct-4 are expressed in cytosol and nucleus, respectively. Therefore, we used fluorescence activated cell sorting (FACS) to analyze the expression pattern of cell-surface Sca-1 and SSEA-1. Single cell suspensions were identified by forward scatter, and immune cells (CD45⁺) were excluded. FACS analysis showed that Sca-1⁺- and SSEA-1⁺- expressing cell populations were two distinct pulmonary cell populations in neonatal mice (Figure 1A). To evaluate the putative stem/progenitor cell population, we analyzed the total cell number of SSEA-1⁺ and Sca-1⁺ cells derived from the lungs of mice of different ages. We found that the number of SSEA-1⁺ cells significantly decreased in an age-dependent manner (Figure 1B, C ). The numbers of SSEA-1⁺ cells at postnatal day 1 and day 7 were 1.7 ± 0.4×10⁵ and 6.0 ± 0.8×10⁵, respectively. In contrast, the Sca-1⁺ cell population significantly increased with age. Compare with neonatal lung section, adult SSEA-1⁺ cells were difficult to detect in whole-mount view (Figure 2 and Figure 3). The frequency of lung SSEA-1⁺ cells presented in the adult mice was far fewer than the frequency seen in neonatal mice by whole-mount staining.

immunofluorescence image showed that adult lung SSEA-1⁺ cells were localized in the BADJ, which was almost the same as neonatal mice (Figure 3).

3.2 Phenotypic characteristics of neonatal SSEA-1

⁺

pulmonary cells

To characterize this potential stem/progenitor cell population, we performed an unbiased FACS-based screen of the SSEA-1⁺ pulmonary cells using a collection of monoclonal antibodies directed against cell surface markers. FACS analysis showed that the neonatal SSEA-1⁺ pulmonary cells expressed epithelial lineages marker-E-cadherin (CD324), while negative for CD31 (endothelial marker), CD34 (hematopoietic stem cell marker), CD90.2, CD73 and CD105 (mesenchymal stem cell; MSC markers) by FACS analysis (Figure 4A). In addition, neonatal SSEA-1⁺ pulmonary cells expressed CD9, CD24, CD26, CD29, CD47, CD54, CD98, CD133, and CD147 (Figure

4A). Lung is a complex organ that requires the specification of various epithelial cell

types for proper homeostasis. To verify the cell lineage of SSEA-1⁺ cells, we checked the expression of p63 (a basal cell marker), T1α (a type I pneumocyte marker), SPC (a type II pneumocyte marker), and CCSP (a Club cell marker) by FACS analysis, RT-QPCR and immunoblotting. Interestingly, SSEA-1⁺ pulmonary cells were negative for p63 and T1α, but positive for SPC (Figure 4B) and CCSP (Figure 4B, C).

Immunofluorescence staining of whole airway tissue mounts revealed that SSEA-1⁺ cells resided in the bronchioles, terminal bronchioles, and the BADJ in the lungs of neonatal mice (Figure 3). Since bronchoalveolar stem cells (BASCs) were defined as CCSP/SPC dual-positive population at the BADJ described previously by Kim and colleagues⁸⁸. Therefore, these results raised the possibility that SSEA-1⁺ cells might be multi-functional and comprise the regenerative cell populations within the airway

3.3 Neonatal lung SSEA-1

⁺

cells possess self-renewal, clonogenicity, and multipotency ability

To test whether pulmonary SSEA-1⁺cells fulfill the criteria for consideration as stem/progenitor cells, we applied repeated sphere formation assay. Pulmonary SSEA-1⁺ single cells were re-suspended in Matrigel-based three-dimensional culture. We found that primary sphere colonies were observed 10-15 days after cell culture (Figure 6A

