Abstract

There is a growing appreciation of the role of lung stem/progenitor cells in the development and perpetuation of chronic lung disease including idiopathic pulmonary fibrosis. Human amniotic epithelial cells (hAECs) were previously shown to improve lung architecture in bleomycin-induced lung injury, with the further suggestion that hAECs obtained from term pregnancies possessed superior anti-fibrotic properties compared with their preterm counterparts. In the present study, we aimed to elucidate the differential effects of hAECs from term and preterm pregnancies on lung stem/progenitor cells involved in the repair. Here we showed that term hAECs were better able to activate bronchioalveolar stem cells (BASCs) and type 2 alveolar epithelial cells (AT2s) compared with preterm hAECs following bleomycin challenge. Further, we observed that term hAECs restored TGIF1 and TGFβ2 expression levels, while increasing c-MYC expression despite an absence of significant changes to Wnt/β-catenin signaling. In vitro, term hAECs increased the average size and numbers of BASC and AT2 colonies. The gene expression levels of Wnt ligands were higher in term hAECs, and the expression levels of BMP4, CCND1 and CDC42 were only increased in the BASC and AT2 organoids co-cultured with hAECs from term pregnancies but not preterm pregnancies. In conclusion, term hAECs were more efficient at activating the BASC niche compared with preterm hAECs. The impact of gestational age and/or complications leading to preterm delivery should be considered when applying hAECs and other gestational tissue-derived stem and stem-like cells therapeutically.

Introduction

Idiopathic pulmonary fibrosis (IPF) is characterized by the continual loss of functional lung parenchyma and its replacement by excessive and disordered extracellular matrix, resulting in an eventual decline in respiratory function [1]. The survival rate for IPF is a bleak median rate of 3 years, and it is the most prevalent of the idiopathic interstitial lung diseases [2]. There is increasing evidence indicating that accelerated alveolar senescence is a major underlying factor in the development and perpetuation of IPF [3]. As a chronic disease that affects the aged population (average age of IPF onset is 66 years) [2], it is likely that stem cell attrition contributes to the aberrant wound healing response. Perinatal stem cells such as umbilical cord-, placenta-, amniotic- and chorionic-derived mesenchymal stromal cells are being investigated in clinical [4,5] and preclinical studies [6] as treatment modalities for IPF. However, information around the impact of donor gestational age on the potency of the isolated cells remains scarce. If perinatal stem cells are to be considered as a cell therapy for IPF, it is prudent to understand the impact of prematurity on cell quality as these differences would inform donor acceptance criteria and key elements in the design of potency assays.

In line with these concepts, we sought to understand the impact of donor gestational age on human amniotic epithelial cells (hAECs) and their ability to activate endogenous lung stem/progenitor cells in the setting of bleomycin-induced lung injury. The alveolar epithelium consists of two main cell types: cuboidal type 2 alveolar epithelial cells (AT2), which secrete high levels of surfactant protein C (SFTPC or SPC), and squamous type 1 alveolar epithelial cells (AT1), which comprise 95% of the gas exchange surface area. The latter express podoplanin (Pdpn) and advanced glycosylation end product-specific receptor (AGER) [7–9]. Genetic lineage-tracing studies have established that AT2 represent lung epithelial progenitors, which proliferate and differentiate during lung maintenance. Following injury, AT2 give rise to AT1 in vivo. In vitro, AT2 give rise to alveolospheres (alveolar-like structures) that consist of both AT1 and AT2 [10]. Bronchioalveolar stem cells (BASCs) reside at the bronchioalveolar duct junctions (BADJ) of adult lungs and express both SPC and Club cell 10-kDa protein (CC10 or CCSP). The BASCs serve as multipotent lung progenitors capable of differentiating into distal AT1 and AT2, as well as proximal bronchiolar lineages following a lung insult [11,12].

Our previous work showed that hAECs isolated from healthy term placenta, released exosomes that could increase BASC and AT2 numbers and this observation coincided with reduced lung inflammation and fibrosis in bleomycin-challenged mice [13]. And while the hAECs have recently entered the clinical testing phase for chronic lung disease in infants [14] and adults [15,16], it is yet unclear how prematurity of birth might influence the overall efficacy of these cells. We previously reported that hAECs from premature births had greater proliferative potential but reduced anti-fibrotic effects [17]. In our present study, we used the bleomycin-induced lung injury murine model to determine if prematurity of hAEC donors would be associated with changes to their ability to activate BASCs, an endogenous stem/progenitor cell population in the lungs. Specifically, we were interested in the differences in cell signaling pathways that underpin functional differences between term and preterm hAECs. Further, we utilized a three-dimensional (3D) organoid air–liquid interface culture system to determine the direct effects of prematurity on the ability of hAECs to support the growth of lung stem/progenitor cells in vitro.

