The Role of Hypoxia in Improving the Therapeutic Potential of Mesenchymal Stromal Cells. A Comparative Study From Healthy Lung and Congenital Pulmonary Airway Malformations in Infants.

Mesenchymal stromal cells (MSCs) play an important role in the field of regenerative medicine thanks to their immunomodulatory properties and their ability to secrete paracrine factors. The use of MSCs has also been tested in children with congenital lung diseases inducing fibrosis and a decrease in lung function. Congenital malformations of the pulmonary airways (CPAM) are the most frequently encountered lung lesion that results from defects in early development of airways. Despite the beneficial properties of MSCs, interventions aimed at improving the outcome of cell therapy are needed. Hypoxia may be an approach aimed to ameliorate the therapeutic potential of MSCs. In this regard, we evaluated the transcriptomic profile of MSCs collected from pediatric patients with CPAM, analyzing similarities and differences between healthy tissue (MSCs-lung) and cystic tissue (MSCs-CPAM) both in normoxia and in cells preconditioned with hypoxia (0.2%) for 24 h. Study results showed that hypoxia induces cell cycle activation, increasing in such a way the cell proliferation ability, and enhancing cell anaerobic metabolism in both MSCs-lung and MSCs-CPAM-lung. Additionally, hypoxia downregulated several pro-apoptotic genes preserving MSCs from apoptosis and, at the same time, improving their viability in both comparisons. Finally, data obtained indicates that hypoxia leads to a greater expression of genes involved in the regulation of the cytoskeleton in MSCs-lung than MSCs-CPAM.

Introduction

Cell therapy is an important field that sees use in tissue engineering and regenerative medicine (Huang et al., 2009). Human mesenchymal stromal cells (MSCs) possess the ability to self-renewal and multilinear differentiation (Minguell et al., 2001; Pittenger and Martin, 2004) and have been isolated from different tissues such as fat, bones (Pittenger and Martin, 2004), bone marrow (Minguell et al., 2001), teeth (Huang et al., 2009), amniotic fluid (Kaviani et al., 2003), and urine (Chun et al., 2012). Thanks to their immunomodulatory properties and their ability to secrete several paracrine factors, they have been used for the treatment of cardiovascular diseases (Thakker and Yang, 2014), nerve injuries (Dadon-Nachum et al., 2011), bone regeneration (Fan et al., 2015) and respiratory disorders (Lou et al., 2021). Furthermore, the use of cell therapy with MSCs was also tested in children with congenital lung diseases (Pelizzo et al., 2020; Tong et al., 2021).

As reported, repeated administration of allogeneic MSCs improved respiratory conditions in children with interstitial lung disease. In particular, in the lung tissue, the regenerative processes are promoted by a pool of MSCs which would appear to be involved in the regeneration and architecture of tissues processes. In conditions such as congenital malformations of the pulmonary airways (CPAM), the therapeutic potential of MSCs ha also been proposed (Pelizzo et al., 2021).

CPAM are congenital pulmonary anomalies histologically characterized by the presence of multiple cysts located in the lung parenchyma. Although considered a rare disease, it is estimated to affect between one in 25,000 and 35,000 live births (Laberge et al., 2001; Duncombe et al., 2002). These lung lesions are the result of embryological damage in the early gestation period causing hyperproliferation and terminal bronchioles dilatation. The absence of normal alveoli induces pulmonary compression and hypoplasia and in some cases also a displacement of the mediastinum (Stocker et al., 1977; Stocker, 2009). CPAM are classified into 5 types according to the size and lesion location. Among these the most recurrent lesions are the type 1 lesion, which occurs in 60–70% of cases with intercommunicating cysts >2 cm, and CPAM types 2 in 15–20% of cases with cystic lesions <2 cm (MacSweeney et al., 2003). It can be asymptomatic or be responsible for recurrent infections or symptoms related to compression of the airways. To date, treatment in symptomatic patients includes surgical therapy with excision of the lesion (Leblanc et al., 2017). In the asymptomatic forms, indeed, the risk of developing infections and the potential development of tumors seem to support resection in childhood (Aslan et al., 2006; Adzick, 2009; Pelizzo et al., 2009; Peters et al., 2013; Gajewska-Knapik and Impey, 2015). MSCs have a crucial role in the microenvironment and regulation of tumor survival, growth, and progression (Pelizzo et al., 2017). However, MSCs could be useful for the lung regenerative process after surgical treatment of congenital pulmonary lesions.

