ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

Background Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. Methods The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and β-Catenin were quantified by Western blot. Results Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho β-Catenin, and BMP4. Conclusions These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

factors, SRY-related high-mobility-group, HMG, box (SOX) proteins, that bind DNA in a sequence-specific manner defined the proximal and distal arrangement during fetal life. Indeed, the fetal lung development starts from a multipotent stem cell population (SOX9 positive) that forms the most, if not all, conducting airways in a mechanism dependent on Wntless (Wnt), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) signaling ( [3][4][5][6][7][8][9][10], reviewed in [11]). In brief, FGF10 directly activates β-Catenin in the distal epithelial progenitors, and induces BMP4 and SOX9 expression, to maintain and keep cells from differentiating into the SOX2 conducting airway epithelium. As the epithelial tube grows towards the distal source of FGF10, progeny from the distal multipotent epithelial progenitor are left behind in the epithelial stalk and once they are out of FGF reach they lose SOX9, β-Catenin and BMP4, start expressing SOX2, and differentiate into conducting airway epithelium ( [3][4][5][6][7][8][9][10], reviewed in [11]). Finally, it is the separate capacity of SOX2 versus SOX9 cells to be differentiated in bronchiolar and alveolar lineages, respectively, that allows the morphological distinction between conducting and respiratory airways at the end of canalicular stage and pulmonary maturation at saccular phase in preparation for the first breath at birth [3,4,[12][13][14].
Neonatal respiratory failure is common in moderate-to-severe cases of congenital diaphragmatic hernia (CDH). CDH is defined by a diaphragmatic defect that allows the herniation of abdominal organs into the thorax and consequently lung hypoplasia, affecting 1 in 3000 live-births [15,16]. These hypoplastic lungs, strictly associated with high rates of mortality and morbidity, demonstrate a reduced number of terminal buds at early developmental stages (embryonic/pseudoglandular); reduced surface areas; and a decrease in distal branching and alveoli (saccular/alveolar) that impaired the efficient gas exchanges [17,18]. Molecularly, the study of the underlying mechanisms demonstrated stage-dependent dynamics for BMP, WNT, or FGF pathways in experimental-CDH ( [19][20][21][22][23], reviewed in [24]) with unexplored SOX2 or SOX9 profiles.
Genome-wide and transcriptome studies in CDH versus normal lungs in humans and rodents described high priority genes and pathways, such as ROBO/SLIT, as potential therapeutic target for the diaphragmatic defect repair [25,26]. Roundabout (Robo) genes encode cellsurface receptors that respond to their secreted ligands, SLIT proteins, in a wide variety of cellular processes. In the lung, ROBO/SLIT knockout is inductor of diaphragm defect, delayed separation of foregut from the body wall [27], poor lung inflation and lethality at birth [27,28]. On the other hand, ROBO/SLIT signaling is described in regulation of cell proliferation or progenitor cell profile in the development of mammary gland [29,30], a branching organ-like lung, and in the central nervous system [31]. These intriguing functions as regulator of progenitor cell profile in development of mammary gland and central nervous system and inductor of the morphological defects in fetal lung development allowed us to hypothesize the ROBO1/2 as regulators of SOX2/SOX9 balance in fetal lung development, particularly in branching morphogenesis.
In this context, considering the intricate morphological, cellular and molecular dynamics during fetal lung development that determine the complex architecture of the lung at birth, the purpose of our investigation was determine the spatiotemporal distribution for receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) from embryonic-to-saccular developmental stages in nitrofen-induced CDH rat model; and to identify the molecular effect of ROBO1 and ROBO2 inhibition in both ex vivo branching morphogenesis and SOX2/SOX9 profiles.

Animal model and experimental design
This study was carried out in strict accordance with FELASA guidelines [32] and with European (European Union Directive 86/609/EEC) regulation. The protocol was approved by the Direção Geral de Alimentação e Veterinária (DGAV 021328).