Materials and methods

Isolation of human amniotic epithelial cells

Amniotic membranes were collected immediately after cesarean sections from term (37.5 ± 0.5 gestational weeks) and preterm (30.4 ± 3.7 gestational weeks) placentae with 8 placentae included per group. Clinical justifications for preterm delivery included intrauterine growth restriction and preeclampsia. All patients gave written informed consent in accordance to human research ethics guidelines set out by Monash Health. Isolation of hAECs was performed as previously described [18,19]. Access to human tissues was obtained from The Ritchie Centre’s Tissue Bank (approved HREC No. 01067B).

Animals

In vivo lung injury was induced in 6- to 8-week-old-female C57BL/6 mice (Monash Animal Services, Victoria, Australia), through intranasal instillation of bleomycin (0.3U each, Bristol Myers Squibb, NY, U.S.A.). After 24 h, mice were administered either 4 million (term or preterm) hAECs suspended in 0.2 ml saline or saline alone via intraperitoneal injection. Animal groups were as follows: (1) control group (saline + saline): saline administered intranasally (vehicle for bleomycin) and intraperitoneally (vehicle for hAECs); (2) saline-treated injured group (bleo + Saline): bleomycin administered intranasally and saline intraperitoneally; (3) injured group treated with term hAECs (bleo + term hAECs): bleomycin administered intranasally and term hAECs intraperitoneally; and (4) injured group treated with preterm hAECs (bleo + preterm hAECs): bleomycin administered intranasally and preterm hAECs intraperitoneally. Group sizes are indicated in each figure legend. Animals were humanely culled 5, 7 or 14 days. The same control group was used for comparison at each time point. All experiments were performed in Monash Medical Centre animal facility and in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose with approval from Monash University Animal Ethics Committee (MMCA2010/34).

Immunofluorescence (IF) staining of lung tissues

Following euthanasia, lungs were perfused with saline and then inflated with 4% (w/v) paraformaldehyde (PFA) at a pressure of 12 cm H2O and fixed for 24 h prior to paraffin embedding. De-paraffinized lung sections (5 μm) were incubated in 1 mg/ml sodium tetrahydroborate (Sigma, Burlington, U.S.A.) in phosphate-buffered saline (PBS) to minimize aldehyde-induced non-specific autofluorescence background. Citrate antigen retrieval was performed prior to blocking in 5% goat serum (Thermo Fisher Scientific, Waltham, U.S.A.). Tissue sections were then incubated overnight with rabbit anti-pro-SPC (1:300, AB3786, MilliporeSigma, Burlington, U.S.A.) and goat anti-CC10 (1:200, SC-9772, Santa Cruz Biotechnology, Dallas, U.S.A.). The slides were then incubated with anti-rabbit 568 (1:200, A10042, Life Technologies, Carlsbad, U.S.A.) and anti-goat 488 (1:200, A21467, Life Technologies, Carlsbad, U.S.A.) at room temperature for 1 h. Nuclear staining was performed using 1 mM 4′,6-diamidino-2-phenylindole (DAPI, 1:5000, MilliporeSigma, Burlington, U.S.A.).

Quantification was performed by a blinded observer as follows: at least 18 terminal bronchioles and 6–10 fields of view of alveolar epithelium from 5 to 8 mice per each group were scored staining for pro-SPC, CC10, β-catenin and DAPI. Fluorescence microscopy and imaging were performed with Nikon C1 Inverted Microscope and NIS-Elements software. The average numbers of BASCs per terminal bronchiole were determined by manual counting while the percentage of AT2 cells and the percentage of positive β-catenin staining were determined using Imaris software (Bitplane, Zurich, Switzerland).

Culture of bronchioalveolar stem cell and AT2 organoids

The establishment of 3D BASC and AT2 organoids was performed as previously described [20]. Briefly, freshly isolated BASCs or AT2s (2 × 103 cells per well) were mixed with Matrigel containing MECs (2 × 104 cells per well) and cultured in triplicate on transwell inserts (6.5 mm, pore size 5.0 μm). At D3, term or preterm hAECs (2 × 105 cells per well) were added to the media in the lower chamber of transwells (n=4). After 14 days in culture, imaging was performed at 100× magnification (Leica AF6000LX, Leica Microsystems, Wetzlar, Germany). Numbers, sizes and phenotypes (i.e., alveolar, bronchiolar and bronchioalveolar) of organoids were counted and measured. The phenotypes of organoids were also confirmed by immunofluorescence staining as previously described [21]. Organoids were sized and enumerated using ImageJ. Organoids were randomly allocated to gene expression analysis and immunofluorescence staining. Organoids of each phenotype were dissociated and subjected to RNA isolation.