and B left). To further clarify the self-renewal capacity of pulmonary SSEA-1

⁺cells, primary spheres were subsequently dissociated to single cell and then re-suspended to Matrigel-based three-dimensional culture. The formation of secondary spheres was observed after culture for 7-10 days (Figure 6B right). Sphere colony assay showed that SSEA-1⁺ cells exert higher sphere-forming ability than SSEA-1^- cells. The sphere formation efficiency of SSEA-1⁺ cells was 1–2 spheres/2,500–5,000 total cells) as determined by limiting dilution assay (Figure 6C). These results indicated that SSEA-1⁺ pulmonary cells might expand through self-renewal. Although SSEA-1⁺ pulmonary cells only expressed SPC (type II pneumocyte marker) when initially isolated from neonatal mice (Figure 4B and Figure 5). However, after culture on Matrigel-coated plate for 15-20 days, the SSEA-1⁺ pulmonary cells differentiated into pro-surfactant protein C⁺ type II pneumocytes and AQP5⁺ type I pneumocytes (Figure 7A). In addition, we investigate that whether SSEA-1⁺ pulmonary cells has the capacity to differentiate into TECs because SSEA-1⁺ cells were located at the BADJ in the lungs of neonatal mice (Figure 3). Immunofluorescence staining of tight junction marker ZO-1 and centrosome marker γ-tubulin showed that SSEA-1⁺ pulmonary cells differentiated into both ciliated and nonciliated cells 15 days after grown in ALI cultures (Figure 7B).

These observations suggested that neonatal SSEA-1⁺ pulmonary cells had the capability to differentiate into both pneumocytic and TEC lineages. Based on these in vitro studies, suggested that neonatal SSEA-1⁺ pulmonary cells are stem/progenitor cells with self-renewing, clonogenic, and multipotent properties.

3.4 SSEA-1

⁺

PSCs reduce TSLP and eotaxin production

Previous study⁹⁹ showed that human lung stem cells repair damaged mouse lung in vivo indicating PSCs might play a protecting role in lung damage. However whether PSCs play a critical role in the process of inflammation still not well understood. To explore the biological functions of neonatal SSEA-1⁺ PSCs, we developed an adult lung epithelial cells and neonatal SSEA-1⁺ PSCs co-culture system in the presence of stimulators. Since TLR4 ligation on airway epithelial cells induces the release of innate cytokines including TSLP, which promote the development of pathogenic Th2 cells and asthmatic inflammation⁴⁴. In addition, IL-4 plays a critical role in the differentiation of Th2 cells, and induces inflammation through stimulating the expression of eotaxin from lung epithelial cells¹⁰⁰. Therefore, we used TLR4 ligand-LPS and IL-4 to stimulate airway epithelial cells to produce TSLP and eotaxin, respectively. ELISA measurements of cell culture supernatant indicated that the primary lung epithelial cells produced high levels of TSLP and eotaxin upon LPS and IL-4 stimulation, respectively. However, the neonatal SSEA-1⁺ PSCs inhibited LPS-induced TSLP and IL-4-induced eotaxin production (Figure 8). To clarify whether the neonatal SSEA-1⁺ PSCs-mediated inhibitory effect was dependent on soluble or cell-cell contact-dependent factors, co-culture of neonatal SSEA-1⁺ PSCs and adult lung epithelial cells were physically separated by a Transwell insert, and found that neonatal SSEA-1⁺ PSCs suppressed

indicated that the SSEA-1⁺ PSCs-mediated inhibitory effect was mainly dependent on soluble factors. To clarify the mechanism of inhibition of inflammation and airway damage by SSEA-1⁺ PSCs, we tested whether SSEA-1⁺ PSCs could inhibit TSLP and eotaxin production in the presence o f CCSP neutralization antibody. We found that anti-CCSP antibody restored SSEA-1⁺ PSCs-induced TSLP but not eotaxin, suggested that CCSP might not to be the predominant pathway for eotaxin inhibition (Figure 10).

3.5 Transplantation of SSEA-1

⁺

PSCs alleviates the severity of asthmatic features

Neonatal SSEA-1⁺ PSCs inhibited TSLP and eotaxin production, therefore, we hypothesized that transplantation of neonatal SSEA-1⁺ PSCs into asthmatic mice might have therapeutic potential. Both SSEA-1 positive and negative fractions isolated from neonatal mice were collected and used for further adoptive transfer studies. To provide a precise area of their niche that would permit the stem/progenitor cells to survive and to investigate the anti-inflammatory effects of this stem/progenitor cell population, SSEA-1⁺ PSCs were intravenously delivered into mice after the second OVA aerosol exposure (Figure 13A). We used pulmonary cells isolated from enhanced GFP transgenic (EGFP-tg) mice to monitor the localization of the SSEA-1⁺ PSCs in the recipient animals. Six days after transfer, anti-GFP-labeled SSEA-1⁺ PSCs were detectable in the lung tissues (Figure 11A and B). Moreover, almost GFP-labeled SSEA-1⁺ PSCs maintained their SSEA-1 expression after repeated allergen challenge in

vivo (Figure 11C). The SSEA-1

⁺ PSCs transplant did not change the anti-OVA IgE titer (Figure 11D). Notably, administration of SSEA-1⁺ PSCs significantly suppressed the invasive AHR to methacholine (Figure 13B) and decreased the infiltration of inflammatory cells into peribronchovascular areas in the OVA-induced asthmatic mice