Female Sprague-Dawley rats (225 g; Charles-River; Spain) were maintained in appropriate cages under controlled conditions and fed with commercial solid food. The rats were mated and checked daily for vaginal plug. The day of plugging was defined as embryonic day (E) 0.5 for time dating purposes. According to the nitrofen-induced CDH rat model [33,34], at E9.5, randomly selected pregnant rats were exposed to 100 mg of nitrofen (2,4-dichlorophenyl-p-nitrophenylether). At different time points (E13.5, E15.5, E17.5, E19.5, and E21.5), fetuses were harvested by cesarean section. After fetal decapitation, a thoracic-laparotomy was performed under a binocular surgical microscope (Leica, Wild M651.MSD, Switzerland) to inspect the diaphragm and harvest the organs.
The fetuses were divided into two groups: a control group (control) with fetuses exposed to olive oil alone, and hypoplastic group (hypoplastic) with those exposed to nitrofen. Regarding this experimental design, the assessment of diaphragmatic defects by surgical inspection at E13.5 and E15.5 is impractical. Therefore, for early gestational age, the hypoplastic group refers to the fetuses exposed to nitrofen (independently of CDH development), while at later gestational ages (E17.5, E19.5, and E21.5), the hypoplastic group refers to fetuses exposed to nitrofen which developed CDH. Lungs were either snapfrozen in liquid nitrogen for protein extraction or fixed in 4% paraformaldehyde for immunohistochemistry. Lungs of fetuses at 13.5 days post-conception were also dissected to perform fetal lung explants cultures and posterior Western blot analysis. GPower 3.1.9.4 (Franz Faul, Universitat Kiel, Germany) was used for sample size calculation. In total, 18 dams and 165 embryonic rats were used in this study.

Immunohistochemistry
Normal and hypoplastic lungs of different gestational ages (E13.5-21.5) were fixed in 4% paraformaldehyde and embedded in paraffin as previously described [35]. 4 µm sections were placed onto glass microscope slides. Heat-induced antigen retrieval was performed with a citrate buffer. Sample sections were blocked with 4% fetal bovine serum for 1 h at room temperature and incubated with primary antibodies [ROBO1 (Cat No. sc25672) and ROBO2 (Cat No. sc16615), 1:200, overnight (ON), 4 ºC, Santa Cruz Biotechnology Inc, USA; SOX2 (Cat No. AF2018) and SOX9 (Cat No. AF3075), 1:100, ON, 4 ºC, R&D systems, USA] diluted in phosphate-buffered saline (PBS1x). Negative control reactions included omission of the primary antibody for which the immunoreactive staining was not observed. Tissue sections for negative control, ROBO1, SOX2, and SOX9 were labeled with a streptavidin-biotin immunoenzymatic antigen detection system (Cat No. TL-125-QHD, Thermo Scientific, USA) according to the manufacturer's instructions and visualized with a diaminobenzidine tetrahydrochloride solution (Cat No. TA-125-QHDX, Thermo Scientific, USA) [36]. Tissues sections for ROBO2 and respective negative control were labeled with biotinylated anti-goat IgG (Cat No. BA-9500, Vector Laboratories, UK) followed by streptavidin-horseradish peroxidase incubation and visualized with a diaminobenzidine tetrahydrochloride solution (DAB, Cat No. TA-125-QHDX, Thermo Scientific, USA). Sections with and without primary antibodies were simultaneously processed and analyzed. The time expended in DAB solution was variable according to the developmental stage, but equally matched between control and hypoplastic sections, allowing the quantification of immunohistochemical signals. The percentage of stained cells per microscopic field was scored in four independent areas per section (four sections per each experimental group) by two blinded observers. Scoring was as follows: 0, 0-1% cells/pulmonary structure; 1, 1-25% cells/pulmonary structure; 2, 25-50% cells/pulmonary structure; 3, 50-75% cells/pulmonary structure; 4, 75-100% cells/pulmonary structure in accordance with [37]. At least three independent experiments were performed for each antibody tested. In each experiment, a different set of slides comprising the whole range of gestational ages was used. Different and unrepeated animal samples were selected for each group (gestational age). Six different animals were examined for each group per studied antibody. All sections were scanned with Olympus BX61 Upright Microscope (Olympus corporation, Japan) and independently evaluated by two investigators.