Quantitative polymerase chain reaction (PCR)

Total RNA was extracted using the RNeasy Mini Kit or Micro Kit (Qiagen, Limburg, Netherlands), and cDNA synthesis was performed using the SuperScript III Kit (Invitrogen, Carlsbad, U.S.A.). The RT-qPCR was performed using the Fluidigm Biomark™ HD system according to manufacturer instructions. All gene expression studies were performed using Taqman Assays (Applied Biosystems, Foster City, U.S.A.). Human Taqman probe sets were used for hAECs and mouse Taqman probe sets used for organoids and mouse tissues. Targeted genes and their assay IDs are listed in Supplementary Table S1. Relative gene expression was determined using the 2−ΔΔCt method and normalized against GAPDH. MicroRNA expressions profiles for term and preterm hAECs (pooled term or preterm hAECs from five cell lines each) were determined using the Taqman OpenArray Human Advanced miRNA Assays. The miRNA expression was normalized against U6 and analysis performed using Diana Tools-mirPath v.3.

Statistical analysis

Statistical analysis was performed using GraphPad Prism statistics software (GraphPad Software Inc, San Diego, CA, U.S.A.). All data were presented as mean ± SD. A one-way analysis of variance (ANOVA) was carried out to compare the mean values of different study groups. Unpaired T-test was carried out to compare the mean values between term and preterm hAECs in culture experiments. Differences were considered significant at P<0.05.

Results

Term hAECs induced activation of BASCs and AT2 following bleomycin challenge

Low engraftment rates of hAECs led us to postulate that their beneficial effects may be associated with an activation of endogenous stem/progenitor cells in the lungs [22,23]. We were also interested in the impact of hAEC administration on BASCs and AT2s given their involvement in alveolar epithelial repair [24], particularly in the context of bleomycin-induced lung injury, where hAEC administration has been previously shown to be protective [25,26].

Identification of BASCs at the terminal bronchioles was performed by immunohistochemical staining for pro-SPC and CC10 (Figure 1A). The average number of BASCs per terminal bronchiole is indicative of the activation status of the niche [10,19]. At D5, no significant changes were observed in the average numbers of BASCs at terminal bronchioles between groups (Figure 1B). At D7, BASC numbers were increased in both saline-treated injured group and term hAEC-treated group compared with the control group, but this increase was only significant in the term hAEC-treated group (Figure 1B, P<0.0001). By D14, the number of BASCs in term hAEC-treated mice returned to numbers that were comparable with healthy controls, suggesting that the bronchioalveolar duct junction stem cell niche had returned to a state of quiescence. Unlike BASCs, the percentage of AT2 population increased dramatically in the term hAEC-treated group on D5, (P<0.0001 compared with control, Figure 2), and was sustained until D7 (P=0.0069, Figure 2). By D14, there were no significant differences between groups (Figure 2).

Immunofluorescence of bronchioalveolar stem cells (BASCs) in mouse lung tissue at D5, D7 and D14 (n=6)

Figure 1
Immunofluorescence of bronchioalveolar stem cells (BASCs) in mouse lung tissue at D5, D7 and D14 (n=6)

(A) Representative images of BASC immunofluorescence staining; scale bar = 20 µm. BASCs are located in the terminal bronchioles and are positive for both pro-surfactant protein C (pro-SPC) and Club Cell 10 kD protein (CC10). (B) The number of BASCs was significantly increased in the term hAEC treated group at D7 and returned to the level comparable with the healthy controls at D14. However, no significant changes of BASC number were observed in the preterm hAEC-treated group across all the time points. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test. (*P<0.05, ****P<0.0001)

Figure 1
Immunofluorescence of bronchioalveolar stem cells (BASCs) in mouse lung tissue at D5, D7 and D14 (n=6)

(A) Representative images of BASC immunofluorescence staining; scale bar = 20 µm. BASCs are located in the terminal bronchioles and are positive for both pro-surfactant protein C (pro-SPC) and Club Cell 10 kD protein (CC10). (B) The number of BASCs was significantly increased in the term hAEC treated group at D7 and returned to the level comparable with the healthy controls at D14. However, no significant changes of BASC number were observed in the preterm hAEC-treated group across all the time points. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test. (*P<0.05, ****P<0.0001)

Percentage of type 2 alveolar epithelial cells (AT2s) in mouse lung tissue at D5, D7 and D14 ( n=5).

Figure 2
Percentage of type 2 alveolar epithelial cells (AT2s) in mouse lung tissue at D5, D7 and D14 ( n=5).

Type 2 alveolar epithelial cells are cells that are positive for pro-surfactant protein C (pro-SPC), but negative for Club Cell 10 kD protein (CC10). At D5, the percentage of AT2 population increased dramatically in the term hAEC treated group, and this change was sustained to D7. By D14, there were no significant differences between groups. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test (**P<0.01, ***P<0.001 ****P<0.0001).

Figure 2
Percentage of type 2 alveolar epithelial cells (AT2s) in mouse lung tissue at D5, D7 and D14 ( n=5).

Type 2 alveolar epithelial cells are cells that are positive for pro-surfactant protein C (pro-SPC), but negative for Club Cell 10 kD protein (CC10). At D5, the percentage of AT2 population increased dramatically in the term hAEC treated group, and this change was sustained to D7. By D14, there were no significant differences between groups. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test (**P<0.01, ***P<0.001 ****P<0.0001).