For this reason, interventions aimed at improving the regenerative capacities of MSCs are needed. Several studies reported that the use of growth factors or exposure to hypoxic conditions improves the properties of MSCs (Khan et al., 2011; Fan et al., 2015). Oxygen (O2) is a substrate needed by cells for energy production and cell metabolism (Wang et al., 2005). Cell proliferation and differentiation are biological processes regulated by O2. Hypoxia preconditioning has been proposed as an approach to improve the therapeutic potential of MSCs; in fact, it has been seen that short-term exposure to hypoxia has an empowering effect leading to increased cell migration and improved proliferation, survival, differentiation and paracrine activities of MSCs (Annabi et al., 2003; Yang et al., 2022).

In this context, MSCs were harvested from infants after surgery for CPAM’s lesions. Healthy lung tissue (MSCs-lung) and cystic tissue (MSCs-CPAM) were considered. In order to investigate the effect of O2 concentration, MSCs-lung and MSCs-CPAM were exposed to in vitro normoxia or hypoxia (0.2% O2) condition for 24 h. Subsequently, analogies and differences in transcriptomic profiles between MSCs-lung or MSCs-CPAM in normoxia against hypoxia were analyzed.

Materials and Methods

Patients

Two male infants with diagnosed CPAM were admitted to the surgical ward for elective surgery including lobectomy. Microscopically, the lesion was diagnosed as CPAM type II by Stocker’s classification. With parental consent, a portion of the lung tissue intended for histological analysis was used for the expansion of MSCs. Two samples were obtained respectively from the “healthy” tissue (called MSCs-lung) and from congenital lung tissue (called MSCs-CPAM). The study was performed according to the Declaration of Helsinki and with the approval of the Institutional Review Board of the “G. Di Cristina” (registration number 87 Civico 2017). Written informed consent was obtained by the parents and/or legal guardian of each infant after receiving information on the study.

Cell Isolation, Culture and Characterization

The MSCs were obtained from pulmonary tissue of two male infants diagnosed with CPAM and submitted to elective surgery, as previously reported (Pelizzo et al., 2017). The cells were expanded until passage 2. Subsequently, the cells were seeded at 2,5 × 106 cells in each Petri dish (150 × 25 mm) and kept in culture with MSCBM (Lonza, Basel, Switzerland) culture medium for 24 h inside an incubator at 37°C with a humidified atmosphere at 5% CO2 in the air before hypoxic treatment.

The cells were characterized as previously reported for:

- proliferative capacity defined as calculated cell count (CCC),

- immunophenotype by flow-cytometry using monoclonal antibodies specific for CD73, CD34, CD90, CD14, CD45, CD31, CD105 class I and class II- HLA (Beckman coulter Milan, Italy),

- osteogenic and adipogenic differentation capacity was evaluated by in vitro adding specific stimuli. In particular, for osteogenic differentation 10−7 M dexamethasone, 50 mg/mL L-ascorbic acid and 5 mM ß-glycerol phosphate were added to α-MEM 10% FBS (Euroclone, Milan, Italy), for adipogenic differentiation 100 mg/ml insulin, 50 mM isobutylmethylxanthine, 0.5 mM indomethacin were also added to culture medium. All reagents were from Sigma-Aldrich (Milan, Italy).

- The capability to enter senescence was evaluated maintaining cells in culture until the number of detached cells was ≤ to the number of plated cells.

- The immunomodulatory capacity was assesed at different ratio (1:2-1:20-1:200) on healthy donor peripheral blood mononuclerar cells (PBMC) activated with phytohemagglutinin (PHA).