Western blot analysis
Normal and hypoplastic lungs from different gestational ages (E13.5-E21.5) were processed for Western blot analysis in accordance with previously described methods [38,39]. Briefly, 15 µg of protein were loaded onto 10% acrylamide mini gels, electrophoresed at 100 V at room temperature, and then transferred to nitrocellulose membranes (HybondTM-C Extra, GE Healthcare Life Sciences, UK). Blots were blocked in 5% bovine serum albumin and probed with primary antibodies to ROBO1 according to the manufacturer's instructions. For loading control, blots were probed with β-Tubulin (1∶150.000; Cat No. ab15568, Abcam, USA). After this, membranes were incubated with a secondary horseradish peroxidase conjugate, developed with Clarity West ECL substrate (Cat No. 1705060, Bio-Rad, USA) and the chemiluminescent signal was captured using the Chemidoc XRS (Bio-Rad, USA) [36].
Quantitative analysis was performed with Quantity One 4.6.5 1-D Analysis Software (Bio-Rad, USA). Three independent experiments were performed (n = 3). In total, nine animals were used in each group (gestational age/condition) per antibody.

Fetal lung explants
Harvesting and dissection of E13.5 lungs were made in PBS under a dissection microscope (Leica MZFLIII, Switzerland). The lungs were transferred to nucleopore membranes (Cat No. TETP01300, Whatman, USA), previously presoaked in Dulbecco's modified Eagle´s medium (DMEM) low glucose (Cat. No SH30021.01, Thermo Scientific, USA) for 1 h, and incubated in a 24-well culture plate (Nunc, Denmark). Floating cultures of the explants were incubated in a complete medium [50% DMEM, 50% nutrient mixture F-12 The fetal lung explants were incubated in a 5% CO 2 incubator at 37 °C for 96 h, and the medium was replaced every 48 h. The branching morphogenesis was monitored daily by photographing the explants. At day 0 (D0: 0 h) and day 4 (D4: 96 h) of culture, the total number of peripheral airway buds (branching) of all lung explants was determined by counting the number of peripheral airway epithelial buds of the developing respiratory tree [37,40].

ROBO1 and ROBO2 inhibitory studies
Ex vivo cultures of normal lung explants were complemented daily with distinct doses of recombinant rat ROBO1 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL, R&D system; Cat No. 1749-RB-050), recombinant human ROBO2 Fc Chimera (0.04, 0.4, 4 and 40 ng/mL, R&D system; Cat No. 3147-RB-050), or recombinant human IgG1 Fc (used as control at higher dose, R&D; Cat No. 110-HG-100). These doses were selected according to the literature [41,42]. Lung explants were obtained in three independent experiments (n ≥ 9 for each dose tested). After 4 days in culture, control, ROBO1 and ROBO2 inhibited lung explants were analyzed for branching morphogenesis in terms of area, external and internal epithelial perimeter, and the number of peripheral airway buds. In addition, SOX2, SOX9, BMP4, total and non-phospho (active) β-Catenin were quantified by Western blot at day 4 in all experimental groups.

Statistical analysis
All quantitative data are presented as mean ± standard error of the mean (SEM). The statistical analysis was performed by two-way ANOVA for lung condition (normal and hypoplastic) and embryonic day (E13.5, E15.5, E17.5, E19.5, and E21.5) on protein expression level. One-way ANOVA was performed for the number of peripheral airway buds and protein expression levels on recombinant rat ROBO1 Fc Chimera (0.04, 0.4, 4, 40 ng/mL), and recombinant human ROBO2 Fc Chimera (0.04, 0.4, 4, 40 ng/mL). The parametric test assumptions were previously verified, and an additional LSD test was used for post-test analysis. Statistical analysis was performed using the statistical software IBM SPSS Statistics 24.0. Statistical significance was confirmed at p < 0.05.