Neither term nor preterm hAECs altered activation of β-catenin in lung tissues

The β–catenin-dependent Wnt signaling pathway (Wnt/β-catenin pathway) plays a vital role in stem cell development, maintenance and self-renewal, and the activation of this pathway leads to the transcription of genes that control cell fate, polarity and migration [27]. Given our observations around hAEC administration on BASC/AT2 activation, β-catenin expression in the lung tissues was measured (Figure 3A), but no significant differences in either nuclear staining or total staining were observed at either D7 or D14 (Figure 3B,C). While these findings may initially suggest that Wnt/β-catenin signaling is not the major driver of hAEC initiated endogenous stem/progenitor cell activation, it is plausible that the transient nature of hAECs entrapment in the lungs [20] may have had an equally transient effect on Wnt/β-catenin signaling that abated by D7. Accordingly, we assessed the transcription of genes related to the Wnt/β-catenin pathway in lung tissues on D14. Here we observed that BMP4, RARRES2, SFRP2, TGFβ2 and WIF1 gene transcription were decreased in saline-treated bleomycin mice compared with control mice (P=0.0042, Figure 4A; P<0.0001, Figure 4B; P=0.0011, Figure 4C; P=0.0096, Figure 4D; P=0.0003, Figure 4E). The gene transcription of TGIF1 in term hAEC treatment groups was comparable with control but remained higher compared to saline and preterm hAEC treated groups (P=0.0129 and P=0.0207, respectively, Figure 4F). There was no difference in MYC levels between term and preterm hAECs (Figure 4G); however, endogenous MYC expression increased following term hAEC treatment (P=0.0143 and P=0.0269, compared with controls and saline treated injured groups, respectively, Figure 4H).

Beta-catenin (β-catenin) immunofluorescence in mouse lung tissue at D7 ( n= 6) and D14 (n=5).

Figure 3
Beta-catenin (β-catenin) immunofluorescence in mouse lung tissue at D7 ( n= 6) and D14 (n=5).

(A) Representative images of β-catenin staining; scale bar = 100 µm. (B and C) There is no significant differences in the total or nuclear β-catenin expression levels across all the groups on both D7 and D14.

Figure 3
Beta-catenin (β-catenin) immunofluorescence in mouse lung tissue at D7 ( n= 6) and D14 (n=5).

(A) Representative images of β-catenin staining; scale bar = 100 µm. (B and C) There is no significant differences in the total or nuclear β-catenin expression levels across all the groups on both D7 and D14.

Changes of Wnt/β-catenin pathway related gene transcription levels in mouse lung tissues on D14 ( n=6), and gene transcription level of MYC in term/preterm hAECs.

Figure 4
Changes of Wnt/β-catenin pathway related gene transcription levels in mouse lung tissues on D14 ( n=6), and gene transcription level of MYC in term/preterm hAECs.

Transcription levels of BMP4 (A), RARRES2 (B), SFRP2 (C), TGFβ2 (D) and WIF1 (E) were decreased in saline-treated bleomycin mice compared with control mice. Gene transcription of TGIF1 (F) in term hAEC treatment groups was comparable with control level and was remained higher than saline and preterm hAEC-treated groups. There was no difference in MYC (G) levels between term and preterm hAECs; however, the level of MYC (H) was increased only in term hAEC treatment groups compared with other groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 4
Changes of Wnt/β-catenin pathway related gene transcription levels in mouse lung tissues on D14 ( n=6), and gene transcription level of MYC in term/preterm hAECs.

Transcription levels of BMP4 (A), RARRES2 (B), SFRP2 (C), TGFβ2 (D) and WIF1 (E) were decreased in saline-treated bleomycin mice compared with control mice. Gene transcription of TGIF1 (F) in term hAEC treatment groups was comparable with control level and was remained higher than saline and preterm hAEC-treated groups. There was no difference in MYC (G) levels between term and preterm hAECs; however, the level of MYC (H) was increased only in term hAEC treatment groups compared with other groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Term hAECs enhanced organoid-forming efficiency of BASCs and AT2s in vitro

Next, we sought to assess if hAECs were able to exert a direct effect on lung progenitor cells. We used an established 3D lung organoid system where BASCs or AT2 were indirectly co-cultured with either term hAECs or preterm hAECs as depicted in Figure 5A. In line with previous reports [20], BASCs can give rise to organoids with three distinct phenotypes: alveolar, bronchiolar and bronchioalveolar, while AT2 cells are only capable of forming alveolar structures (Figure 5B). In keeping with their morphology, alveolar organoids expressed pro-SPC, bronchiolar organoids expressed CC10, and bronchioalveolar organoids were found to express pro-SPC and CC10 (Figure 5C). Our results indicate that term hAECs significantly enhanced the proliferation and organoid-forming efficiency of both BASCs and AT2 in vitro, compared with preterm hAECs. This was reflected in the increased sizes of BASC and AT2 organoids formed (Figure 5D,E) as well as the number of BASC organoids formed (Figure 5F,G). However, there was no significant change in the lineage commitment of lung progenitors (Figure 5H).

