Perinatal origins of bronchopulmonary dysplasia—deciphering normal and impaired lung development cell by cell

Understanding the lung composition on single-cell level has been a focus of substantial scientific research for more than a decade. During this period, more than 300 single-cell RNA sequencing (scRNA-seq) or single-nuclei RNA sequencing (snRNA-seq) human or animal lung datasets have been published. However, only a small fraction of these is dedicated to late lung development, and even fewer to the pathogenesis of BPD.

First exploratory scRNA-seq analysis of postnatal developing lung in mice were performed by Cohen et al., constructing a detailed single-cell map of the developing lung and lung progenitor cell populations covering the period from embryonic day (E)12.5 to postnatal day (P)7 [222]. The study identified 10 non-immune and 12 immune cell populations, revealing dynamic changes in population sizes, particularly during the pseudoglandular (E12.5) and canalicular stage (E18/19) of lung development. This data were further expanded in another study, characterizing lung immune (CD45⁺) cells between E18.5 and P21 [223]. When comparing the prenatal and postnatal immune cells authors identified a gradual increase in macrophage heterogeneity, as well as rapid increase in the proportion of lymphoid populations (2% vs 60% of all immune cells, respectively). Additional studies have focused on the developmental changes and postnatal adaptation in lung epithelial, endothelial, and stromal populations [224‐227].

Several scRNA-seq studies have contributed to establishing a cell atlas of human fetal lung development [228‐231]. No study to date has analysed lungs of BPD patients, although the scRNA-seq analysis of TA-derived cells to establish novel biomarkers and aid in stratifying BPD into endotypes has recently been proposed [232]. In contrast to the lack of studies in humans, few studies have explored the BPD pathogenesis at the single-cell level in the neonatal mouse hyperoxia model [151, 183, 226]. As mentioned above, the expression levels of angiogenic markers FOXF1 and c-KIT are decreased in the lungs of BPD patients [183]. This was confirmed by scRNA-seq in adult mouse lungs where hyperoxia exposure for first 7 days of life decreased the number of c-Kit⁺ endothelial cells (ECs) progenitors. Importantly, authors showed that adoptive transfer of c-Kit⁺ ECs improved lung angiogenesis and alveolarization in developing hyperoxia-exposed mice [183]. Substantial expression changes in all cell compartments were observed in the largest to date study of hyperoxia-exposed developing mice by Hurskainen et al., profiling over 66.000 lung cells at P3, P7, and P14 [151]. In this study, hyperoxia caused gradual changes in cell composition and expression patterns, particularly after 7 days of exposure. Within the stroma, authors identified transcriptomic shifts in myofibroblasts, pericytes, and Col13a1⁺ fibroblast, which were also among the most active signal senders and receivers in the hyperoxic lung. The study further revealed a substantial depletion of gCap (general capillary) cells and an increase in number of Car4⁺ aCap cells (aerocytes) after hyperoxia exposure [151]. The gCap cells were previously identified as putative distal lung vascular progenitors and regulators of capillary homeostasis, vasomotor tone, and repair [233]. The depletion of gCap ECs may contribute not only to the developmental injury, but also to the lack of repair capacity and an increased susceptibility to lung injury later in life, implying potential benefits of EC-derived cell therapies in BPD [151, 234]. In parallel, the Car4⁺ aCap cells showed pathological gene expression characterized by pro-inflammatory and anti-angiogenic markers. This is in agreement with recent reports, that aCap cells might contribute to septation [227] and revascularization following injury [235]. Moreover, the study highlighted the importance of inflammation in hyperoxia injury, with majority of the impacted transcriptional programs related to inflammatory response [151]. Finally, a recent study explored the long-term implication of neonatal hyperoxia [236]. Authors mapped lung cell populations in developing (P7) and adult (P60) mice previously exposed to hyperoxia for the first 3 days of life and identified persistent changes to AT2 subpopulations, predicting lasting perturbations to lung architecture and function [236].

While scRNA-seq studies so far have provided us with a somewhat complete map of the postnatally developing mouse lung, presently only a small portion of data derived from these studies have contributed to our understanding of BPD pathogenesis. As such, parallel studies in humans are still needed and further studies of BPD lungs are necessary to improve our interpretation of data obtained from animal studies. Finally, additional techniques, such as lineage tracing and spatial transcriptomics should be used to complement scRNA-seq to further investigate the role of newly identified cell populations, particularly rare cell subtypes and potential putative progenitors.