Hypoxic Treatment

The cultured cells of both MSC-lung and MSCs-CPAM were seeded in a 6-multiwell plate with a density of 8 × 104 for each well. After seeding cells were maintained for 24 h in the standard medium MSCBM (Lonza) in incubator at 37°C with a humidified atmosphere at 5% CO2. Then cultured cells of MSC-lung and MSCs-CPAM were subjected to hypoxic treatment by inserting the plates into the ProOx Model P110 (BioSpherix, New York, NY, United States) hypoxia chamber for 24 h at 0.2% of hypoxia. Cells were observed under inverted light microscopy (Leica Microsystem, Milan, Italy) to evaluate the morphological features in normoxic and hypoxic conditions. Cultured cells of MSC-lung and MSCs-CPAM were maintained in incubator with normoxic condition and used as control cells.

RNA Extraction

RNA isolation was performed using the Total Exosome RNA and Protein Isolation Kit (catalog # 4478545; Thermo Scientific, Rockford, IL, United States) following the manufacturer’s instruction. The RNA quality and concentration were measured using Eppendorf BioSpectrometer fluorescence. TruSeq RNA Exome protocol (Illumina, San Diego, CA, United States) was used for library preparation. 3 batches were analyzed by RNA sequencing. Concisely, each sample RNA extracted was fragmented at 94 C for 8 min and then was synthesized the first strand of cDNA using the SuperScript II Reverse Transcriptase (Invitrogen, Milan, Italy). Subsequently, the second strand of cDNA was synthesized and purified using AMPure XP beads (Beckman Coulter, Brea, CA, United States). In the following step, in order to allow the adaptor ligation, the 3′ ends of the cDNA were adenylated and then was performed the indexing adapter ligation. The library was purified with AMPure XP beads. In order to enrich those fragments of DNA that have adaptors on both ends, and also to increase the quantity of library DNA a first PCR amplification step was carried out. The library has been validated through the Agilent Technologies 2,100 Bioanalyzer and then, 200 ng of each DNA library were combined for performing the first hybridization step. Magnetic beads coated with streptavidin were used to capture probes hybridized to the target regions, in order to eliminate nonspecific binding. After the enriched libraries were eluted from the beads, the second cycle of hybridization was performed, in order to obtain a wide specificity of regions of capture. Finally, the libraries were purified using the AMPure XP bead and then amplified. The final library was quantified and certified with the Agilent High Sensitivity Kit through a bioanalyzer. Subsequently, the library was normalized and was loaded for clustering on a MiSeq Flow Cell and then sequenced with a MiSeq Instrument (Illumina).

RNA-Seq Inspection

After the sequencing, the quality of the demultiplexed samples was verified using the software fastQC (version 0.11.5, Babraham Institute, Cambridge, United Kingdom) along with Trimmomatic (version 0.38, Usadel Lab, Aachen, Germany) (Bolger et al., 2014) to drop adapters or bad bases. Each read was mapped to the relative gene of the human reference genome version GRCh38 through the spliced transcripts alignment to a reference (STAR)RNA-seq aligner (version 2.7.3a, New York, NY, United States) (Dobin et al., 2013). Then, the htseq-count (version 0.6.1p1, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany) package (Anders et al., 2015) in python was used to count the transcripts and the differentially expressed genes (DEGs) for both the comparisons were analyzed in R (version 3.6.3, R Core Team) using the Bioconductor package DESeq2 (Love et al., 2014).

Western Blotting

Proteins extracted from all experimental groups were processed as previously described (Soundara Rajan et al., 2017). Briefly, 30 μg of proteins were resolved on SDS–PAGE gel and subsequently transferred to nitrocellulose sheets using a semidry blotting apparatus. Sheets were saturated for 120 min at room temperature in blocking buffer (1×TBS, 5% milk, 0.1% Tween- 20) and incubated overnight at 4°C in blocking buffer containing primary antibodies Bcl-2 (1:750; Santa Cruz Biotechnology, Dallas, TX, United States), Ki-67 (1:500; BioGenex, Fremont, CA, United States), Cleaved caspase-3 (1:1,000; Cell Signalling Technology, Milan, Italy), Cleaved caspase-8 (1:400; Cell Signalling Technology), p53 (1:400; Merck Millipore, Milan, Italy) and ITGA4 (1:400; Abnova, DBA, Milan, Italy). β-Actin (1:750; Santa Cruz Biotechnology) was used as a loading control. After four washes in TBS containing 0.1% Tween-20, samples were incubated for 60 min at room temperature with peroxidase-conjugated secondary antibody diluted as 1:1,000 in 1 × TBS that contained 2.5% milk and 0.1% Tween-20. Bands were visualized and quantified by the ECL method with Alliance 2.7 (UVItec Limited, Cambridge, United Kingdom).