Bronchioalveolar stem cells and AT2s in vitro culture (n=4).

Figure 5
Bronchioalveolar stem cells and AT2s in vitro culture (n=4).

(A) Diagram of BASC and AT2 co-culture. (B) Representative images of alveolar, bronchiolar and bronchioalveolar organoids after 2 weeks of culture; scale bar = 50 μm. (C) Representative images of alveolar, bronchiolar and bronchioalveolar organoid immunofluorescence staining; scale bar = 50 μm. (D and E) Area of BASC and AT2 colonies. Term hAECs significantly enhanced the size of both BASCs and AT2 in vitro, compared with preterm hAECs. (F and G) Number of BASC and AT2 colonies. Both term and preterm hAECs significantly increased the number of both BASCs and AT2 in vitro, and term hAECs increased the number of BASCs compared with preterm hAECs. (H) The percentage of each type of colony. There is no difference between groups. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 5
Bronchioalveolar stem cells and AT2s in vitro culture (n=4).

(A) Diagram of BASC and AT2 co-culture. (B) Representative images of alveolar, bronchiolar and bronchioalveolar organoids after 2 weeks of culture; scale bar = 50 μm. (C) Representative images of alveolar, bronchiolar and bronchioalveolar organoid immunofluorescence staining; scale bar = 50 μm. (D and E) Area of BASC and AT2 colonies. Term hAECs significantly enhanced the size of both BASCs and AT2 in vitro, compared with preterm hAECs. (F and G) Number of BASC and AT2 colonies. Both term and preterm hAECs significantly increased the number of both BASCs and AT2 in vitro, and term hAECs increased the number of BASCs compared with preterm hAECs. (H) The percentage of each type of colony. There is no difference between groups. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Term hAECs increase transcription of Wnt/β-catenin related genes in 3D lung organoids

Given that the expression levels of Wnt/β-catenin signaling target genes were changed in hAEC-treated bleomycin challenged mice, we next examined the expression levels of Wnt/β-catenin pathway related genes in the organoids to determine if this pathway was involved in term hAEC induced BASC and AT2 proliferation. Both term and preterm hAECs reduced gene transcription of FZD9 in alveolar organoids generated from AT2s (P=0.0175 and P=0.0194 respectively, Figure 6A), and BASCs (P=0.0167 and P=0.0196 respectively, Figure 6A). In contrast, only term hAECs increased the gene transcription of CCND1 in alveolar colonies arising from AT2 and BASCs (P=0.017 and P=0.0135 respectively, Figure 6B). Furthermore, BMP4 and CDC42 transcriptional levels were only increased in alveolar organoids from BASCs co-cultured with term hAECs (P=0.0129 and 0.0224 respectively, Figure 6C,D). In BASC-derived bronchioalveolar organoids, FZD9 transcription was decreased only when co-cultured with preterm hAECs (P=0.0357, Figure 6A). There was no change in transcription levels of Wnt/β-catenin related genes in BASC-derived bronchiolar organoids across the groups.

Changes of Wnt/β-catenin pathway related gene transcription levels in BASC and AT2 organoids ( n=4).

Figure 6
Changes of Wnt/β-catenin pathway related gene transcription levels in BASC and AT2 organoids ( n=4).

(A) Gene transcription of FZD9 in alveolar organoids was decreased when co-cultured with either term or preterm hAECs. (B) Gene transcription of CCND1 in alveolar organoids was elevated only when co-cultured with term hAECs. (C and D) Bone morphogenetic protein 4 and CDC42 transcriptional levels were increased in term hAEC group in BASC formed alveolar organoids (*P<0.05).

Figure 6
Changes of Wnt/β-catenin pathway related gene transcription levels in BASC and AT2 organoids ( n=4).

(A) Gene transcription of FZD9 in alveolar organoids was decreased when co-cultured with either term or preterm hAECs. (B) Gene transcription of CCND1 in alveolar organoids was elevated only when co-cultured with term hAECs. (C and D) Bone morphogenetic protein 4 and CDC42 transcriptional levels were increased in term hAEC group in BASC formed alveolar organoids (*P<0.05).

Term hAECs express higher WNT ligands than preterm hAECs

Having observed differential expression of downstream Wnt targets in the organoids and mouse lung tissues, we next asked if this was attributed to differential expression of Wnt ligands in the term and preterm hAECs. Gene expression analysis revealed that both term and preterm hAECs expressed the Wnt ligands WNT2, WNT3, WNT5A, WNT6, WNT 7B and WNT11 to a similar extent. However, WNT1, WNT4, WNT10A and WNT16 were only expressed in term hAECs not preterm hAECs, and gene expressions levels of WNT2B, WNT5B, WNT7A and WNT10B were significantly higher in term hAECs compared with preterm hAECs (Figure 7). WNT3A, WNT8A, WNT8B, WNT9A and WNT9B were not detected in either term or preterm hAECs.