To further explore these molecular profiles across hypoplastic versus normal fetal lung development, in vivo spatiotemporal distributions for ROBO1, ROBO2, SOX2, and SOX9 were analyzed by immunohistochemistry and quantified by pulmonary structure.
Concerning SOX9, the immunohistochemistry assay identified an initial SOX9 expression in epithelium at E13.5 and E15.5 and mesenchyme at E15.5 that give rise to wide staining in all pulmonary structures in normal ( Fig. 5aA-aH) and hypoplastic lungs (Fig. 5aa-ah). Indeed, SOX9 was detected in bronchi at E17.5, E19.5, and E21.5; primordia of bronchiole at E17.5; terminal bronchiole and BADJ at E19.5 and E21.5; and in alveolar duct at E21.5 in normal (Fig. 5aC-aH), and hypoplastic lungs (Fig. 5ac-ah) for which the more significant differences were observed after quantification of immunohistochemical signals (Fig. 5b). Particularly in experimental-CDH, SOX9 was downregulated in primordia of bronchiole at E17.5; terminal bronchiole at E19.5 and E21.5; BADJ at E19.5; and mesenchyme at E17.5 and E19.5 (Fig. 5b).  These new findings urged us to explore the function of ROBO1 and ROBO2 in fetal lung development, particularly in branching morphogenesis and in SOX2/ SOX9 expression profiles. For that, lung explant cultures at E13.5, previous reported as comparable to the in vivo lung both in structure and function, were selected as a model, in which restricted inhibition of ROBO1 or ROBO2 was performed followed by branching morphogenesis analysis and SOX2, SOX9, total and non-phospho (active) β-Catenin and BMP4 quantification at day 4.

Discussion
Despite the various distinct models used for the study of CDH, the nitrofen-induced CDH rat model is the most extensively used, particularly for the study of cellular and molecular mechanisms at early developmental stages since it produces diaphragmatic hernias and abnormal lungs. In fact, the nitrofen administration to pregnant dams midgestation causes developmental anomalies that reasonably replicate the major abnormalities and the pathophysiology described in human CDH, namely the specific location and extent of the diaphragmatic defects, lung hypoplasia, pulmonary hypertension, cardiovascular and skeletal defects (reviewed in [24], reviewed in [43]). On the other hand, the surgical model is employed to test promising antenatal therapies since it better mimics the later human developmental stage, alveolarization, and the cardiopulmonary transition at birth (reviewed in [24], reviewed in [43]). Our research aimed to determine the cellular/molecular alterations in CDH context at early developmental stages, embryonic-to-saccular, and thus the nitrofen-induced CDH rat model was the selected strategy.
Our findings demonstrated changes in spatiotemporal distribution and relative expression levels for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) in experimental-CDH versus normal lungs, according to pulmonary structure and developmental stage. In brief, the overexpression of ROBO2 and SOX2 in epithelium and the new ROBO2+ cells in mesenchyme defined the undifferentiated tissues in CDH. In contrast, at the time the first pulmonary structures are formed, distinct molecular profiles for ROBO1, ROBO2, SOX2 and SOX9 were visualized in epithelial cells (Fig. 7a), evidencing distinct capacities to react to molecular and environment signals in a hypoplastic lung.