Lung constitutes a quiescent organ, with the turnover time gradually decreasing along the proximal–distal axis [237, 238]. Following injury, lung cells are typically activated by their microenvironment and directed to participate in remodelling or repair [237, 239, 240]. The same injurious stimuli can damage or inhibit stem cells' ability to differentiate, leading to decreased, incorrect, or inappropriately timed production of particular cell populations, contributing to the development of lung disease [241]. The role of stem cells in the dysplastic pulmonary growth, premature lung aging, and pathogenesis of BPD has previously been proposed [241]. However, while multiple populations of lung endothelial, epithelial, and stromal stem or progenitor cells have been described, relatively little is known about their role in late lung development or BPD [237]. Particularly of interest are questions why resident stem cells lose their function and whether it is more feasible to restore their progenitor potential, or rather supplement the injured lung with undamaged, exogenous, therapeutic stem cells.

Epithelial stem cells are the most studied putative progenitors in the lung [242‐244]. These include proximal airways basal cells, secretory cells, bronchial alveolar stem cells (BASC), and distal AT2 cells. The fate of the progenitor basal cells, characterized by the expression of luminal cytokeratin KRT8, seems to be largely guided by the NOTCH signaling. Low levels of NOTCH expression predispose basal cells toward the secretory phenotype, while high levels lead to differentiation into goblet cells, and the absence of NOTCH results in the ciliated phenotype [245‐249]. Although the progenitor capacity of BASC cells has been demonstrated in mice, their exact role in postnatal lung growth and even their existence in human lungs still remain controversial [237, 244, 250]. On the other hand, the role of distal AT2 cells in lung repair is well-established and has been broadly studied. Lineage-tracing and scRNA-seq studies shown, that (alveolar type) AT1 and AT2 cells originate from a common bipotent progenitor. In humans, the AT1/AT2 progenitors were reported in developing lungs at gestational week 15 [230]. Studies in mice suggest that the bipotent AT1/AT2 population splits into independent cell lines by E18.5 [251, 252]. AT2 cells were repeatedly shown to self-renew, differentiate into AT1 cells, and exhibit repair capacities even in matured lungs [253‐256]. In regard to BPD pathogenesis, increased compensatory AT2-to-AT1 trans-differentiation was shown in the developing hyperoxia-exposed rats [257]. Early postnatal hyperoxia-exposure in mice resulted in reduced AT2 proliferation which persisted for up to 2 months [167]. Contradicting observations were however made in premature ventilated baboons, where AT2 hyperproliferation was observed [164], perhaps indicating that the nature, intensity, and timing of the injurious stimulus are critical in determining the way progenitor populations respond. Indeed, the molecular mechanisms involved in the AT2 progenitor capacity are largely unknown. Among the proposed pathways are the WTN, EGFR, and KRAS signaling pathways [256, 258]. Further, reports of progenitor-like AT1 cells [259] and the AT1-to-AT2 trans-differentiation also exist [260], and a specific Hopx⁺ AT1 population was shown to generate new AT2 cells in an adult mice post-pneumonectomy [261]. Finally, the progenitor role of so-called respiratory airway secretory cells (RAS) was also recently revealed [262]. RAS, which are located in human, but not mice proximal airways, differentiate exclusively into AT2 cells, a process regulated by NOTCH and WNT signalling. While this study explored the potential role of RAS in adult lung disease, future studies are needed to reveal their role in the neonatal lung.

In comparison to epithelial cells, less is known in regard to lung resident endothelial and mesenchymal stem cells. The rational for the search for endothelial progenitor cells (EPCs) is based in the hypothesis that the lung development is driven by pulmonary vessel formation [56, 263]. Numerous studies support the existence of resident EPCs in the postnatally developing lungs and a defective lung vascularization can be found in both, BPD patients and animal models of BPD [170, 172, 176, 177]. Moreover, inhibition of vessel formation in developing animals stunts lung development and results in alveolar hypoplasia [172, 264‐266]. Importantly, pro-angiogenic interventions proved effective in improving lung alveolarization in animal BPD models [172, 267‐269]. Reduction in number of resident and circulating EPCs was observed in murine BPD model [174], and the hyperoxia exposure decreased proliferation in human fetal lung endothelial colony-forming cells (ECFCs) in vitro [234]. Importantly, intravenously administered human cord blood-derived ECFCs were effective in restoring lung function, alveolar and vascular growth, and colony-formic capacity of resident ECFCs in hyperoxia-exposed developing mice [234]. Finally, some efforts have recently been made to identify markers of resident lung EPCs. Among the promising proposed candidates are above-mentioned markers FOXF1 and c-KIT, which expression is decreased in the lungs of hyperoxia-exposed rodents and BPD patients alike [151, 183].