Changes in Wnt ligand transcription levels in both term and preterm hAECs ( n=8).

Figure 7
Changes in Wnt ligand transcription levels in both term and preterm hAECs ( n=8).

Gene expressions levels of WNT2B, WNT5B, WNT7A and WNT10B were significantly higher in term hAECs compared with preterm hAECs. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test (*P<0.05; **P<0.01).

Figure 7
Changes in Wnt ligand transcription levels in both term and preterm hAECs ( n=8).

Gene expressions levels of WNT2B, WNT5B, WNT7A and WNT10B were significantly higher in term hAECs compared with preterm hAECs. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test (*P<0.05; **P<0.01).

miRNAs are differentially expressed between term and preterm hAECs

Comparing miRNA expression between term and preterm hAECs, we observed that 28 miRNAs were only expressed in term hAECs, 40 miRNAs were only expressed in preterm hAECs, and 345 miRNAs were co-expressed in both preterm and term hAECs (Figure 8). Of the co-expressed miRNAs, 101 miRNAs that were expressed at 2-fold or higher (Supplementary Table S2) in term hAECs compared with preterm hAECs. A posteriori analysis of pathways union identified the pathways significantly targeted by the most up-regulated 22 miRNAs (expressed 4-fold higher in term hAECs compared with preterm hAECs, Supplementary Table S3). When significantly over-represented pathways were ranked according to the numbers of miRNA targeted, we noted that a number of signaling pathways associated with stem cell development, maintenance and self-renewal were identified. These included the Hippo, p53, TGF-β, regulating pluripotency of stem cells and Wnt signaling pathways.

Venn diagram showing overlap of miRNAs expressed in term and preterm hAECs.

Figure 8
Venn diagram showing overlap of miRNAs expressed in term and preterm hAECs.

There were 345 miRNAs co-expressed in both preterm and term hAECs, while 28 miRNAs were only expressed in term hAECs and 40 miRNAs were only expressed in preterm hAECs.

Figure 8
Venn diagram showing overlap of miRNAs expressed in term and preterm hAECs.

There were 345 miRNAs co-expressed in both preterm and term hAECs, while 28 miRNAs were only expressed in term hAECs and 40 miRNAs were only expressed in preterm hAECs.

Of the co-expressed miRNAs, 19 miRNAs that were expressed 2-fold lower (Supplementary Table S4) in term hAECs compared with preterm hAECs. A posteriori analysis of pathways union was applied (Supplementary Table S5). We noted that Hippo, p53, TGF-β, regulating pluripotency of stem cells and PI3K-Akt signaling pathways associated with stem cell development, maintenance and self-renewal were identified.

Since Hippo signaling pathway was in both pathways unions with the greatest significance, we also noted that Hippo-related genes SMAD3, SMAD7, ID2, and YAP1 were associated with up-regulated miRNAs. Connective tissue growth factor (CTGF) was associated with downregulated miRNAs, and LATS1, LATS2, SMAD2, SMAD4, and BIRC5 were associated with both up-regulated and down-regulated miRNAs. We proceeded to assess the transcription of these genes and observed that YAP1 and LATS1 gene transcription were increased in term hAEC treated bleomycin mice compared with control mice (P<0.05, Figure 9A,B), and gene transcription of BIRC5 were increased in preterm hAEC-treated group compared with control group (P<0.05, Figure 9C).

Changes in Hippo pathway related gene transcription levels in mouse lung tissues on D14 ( n=6).

Figure 9
Changes in Hippo pathway related gene transcription levels in mouse lung tissues on D14 ( n=6).

Yes-associated protein 1 (YAP-1, A) and LATS1 (B) gene transcription were increased in term hAEC-treated bleomycin mice compared with control mice, and gene transcription of BIRC5 (C) were increased in preterm hAEC-treated group compared with control group. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test (*P<0.05).

Figure 9
Changes in Hippo pathway related gene transcription levels in mouse lung tissues on D14 ( n=6).

Yes-associated protein 1 (YAP-1, A) and LATS1 (B) gene transcription were increased in term hAEC-treated bleomycin mice compared with control mice, and gene transcription of BIRC5 (C) were increased in preterm hAEC-treated group compared with control group. Statistical significance was determined with one-way ANOVA accompanied by the Bonferroni post-hoc test (*P<0.05).

Discussion

The initial discovery of the therapeutic potential of hAECs has moved into the sphere of clinical translation where the safety and tolerability of these allogeneic perinatal stem-like cells are being evaluated across a number of clinical indications. While we and others have shown that hAECs isolated from term, healthy placentae can have potent pro-regenerative potential, there is little information on the impact of prematurity of the donor on the quality of the cells. In the present study, we describe key differences between hAECs obtained from term and preterm pregnancies when applied to a bleomycin model of lung injury, with specific focus on their impact on endogenous lung/progenitor cells. Term hAECs had greater capacity to activate endogenous stem/progenitor cells without significant changes to Wnt/βcatenin signaling. Both term and preterm hAECs mitigated the reduction of BMP4 and WIF1 gene expression levels in response to bleomycin challenge. Furthermore, only term hAECs restored TGIF1 and TGFβ2 expression levels, while increasing c-MYC expression. In an in vitro co-culture system, term hAECs increased the average size as well as number of BASC and AT2 colonies without significant changes to lineage commitment. These observations were associated with differential expression of Wnt ligands in the term and preterm hAECs, and Fzd receptors in the BASC and AT2 organoids.