Morphologic defects, such as decreased number of terminal buds at early and reduced air space and thicker septa at later developmental stages, characterize a CDH (See figure on next page.) Fig. 6 Effect of ROBO1 or ROBO2 functional impairment in branching morphogenesis. a The upper panel is representative of untreated lung explants (0 ng/mL) at D0; the bottom panel represents lung explants treated with recombinant IgG protein (0 ng/mL) and several doses of recombinant ROBO1 or ROBO2 proteins at day 4 (D4). b Morphometric analysis of the number of peripheral airway buds of fetal rat lung explants treated with increasing concentrations of recombinant ROBO1 (black) and ROBO2 (gray) proteins. Results are expressed as the D4/D0 ratio. c Examples of representative blots are showed for each analysed protein. Protein expression levels of d ROBO; e SOX2; f SOX9; g total β-Catenin; h non-phospho (active) β-Catenin; and i BMP4 in normal explant cultures treated with recombinant rat ROBO1 Fc Chimera for ROBO1 inhibition (black line) or recombinant human ROBO2 Fc Chimera for ROBO2 functional impairment (gray line). Recombinant human IgG Fc was used as control (IgG). n ≥ 9 per protein/condition. Each lane represents a pooled-tissue sample, and relative expression was determined against β-Tubulin and IgG. The data are presented as means ± SEM. p < 0.05: α vs. IgG lung in both human infant and animal models. Underlying these morphological anomalies, our in vivo data demonstrated the impairment of proximodistal patterning from pseudoglandular-to-saccular stages (Fig. 7a), contributing to a better understanding of CDH pathogenesis. Indeed, the previous studies showed the proximodistal patterning formed at pseudoglandular stage building the functional units, i.e. the conducting airways, and respiratory airways at canalicular and saccular phases in preparation for the first breath at birth [3,4,[12][13][14]. In addition, severe defects in branching morphogenesis and epithelial cell differentiation were observed after SOX2 [4,13] or SOX9 [3] knockout. Our results also exposed distinct expression profiles suggesting three independent zones (bronchi and terminal bronchiole; BADJ; and alveolar duct) in a hypoplastic lung, able to distinctly react to molecular or environmental signals at E21.5. This study is the first to present SOX2/SOX9 analysis by pulmonary structure. In the adjacent literature, the molecular regulators of SOX2/SOX9 profiles (WNT, FGF, or BMP) have been confusedly reported in a separate studies with undefined conclusions ( [19][20][21][22][23], reviewed in [24]), because of the limited information that can be taken from the protein quantification in the whole lung. Indeed, when we are looking for formation of the conducting and respiratory airways defined by proximodistal patterns, molecular and cellular analysis by pulmonary structure could be relevant, particularly in pathologic scenarios, since distinct mechanisms can be at work. Therefore, our investigation provides novel observations concerning the impairment of SOX2 versus SOX9 profiles and the morphological defects in experimental-CDH. In this context, research revealing the mechanisms underlying SOX2/SOX9 expression in fetal lung development is of paramount importance since it allows the manipulation of branching morphogenesis and later the formation of conducting and respiratory airways. Using ex vivo lung explants cultures, we detected the ROBO2 inhibition as stimulator of fetal lung growth through activation of β-Catenin and BMP4 that promote SOX9 instead of SOX2 profiles. In contrast, the decrease of BMP4 and SOX2 increase visible after ROBO1 inhibition determines unaffected branching morphogenesis (Fig. 7b). Importantly, lung explant cultures are described as a useful method in the study of branching morphogenesis since they maintain the native physiological interaction between cells in the developing lung. Simultaneously, molecular analysis of the proximal and distal network in terms of SOX2, SOX9, BMP4 and active β-Catenin (target of Wnt pathway) (reviewed in Fig. 7 a Overview of the main changes in spatiotemporal distribution of ROBO1, ROBO2, SOX2 and SOX9 at pseudoglandular (E17.5) and saccular stages (E21.5) in hypoplastic (hyp) fetal lungs (lower panel). w/o without b A proposed model of ROBO regulation of ex vivo branching morphogenesis through BMP4, β-Catenin, SOX2 and SOX9 [11]) allowed us to validate our ex vivo results regarding the molecular effects of ROBO2 inhibition in fetal lung growth and SOX9/SOX2 balance, since the proximodistal patterning is dependent on BMP4 and β-Catenin working in gradient in the distal tip of the lung (Fig. 7a).