Statistical Analysis

To select the DEGs and reject the genes that did not differ statistically we used in DESeq2 the Welch’s test applied to the negative binomial distribution. In detail, we corrected the p-value in q-value using the post-hoc Benjamini–Hochberg procedure and a threshold level of 0.05. Thus, only genes whose q-value was lower than 0.05 were defined as DEGs.

Furthermore, for both MSCs-lung and MSCs-CPAM comparisons we enriched the DEGs in both the comparison with the KEGG pathways using the clusterProfiler Bioconductor package (Yu et al., 2012). We used the default statistics test and we set 0.05 as the threshold. Even in this case, we used the false discovery rate post-hoc Benjamini–Hochberg to correct the p-value and drop the false positive pathways.

For protein expression the experiments were performed in triplicate. All data are expressed as mean ± standard deviation. Statistical analyses were performed using the Student’s unpaired t-test for comparisons of two groups and one-way ANOVA followed by the Bonferroni method for comparisons of three or more groups. Values of p < 0.05 were considered statistically significant.

Results

Characterization of MSCs-Lung and MSCs-CPAM

MSCs-lung and MSCs-CPAM were successfully isolated and expanded from lung samples of both patients. As already reported they were plastic adherent, showed spindle shape morphology and high proliferative capacity, reaching ≥80% confluence in less than 5 days.

Cells were negative for CD34, CD14, CD45, and HLA-DR and positive for CD73, CD90, CD105, and HLA-I.

They did not differentiate into adipocytes or osteocytes, since lipid droplet formation or AP activity and calcium deposition was not observed.

MSCs-lung ceased to growth at P14 for both lines, while MSCs-CPAM at P19 and P16.

Moreover, they showed a dose dependent in vitro immunosuppressive activity on activated PBMC.

Culture of MSCs-Lung and MSCs-CPAM

Cells were incubated under normoxic conditions and under hypoxic conditions. Cells morphology was recorded after hypoxic treatment using a light microscope. After hypoxic treatment, several spindle-shaped fibroblast-like cells were observed and their numbers evidently increased. In contrast to normoxic conditions, hypoxic conditions promoted the proliferation in MSCs-lung and MSCs-CPAM (Figure 1).

FIGURE 1

MSCs-lung and MSCs-CPAM morphology observations under inverted light microscopy. (A,C) Normoxic controls. (B,D) Hypoxia samples. Scale bar = 10 µm.

Transcriptomic Analysis

We focus our analysis on the DEGs observed in the two cell types (MSCs-lung or MSCs-CPAM) in the hypoxia condition compared to the normoxia condition. Thus, the analyzed comparisons are:

- MSCs-lung-Normoxia against MSCs-lung-Hypoxia

- MSCs-CPAM-Normoxia against MSCs-CPAM-Hypoxia.

The comparison of MSCs-lung-Normoxia against MSCs-lung-Hypoxia highlighted 2,481 DEGs (1,361 upregulated, 1,120 downregulated). On the other hand, the comparison of MSCs-CPAM- Normoxia against MSCs-CPAM-Hypoxia highlighted 2,888 DEGs (1,501 upregulated, 1,387 downregulated). The Venn diagram in Figure 2 summarizes the similarity and differences in DEGs among MSCs-lung and MSCs-CPAM in normoxia or hypoxia conditions.

FIGURE 2

Venn diagram of MSCs-lung and MSCs-CPAM in the different normoxia and hypoxia conditions. All the comparisons found 606 genes as different in statistical manner (the bottom intersection).

In order to observe the biological differences between MSCs-lung and MSCs-CPAM, we searched for the common enriched pathways. We focused our attention on the pathways “Cell cycle” (hsa04110), “p53 signaling pathway” (hsa04115), “Glycolysis/Gluconeogenesis” (hsa00010). The analysis of these pathways allowed us to observe 3 different processes involved in increasing the proliferative capacity of MSCs and which could support their role in tissue repair and regeneration. Indeed, treatment with hypoxia seems to increase their ability to proliferate through the activation of the cell cycle (Figure 3A) and inhibition of the apoptosis (Figure 3B), which is parallelly supported by the activation of the cellular metabolism (Figure 3C).

