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Rationale: The chronic lung disease bronchopulmonary dysplasia (BPD) remains the most common complication of extreme prematurity (<28 weeks of gestation). Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) represent an opportunity for autologous cell-therapy, as UC-MSCs have been shown to improve lung function and structure in experimental BPD. However, characterization and repair capacity of UC-MSCs derived from donors with pregnancy-related complications associated with prematurity remain unexplored., Objectives: To characterize UC-MSCs' transcriptome and determine if pregnancy-related complications (preeclampsia and chorioamnionitis) alter their therapeutic potential., Methods: Single-cell RNA sequencing (scRNA-seq) was used to compare the transcriptome of UC-MSCs derived from five term donors, 16 preterm donors, and human neonatal dermal fibroblasts (HNDFs, control cells of mesenchymal origin), and correlated with their therapeutic potential in experimental BPD. Using publicly available neonatal lung single-nuclei RNA sequencing data, we also determined putative communication networks between UC-MSCs and resident lung cell populations., Measurements and Main Results: Most UC-MSCs displayed a similar transcriptome despite of their pregnancy-related conditions and mitigated hyperoxia-induced lung injury in newborn rats. Conversely, HNDFs, one term and two preeclampsia preterm UC-MSC donors exhibited a distinct transcriptome enriched in genes related to fibroblast function and senescence and were devoid of therapeutic benefit in hyperoxia-induced BPD. Conversely, therapeutic UC-MSCs displayed a unique transcriptome active in cell proliferation and distinct cell-cell interactions with neonatal lung cell populations, including NEGR and NRNX pathways., Conclusion: Term and preterm UC-MSCs are lung protective in experimental BPD. scRNA-seq allows to identify donors with a distinct UC-MSC transcriptome characteristic of reduced therapeutic potential.

Rationale: The chronic lung disease bronchopulmonary dysplasia (BPD) is the most severe complication of extreme prematurity. BPD results in impaired lung alveolar and vascular development and long-term respiratory morbidity, for which only supportive therapies exist. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) improve lung structure and function in experimental BPD. Results of clinical trials with MSCs for many disorders do not yet match the promising preclinical studies. A lack of specific criteria to define functionally distinct MSCs persists. Objectives: To determine and correlate single-cell UC-MSC transcriptomic profiles with therapeutic potential. Methods: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs; control cells of mesenchymal origin) transcriptomes were investigated using single-cell RNA sequencing (scRNA-seq) analysis. The lung-protective effect of UC-MSCs with a distinct transcriptome and control HNDFs was tested in vivo in hyperoxia-induced neonatal lung injury in rats. Measurements and Main Results: UC-MSCs showed limited transcriptomic heterogeneity but were different from HNDFs. Gene Ontology enrichment analysis revealed distinct (progenitor-like and fibroblast-like) UC-MSC subpopulations. Only treatment with progenitor-like UC-MSCs improved lung function and structure and attenuated pulmonary hypertension in hyperoxia-exposed rat pups. Moreover, scRNA-seq identified major histocompatibility complex class I as a molecular marker of nontherapeutic cells and associated with decreased lung retention. Conclusions: UC-MSCs with a progenitor-like transcriptome, but not with a fibroblast-like transcriptome, provide lung protection in experimental BPD. High expression of major histocompatibility complex class I is associated with reduced therapeutic benefit. scRNA-seq may be useful to identify subsets of MSCs with superior repair capacity for clinical application.

Rationale: Bronchopulmonary dysplasia, a chronic respiratory condition originating from preterm birth, is associated with abnormal neurodevelopment. Currently, there is an absence of effective therapies for bronchopulmonary dysplasia and its associated brain injury. In preclinical trials, mesenchymal stromal cell therapies demonstrate promise as a therapeutic alternative for bronchopulmonary dysplasia. Objectives: To investigate whether a multifactorial neonatal mouse model of lung injury perturbs neural progenitor cell function and to assess the ability of human umbilical cord-derived mesenchymal stromal cell extracellular vesicles to mitigate pulmonary and neurologic injury. Methods: Mice at Postnatal Day 7 or 8 were injected intraperitoneally with LPS and ventilated with 40% oxygen at Postnatal Day 9 or 10 for 8 hours. Treated animals received umbilical cord-mesenchymal stromal cell-derived extracellular vesicles intratracheally preceding ventilation. Lung morphology, vascularity, and inflammation were quantified. Neural progenitor cells were isolated from the subventricular zone and hippocampus and assessed for self-renewal, in vitro differentiation ability, and transcriptional profiles. Measurements and Main Results: The multifactorial lung injury model produced alveolar and vascular rarefaction mimicking bronchopulmonary dysplasia. Neural progenitor cells from lung injury mice showed reduced neurosphere and oligodendrocyte formation, as well as inflammatory transcriptional signatures. Mice treated with mesenchymal stromal cell extracellular vesicles showed significant improvement in lung architecture, vessel formation, and inflammatory modulation. In addition, we observed significantly increased in vitro neurosphere formation and altered neural progenitor cell transcriptional signatures. Conclusions: Our multifactorial lung injury model impairs neural progenitor cell function. Observed pulmonary and neurologic alterations are mitigated by intratracheal treatment with mesenchymal stromal cell-derived extracellular vesicles.