The existence of a BASC population was first described in 2005, as a subpopulation of lung stem/progenitor cells arising from the bronchioalveolar duct junction [11]. They are resistant to bronchiolar and alveolar damage but proliferate during epithelial cell renewal. They are also responsible for maintaining bronchiolar Club cells and alveolar cells in the distal lung [11]. Indeed, recent RNA sequencing of isolated BASCs has shown that they bear a transcriptionally distinct profile that co-expresses bronchiolar and alveolar epithelial genes, and while generally quiescent during homeostasis, the BASC population is the major source for regeneration of distal lung epithelial [28].

In the present study, we observed that BASCs were activated earlier in mice that received term hAECs compared with preterm hAECs. Where injured mice were administered the vehicle (saline), significant BASC activation was seen at D14, this was seen at D7 in mice administered term hAECs. In comparison, mice administered preterm hAECs achieved an attenuated BASC activation response at D14 (Figure 1). Furthermore, we observed significant AT2 expansion only in mice that received term hAECs (D5 and 7, Figure 2). These findings indicate that there are inherent differences between hAECs isolated from term pregnancies compared with preterm pregnancies. Indeed, we had previously reported that hAECs from preterm pregnancies had limited antifibrotic and anti-inflammatory potential [17]. We had proposed in this earlier study that this finding was in large part due to the lower levels of immunomodulatory HLA-G in preterm hAECs [17]. In the present study, we report that term hAECs also have superior ability to activate endogenous stem/progenitor cells in the lungs compared with their preterm counterparts.

In particular, the BASCs and AT2 cells, function as facultative stem cells that can be induced to proliferate and differentiate in response to injury [29]. That term hAECs but not their preterm counterparts were able to induce an early expansion of the BASC population (D7 compared with D14 expansion seen in untreated, spontaneously recovering animals) suggests that the pro-regenerative potential of hAECs is associated with the gestational age of the fetus. We saw an initial early expansion of BASCs at D7 in animals given term hAECs, and this expansion was self-limiting by D14 (Figure 1). Similarly, term hAECs triggered a self-limiting expansion of AT2 cells at D5 which returned to quiescence by D14 (Figure 2). This suggests that any influence that term hAECs may have on the BASC and AT2 population is transient. This bodes well for future clinical applications where one may be concerned that progenitor cells could become constitutively activated and predisposed to tumorigenesis. And while Wnt/β-catenin signaling has been implicated in lung injury and repair, we did not observe any changes to β-catenin nuclear localization between groups (Figure 3).

In the present study, we observed reduction in transcriptional levels of BMP4, RARRES2, SFRP2 and TGFβ2 in total RNA from lungs of mice 14 days following bleomycin challenge (Figure 4). These were not restored to normal levels by administration of either term or preterm hAECs. However, bleomycin-related reduction in BMP4 transcription was partially mitigated by term and preterm hAECs such that transcriptional levels were not significantly different from controls. These findings suggest that hAECs may support BASC proliferation through the reported BMP4–nuclear factor of activated T cells c1 (NFATc1)–thrombospondin-1 (TSP1) axis [20].

We observed that only the term hAECs were able to mitigate bleomycin-induced suppression of WIF1 and TGIF1 (Figure 4). The significant increase in WIF1, TGIF and MYC transcription at 14 days post-bleomycin challenge in mice given term hAECs, coincides with the return of BASCs to levels of quiescence— suggesting that WIF1 and TGIF may also regulate the quiescence and self-renewal of BASCs. Wnt inhibitor factor 1 (WIF1) binds to Wnt proteins and sequesters them to the extracellular space, thereby inhibiting their binding to a receptor [30]. While TGIF inhibits the TGFβ signaling pathway by associating with Smad2 and recruiting co-repressors, and is able to regulate quiescence and self-renewal of haematopoietic stem cells [31]. Since there was no difference in MYC transcriptional levels between term and preterm hAECs, the difference in MYC levels in hAEC treated mice was likely a reflection of endogenous MYC, which plays a critical role in maintaining the self-renewal capacity of BASCs [32].