FIGURE 3

Heatmap of genes involved in the activation of the cell cycle (A), inhibition of the apoptosis (B) or activation of the cellular metabolism (C) in the comparison of MSCs-lung-Normoxia against MSCs-lung-Hypoxia or MSCs-CPAM-Normoxia (left column of each comparison) against MSCs-CPAM-Hypoxia (right column of each comparison). The green scale is related to downregulated genes whereas the red palette represents upregulated ones. NA value is put when the difference is not statistically relevant in the comparison. All fold changes are rounded to the second decimal digit.

Additionally, literature shows that the MSCs have a potential role in tissue repair (Parekkadan and Milwid, 2010). The gene ontology biological process term “Epithelium development” collects all the genes involved in the progression and formation of the epithelium to the mature structure (Attrill et al., 2019). It is known that MSCs can graft as lung epithelium in order to carry out structural repair of the damaged lung (Weiss and Finck, 2010), and the GO “epithelium development” has been identified as involved in the processes of wound healing and epithelial regeneration in other pathological conditions (Pokrywczynska et al., 2019). Thus, we enriched the KEGG pathways all DEGs of the two comparisons MSCs-lung and MSCs-CPAM included in this biological term. The pathways enriched in both the two comparison are “Regulation of actin cytoskeleton (hsa04810),” “Focal adhesion (hsa04510),” and “PI3K-Akt signaling pathway (hsa04151),” were common pathways observed. The reorganization of the cytoskeleton, the cellular adhesion and the PI3Ks signaling are pathways involved in the migratory activity and in the tissue regeneration processes (Figure 4). This consideration is in line with the cell cycle activation since and cell proliferation. Interestingly, the MSCs-lung seems more inclined to trigger the activation of these processes than the MSCs-CPAM.

FIGURE 4

Heatmap of genes involved in the gene ontology term “Epithelium development” and observed in the KEGG pathways “Regulation of actin cytoskeleton,” “Focal adhesion” or “PI3K-Akt signaling pathway” in the comparison of MSCs-lung-Normoxia against MSCs-lung-Hypoxia (left column of each comparison) or MSCs-CPAM-Normoxia against MSCs-CPAM-Hypoxia (right column of each comparison). The green scale is related to downregulated genes whereas the red palette represents upregulated ones. NA value is put when the difference is not statistically relevant in the comparison. All fold changes are rounded to the second decimal digit.

Protein Expression

Bcl-2, Ki-67, Cleaved caspase-3, Cleaved caspase-8, p53 and ITGA4 were investigated by Western blot analysis. The expression of Ki-67 and Cleaved caspase-3 was significantly increased in MSCs-CPAM cultured under Hypoxic conditions when compared to the MSCs-CPAM culture maintained under standard atmosphere parameters. On the other hand, Bcl-2 was down regulated in MSCs-CPAM maintained in hypoxic incubator in comparison with normoxic conditions. Cleaved caspase-8 and p53 were downregulated in MSCs-CPAM under hypoxic culture conditions when compared to the normoxic maintained culture. ITGA4 showed an increase expression in MSCs-lung and MSCs-CPAM under hypoxic conditions (Figure 5).

FIGURE 5

MSCs-lung and MSCs-CPAM protein expression under Normoxic (N) and Hypoxic (H) conditions. (A) Specific band of investigated markers Bcl-2, Ki-67, Cleaved caspase-3, Cleaved caspase-8, p53 and ITGA4. Beta-actin was used as housekeeping protein. (B) Densitometric analyses of the studied markers. *

MSCs-lung and MSCs-CPAM protein expression under Normoxic (N) and Hypoxic (H) conditions. (A) Specific band of investigated markers Bcl-2, Ki-67, Cleaved caspase-3, Cleaved caspase-8, p53 and ITGA4. Beta-actin was used as housekeeping protein. (B) Densitometric analyses of the studied markers. *