Our in vivo observations also showed the overexpression of ROBO2 in lung hypoplasia, whereas the significant decrease of ROBO2 promotes the ex vivo branching morphogenesis, indicating a concordant role for ROBO2 in in vivo and ex vivo branching morphogenesis, respectively. In addition, there is similar inhibition of ROBO1 and overexpression of ROBO2 and SOX9 in bronchi and primordia of bronchiole with loss of SOX2+ cells only in primordia of bronchiole in experimental-CDH (Fig. 7a). This contrasts with the consistent opposite effect on SOX2 and SOX9 expression triggered by ROBO2 inhibition in ex vivo branching morphogenesis (Fig. 7b), providing evidence of the complexity of the in vivo model. Finally, genetic studies showing the impairment of SOX2 [44] and ROBO/SLIT signaling [25,26] in human patients with CDH further validate the investigation of these targets in fetal lung development and particularly in CDH.
The literature has demonstrated a close relationship between epithelial and vascular development during fetal lung morphogenesis. Indeed, the downregulation of BMP, Wnt or transforming growth factor beta (TGFβ) signaling was previously associated not only with lung hypoplasia but also with pulmonary vascular remodeling, responsible for the development of persistent pulmonary hypertension at birth in a nitrofen-induced CDH rat model (reviewed in [21,24,[45][46][47][48]). These observations are particularly relevant for in vivo studies since the development of the fetal lung and cardiovascular system cannot be separated when they divide the same molecular regulators, like BMP4. Our results showed that ROBO2 inhibition increases BMP4 expression and also branching morphogenesis. Thus, it is acceptable to suppose a simultaneous molecular effect of ROBO2 inhibition on epithelial and vascular branching, and consequent reversion of pulmonary hypoplasia and pulmonary hypertension in an experimental CDH context. In addition, the literature showed another SOX family member, SOX7, deleted in human infants with CDH [44] with functions in diaphragm formation [49], cardiovascular development [50] and in regulation of lineage decisions in cardiovascular progenitor cells [51] These functions are also identified for ROBO/SLIT signaling in fetal lung development, suggesting a potential association between SOX7 and ROBO/SLIT pathways.
Despite all the new findings reported here, we cannot ignore the limitations of our study regarding the in vivo applicability of this target and the challenge of treating a fetus patient, where several dysfunction pathways can be simultaneously targeted with one single treatment. Indeed, the careful analysis of our results suggests a limited applicability of ROBO2 in CDH. Although our observations report an upstream target that forms SOX9 instead of SOX2 progenitor cells at pseudoglandular stage, the literature showed distal and proximal progenitors giving rise to alveolar and bronchiolar lineages, respectively, in the next developmental stages in a mechanism dependent on both molecular and environment signals. In this context, the differentiated profile of the epithelial progenitor cells become unpredictable in a pathologic scenario, like CDH. Indeed, previous studies reported dysregulated factors involved in differentiation of airway epithelium in a nitrofen-induced CDH rat model (reviewed in [49]). Now, we describe cellular problems regarding the major coordinators of bronchiolar and alveolar differentiation, reinforcing the unanswered question: is the CDH pathogenesis a problem of differentiated or undifferentiated tissues?

Conclusion
Our data provide the first evidence that receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) are affected in experimental-CDH from embryonic-to-saccular stages and identified the ROBO2 inhibition as stimulator of branching morphogenesis and regulator of SOX2/SOX9 profiles through β-Catenin and BMP4 that could play important roles not only in stimulating in vivo lung growth, but also proximodistal patterning. Although ROBO2 inhibitors could be a promising therapeutic strategy for treatment of pulmonary hypoplasia related to CDH, more studies are necessary to carefully evaluate the differentiated capacity of these epithelial progenitor cells in the in vivo CDH context and the potential improvement in both lung morphological defects and neonatal respiratory function.