Although the lung has extensive regenerative capacity, some diseases affecting the distal lung result in irreversible loss of pulmonary alveoli. Hitherto, treatments are supportive and do not specifically target tissue repair. Regenerative medicine offers prospects to promote lung repair and regeneration. The neonatal lung may be particularly receptive, because of its growth potential, compared to the adult lung. Based on our current understanding of neonatal lung injury, the ideal therapeutic approach includes mitigation of inflammation and fibrosis, and induction of regenerative signals. Cell-based therapies have shown potential to prevent and reverse impaired lung development. Their mechanisms of action suggest effects on both, mitigating the pathophysiological processes and promoting lung growth. Here, we review our current understanding of normal and impaired alveolarization, provide some rationale for the use of cell-based therapies and summarize current evidence for the therapeutic potential of cell-based therapies for pulmonary regeneration in preterm infants., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

During late lung development, alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia with validation of some of the findings in lungs from BPD patients. We observe dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, stromal fibroblasts, capillary endothelium and macrophage populations. Pathway analysis and predicted dynamic cellular crosstalk suggest inflammatory signaling as the main driver of hyperoxia-induced changes. Our data provides a single-cell view of cellular changes associated with late lung development in health and disease.

Background: Bronchopulmonary dysplasia (BPD), the most common complication of prematurity, arises from various factors that compromise lung development, including oxygen and inflammation. Hyperoxia has been used to mimic the disease in newborn rodents. The use of a second hit to induce systemic inflammation has been suggested as an added strategy to better mimic the inflammatory aspect of BPD. Here we report a novel 2 hit (2HIT) BPD model with in-depth characterization of the innate immune response, enabling mechanistic studies of therapies with an immunomodulatory component., Methods: C57BL/6N mice were exposed to 85% O 2 from postnatal day (P)1 to P7, and received postnatally (P3) Escherichia coli LPS. At various timepoints, immune activation in the lung and at the systemic level was analyzed by fluorescence-activated cell sorting (FACS), and gene and protein expressions., Results: 2HIT mice showed fewer alveoli, increased lung compliance, and right ventricular hypertrophy. A transient proinflammatory cytokine response was observed locally and systemically. Type 2 anti-inflammatory cytokine expression was decreased in the lung together with the number of mature alveolar macrophages. Simultaneously, a Siglec-F intermediate macrophage population emerged., Conclusion: This study provides long-term analysis of the 2HIT model, suggesting impairment of type 2 cytokine environment and altered alveolar macrophage profile in the lung., Impact: We have developed a novel 2HIT mouse BPD model with postnatal LPS and hyperoxia exposure, which enables mechanistic studies of potential therapeutic strategies with an immunomodulatory component. This is the first report of in-depth characterization of the lung injury and recovery describing the evolution of the innate immune response in a standardized mouse model for experimental BPD with postnatal LPS and hyperoxia exposure. The 2HIT model has the potential to help understand the link between inflammation and impaired lung development, and will enable testing of new therapies in a short and more robust manner.

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Sepsis is the main cause of morbidity and mortality in neonates. Mesenchymal stromal cells (MSCs) are potent immune-modulatory cells. Their effect in neonatal sepsis has never been explored. We hypothesized that human umbilical cord-derived MSCs (hUC-MSCs) improve survival in experimental neonatal sepsis. Sepsis was induced in 3-day-old rats by intravenous injection of Escherichia coli (5 × 10 5 /rat). One hour after infection, rats were treated intravenously with normal saline, hUC-MSCs, or with interferon-γ preconditioned hUC-MSCs (10 7 cells/kg). Eighteen hours after infection, survival, bacterial counts, lung neutrophil and macrophage influx, phagocytosis and apoptosis of splenocytes plasma, and LL-37 concentration were evaluated. Animals were observed for survival for 72 h after E. coli injection. Treatment with either hUC-MSCs or preconditioned hUC-MSCs significantly increased survival (hUC-MSCs, 81%; preconditioned hUC-MSCs, 89%; saline, 51%; P < 0.05). Both hUC-MSCs and preconditioned hUC-MSCs enhanced bacterial clearance. Lung neutrophil influx was decreased with preconditioned hUC-MSCs. The number of activated macrophages (CD206 + ) in the spleen was increased with hUC-MSCs and preconditioned hUC-MSCs; preconditioned hUC-MSCs increased the phagocytic activity of CD206 + macrophages. hUC-MSCs and preconditioned hUC-MSCs decreased splenocyte apoptosis in E. coli infected rats. Finally, LL-37 plasma levels were elevated in neonatal rats treated with hUC-MSCs or preconditioned hUC-MSCs. hUC-MSCs enhance survival and bacterial clearance in experimental neonatal sepsis. hUC-MSCs may be an effective adjunct therapy to reduce neonatal sepsis-related morbidity and mortality.

ShcA is an important mediator of ErbB2- and transforming growth factor β (TGF-β)-induced breast cancer cell migration, invasion, and metastasis. We show that in the context of reduced ShcA levels, the bone morphogenetic protein (BMP) antagonist chordin-like 1 (Chrdl1) is upregulated in numerous breast cancer cells following TGF-β stimulation. BMPs have emerged as important modulators of breast cancer aggressiveness, and we have investigated the ability of Chrdl1 to block BMP-induced increases in breast cancer cell migration and invasion. Breast cancer-derived conditioned medium containing elevated concentrations of endogenous Chrdl1, as well as medium containing recombinant Chrdl1, suppresses BMP4-induced signaling in multiple breast cancer cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)