The best described and most attractive among the somatic stem cells are mesenchymal stromal cells (MSCs), which can be easily isolated from bone marrow (BM-MSCs) or umbilical cord (UC-MSCs). Several studies have demonstrated the therapeutic properties of exogenous MSCs in experimental BPD, where UC- and BM-MSCs restored lung architecture and function, and attenuated inflammation and PH in developing rodents [270‐274]. This evidence prompted further interest in MSC-based cell therapies for BPD and selected approaches are currently in early phase clinical trials [275‐278]. Besides the cell-based therapies, MSC research further encompasses the study of cell-derived products, mainly extracellular vesicles (EVs). EVs represent a heterogenous population with smaller EVs (30–100 nm) also being referred to as exosomes [279]. Multiple studies have shown their role in cell communication during both, organ homeostasis and disease [280]. Human UC-MSC-derived EVs were shown to improve alveolar and vascular development, as well as lung function and RVH in hyperoxia-exposed developing mice [281‐283]. Similar results were further observed in studies employing EVs from amniotic fluid-derived [284] and Wharton’s Jelly-derived MSCs [285]. Studies indicate that EVs exert their mostly anti-inflammatory effects by promoting an immunosuppressive CCR2-associated myeloid cell phenotype [283]. Similarly, antenatal delivery of BM-MSCs-derived EVs benefited rats with endotoxin-induced chorioamnionitis, resulting in reduced cytokine levels and improved lung growth and mechanics [286]. Finally, UC-MSCs-derived EVs protected lung architecture, vessel formation and inflammatory modulation in LPS-injected and mechanically ventilated developing mice [287].

In addition to exogenous MSCs, the notion of the resident lung MSC (L-MSC) population come from reports of MSCs in TAs from prematurely born infants, where their presence was identified as indicator of BPD morbidity and severity [288‐290]. Resident L-MSCs were also described in human fetal lungs (gestational week 15–17) [291] and hyperoxia-exposed developing rodents [292, 293]. Hyperoxia exposure increased the number of L-MSCs and triggered expression of pro-inflammatory, pro-fibrotic, and anti-angiogenic genes [151, 293]. A scRNA-seq cell communication analysis revealed inflammatory signals from immune populations as main drivers of hyperoxia-induced changes in L-MSCs [293]. Importantly, hyperoxia-exposed human fetal L-MSCs exhibited decreased colony-forming capacity [291], while L-MSCs isolated from hyperoxia-exposed animals had decreased ability to support angiogenesis [292]. Although minimal criteria for MSCs characterization have been officially established [294], the definition remains rather crude and the identification of organ-specific MSCs, including the L-MSCs, lacks standardization. As a result, no L-MSC-specific marker has been accepted to date, although few markers have been proposed [292, 293, 295‐297]. Notable among these is LY6A, also known as SCA-1 (stem cell antigen 1) [295, 297]. Recent studies have shown that L-MSCs may constitute a rather heterogenous population and their study might require more advanced methods, such as scRNA-seq or sc-proteomics [292, 293]. Another recently emerging candidate resident MSC population are the Gli-1⁺ repair-supportive mesenchymal cells [298, 299]. Progenitor properties of Gli-1⁺ cells were previously described in other organs, including bones [300, 301], teeth [300], and liver [302]. In the lung, Gli-1⁺ cells co-express Acta2, Fgf10 and Pdgfra, thus resembling alveolar fibroblasts [225, 298]. In mice Gli-1⁺ MSCs were shown to aid epithelial regeneration following naphthalene-induced airway injury [298], and were shown to be increased in bleomycin-induced lung fibrosis in mice [303]. A more detailed characterization of all types of lung resident stem cell populations will clearly be of essence in understanding their role in normal and impaired lung development and regeneration.