BAL fluid were significantly decreased in the SSEA-1⁺ PSCs-treated group compared to untreated group (Figure 13C and D). ELISA showed that neonatal SSEA-1⁺ PSCs-treatment significantly inhibited the secretion of eotaxin, TSLP, IL-4, IL-5, and IL-13 in the BAL fluid of OVA-induced asthmatic mice (Figure 14). To confirm the beneficial effects of SSEA-1⁺ PSCs in asthmatic mice, we examined the effect of transplantation of SSEA-1⁺ PSCs into mice before they received the OVA inhalation challenge. We found that both the cytokine profile of the BAL fluid and the inflammatory cell infiltration were decreased in the SSEA-1⁺ PSCs-treated group (Figure 12).

3.6 Transplantation of SSEA-1

⁺

PSCs increase Foxp3

⁺

Treg population

We further evaluate whether transplantation of SSEA-1⁺ PSCs modulate airway inflammation through regulatory T cells (Treg). We developed allergic asthma model in Foxp3-GFP reporter mice to address this hypothesis. After 7-day consecutive challenge of OVA, thoracic and cervical draining lymph nodes (LN) were collected. Total cell number in thoracic LN but not in cervical LN was increased in asthmatic mice compared with that of healthy mice (Figure 17A and B). Analysis of cell composition by FACS showed that a significantly increased percentage and cell number of thoracic LN Foxp3-GFP⁺ Treg in SSEA-1⁺ PSCs-treated asthmatic mice (Figure 17C). In contrast, Treg population and cell number in cervical LN was not significant difference among these groups (Figure 17D). These data indicated that SSEA-1⁺ PSCs increased Foxp3⁺ Treg cells in inflamed lung draining LN, and may subsequently prevent the recruitment of inflammatory cells during the disease development of asthma.

3.7 Transplantation of SSEA-1

⁺

PSCs preserves the epithelium and inhibits

The levels of CCSP in both the BAL fluid and blood serum were lower in patients with asthma compared with healthy controls^{101, 102}. Transplantation of SSEA-1⁺ PSCs reduced the severity of asthma. Therefore, we hypothesized that SSEA-1⁺ PSCs might reduce asthmatic injury through regulating the expression of CCSP.

Immunofluorescence staining showed that CCSP was weakly expressed in asthmatic lung tissue compared with healthy lung tissue (Figure 18A). In the asthmatic model, CCSP was strongly expressed in the SSEA-1⁺ PSCs recipients (Figure 18A).

Furthermore, RT-QPCR confirmed the transcript of CCSP in these groups and obtained consistent results (Figure 18B). Therefore, Transplantation of neonatal SSEA-1⁺ PSCs preserves CCSP secretion and thereby reduces the severity of asthma.

It has been suggested that the dysregulated proliferation of epithelial cells in asthma contributes to airway remodeling^{103, 104}. To test whether SSEA-1⁺ PSCs regulate the proliferation of lung structural cells, we adoptively transferred SSEA-1⁺ PSCs into asthmatic mice and exposed to BrdU 18 hours before sacrifice (Figure 19A). BrdU immunostaining showed that the proliferation in the lung was increased in asthmatic mice (Figure 19B), which is consistent with observations in patients with asthma¹⁰³. Interestingly, cell proliferation in the lung was dramatically inhibited in SSEA-1⁺ PSCs-treated asthmatic mice (Figure 19C). Immunofluorescence staining confirmed that main BrdU-incorporated cells were expressed T1α (type I pneumocytes marker) (Figure 20), but not expressed CD3 or thyroid transcription factor-1 (data not shown).

These observations suggested that the proliferative rate of type I pneumocytes was increased under repeated allergen-challenge condition.

在文檔中 分離與鑑定SSEA-1+肺部前驅幹細胞治療過敏性氣喘 (頁 32-40)