When we assessed the expression of Wnt target genes in BASC and AT2 organoids, we observed significant changes in gene expression only in alveolar organoids from both AT2 and BASC origins that were co-cultured with term hAECs. Here we found that term hAECs were better able to influence these Wnt target genes involved in alveolar epithelium regeneration. They enhanced the expression of BMP4 and CDC42 in BASC induced alveolar organoids, and the expression of cell cycle related gene CCND1 in both AT2 and BASC induced alveolar organoids. A previous report showed that BMP4 induced lung endothelial cell activation directs BASC differentiation to the alveolar lineage through BMP4–NFATc1–TSP1 axis in vivo [20]. Cell division control protein 42 (CDC42) is an activator of the Fzd promoter, plays a vital role in lung epithelial cell proliferation, and is essential for formation and maintenance of the respiratory tract [33]. Interestingly, FZD9 expression was down-regulated in both term and preterm hAEC co-cultures. Frizzled-9 (FZD)9 acts as a tumor suppressor through the non-canonical Wnt pathway when bound to WNT7A [34]. Restoration of WNT7A and FZD9 have been shown to inhibit the growth of murine lung carcinoma cells and human non-small cell lung cancer cells [35]. Down-regulation of FZD9 in lung organoids may reflect the pro-proliferative effect of hAECs on the BASC and AT2 organoids. Furthermore, the differences in gene transcription of Wnt ligands between term and preterm hAECs may be partly due to corticosteroid exposure, commonly administered when preterm delivery is imminent and thus warrants further investigation.

The majority of miRNA content were similar between term and preterm donors. When commonly expressed miRNAs were assessed, we noted that 101 miRNAs were ≥2-folds more abundant in term hAECs. Three of the 22 miRNAs (miR-302d, miR-1260b, miR-1291) that were expressed at 4-folds higher levels in term hAECs compared with preterm hAECs were associated with 34 genes of the Wnt signaling pathway. Two of the 19 miRNAs (miR-302d, miR-1260b, miR-1291) that were expressed 2-fold lower in term hAECs compared with preterm hAECs were associated with 27 genes of the Wnt signaling pathway. Five of the down-regulated miRNAs in term hAECs associated with 55 genes related to the Hippo signaling pathway, which regulates lung epithelial progenitor cell emergence and differentiation [36]. Interestingly, YAP1, which is crucial in regulating lung embryonic and adult stem cell differentiation [37], was increased in animals treated with term hAECs.

Taken together, our study suggests that the gestational age and possibly the pregnancy related complications that resulted in the premature delivery, of hAECs and potentially other perinatal stem and stem-like cells, can impact their regenerative potential. Differences in the ability of hAECs from term and preterm donors to activate lung stem cell niches, resulted in observable differences in their ability to enhance proliferation of resident stem/progenitor cells, and consequently augment their regenerative potential. It is important to note that this increase in endogenous stem/progenitor cell activity was transient, and reflective of the previous reports on the transient presence of hAECs following cell administration. The present study provides a better understanding of the mechanisms by which hAECs exert their regenerative actions, as well as indications to potency tests that should be implemented prior to clinical use of hAECs and other modalities of cell therapy. The present study also suggests that complications of pregnancy may impact quality of perinatal stem cells and thus necessitates further research as the banking of these perinatal stem cells become increasingly commonplace.

Clinical perspectives

  • Information around the impact of donor gestational age on the potency of the isolated perinatal cells remains scarce. We previously showed that human amniotic epithelial cells (hAECs) from term healthy pregnancy but not preterm pregnancy improves lung architecture in bleomycin-induced mouse lung injury. Here, we sought to understand the impact of donor gestational age on hAECs and their ability to activate endogenous lung stem/progenitor cells.

  • We observed that term hAECs were better able to activate bronchioalveolar stem cells (BASCs) and type 2 alveolar epithelial cells (AT2s) compared with preterm hAECs in the context of bleomycin-induced lung injury.

  • Our study suggests that the impact of gestational age on hAECs and potentially other perinatal stem and stem-like cells should be considered in clinical translation.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the National Health and Medical Research Council project [grant number GNT1083744].

Open Access

Open access for this article was enabled by the participation of Monash University in an all-inclusive Read & Publish pilot with Portland Press and the Biochemical Society under a transformative agreement with CAUL.

Author Contribution

D.Z., R.S., S.T.C., J.T., M.S. and R.L. performed research, collected and analysed data presented in this manuscript. D.Z., M.S. G.D.K., R.S. and R.L. wrote the manuscript. K.T.L., C.K., E.M.W. and R.L. conceived and designed the research.

Abbreviations

     
  • AT2

    type 2 alveolar epithelial cell

  •  
  • BASC

    bronchioalveolar stem cell

  •  
  • FZD9

    Frizzled-9

  •  
  • hAEC

    human amniotic epithelial cell

  •  
  • IPF

    idiopathic pulmonary fibrosis

  •  
  • NFATc1

    nuclear factor of activated T cells c1

  •  
  • TSP1

    thrombospondin-1

  •  
  • WIF1

    Wnt inhibitor factor 1

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This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY-NC-ND). Open access for this article was enabled by the participation of Monash University in an all-inclusive Read & Publish pilot with Portland Press and the Biochemical Society under a transformative agreement with CAUL.

Supplementary data