Single-Cell RNA Sequencing Reveals Repair Features of Human Umbilical Cord Mesenchymal Stromal Cells.

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 profile with therapeutic potential. METHODS: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs, control cells of mesenchymal origin) transcriptomes were investigated by single-cell RNA sequencing analysis (scRNA-seq). 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 the 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 non-therapeutic 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.

A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency.

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 1011 vg donor template and 1 × 1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 1012 vg SP-B-donor template and 5 × 1011 vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

Neonatal hyperoxia in mice triggers long-term cognitive deficits via impairments in cerebrovascular function and neurogenesis.

Preterm birth is the leading cause of death in children under 5 years of age. Premature infants who receive life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD), a chronic lung disease. Infants with BPD are at a high risk of abnormal neurodevelopment, including motor and cognitive difficulties. While neural progenitor cells (NPCs) are crucial for proper brain development, it is unclear whether they play a role in BPD-associated neurodevelopmental deficits. Here, we show that hyperoxia-induced experimental BPD in newborn mice led to lifelong impairments in cerebrovascular structure and function as well as impairments in NPC self-renewal and neurogenesis. A neurosphere assay utilizing nonhuman primate preterm baboon NPCs confirmed impairment in NPC function. Moreover, gene expression profiling revealed that genes involved in cell proliferation, angiogenesis, vascular autoregulation, neuronal formation, and neurotransmission were dysregulated following neonatal hyperoxia. These impairments were associated with motor and cognitive decline in aging hyperoxia-exposed mice, reminiscent of deficits observed in patients with BPD. Together, our findings establish a relationship between BPD and abnormal neurodevelopmental outcomes and identify molecular and cellular players of neonatal brain injury that persist throughout adulthood that may be targeted for early intervention to aid this vulnerable patient population.

Single-Cell RNA Sequencing-Based Characterization of Resident Lung Mesenchymal Stromal Cells in Bronchopulmonary Dysplasia.

Late lung development is a period of alveolar and microvascular formation, which is pivotal in ensuring sufficient and effective gas exchange. Defects in late lung development manifest in premature infants as a chronic lung disease named bronchopulmonary dysplasia (BPD). Numerous studies demonstrated the therapeutic properties of exogenous bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) in experimental BPD. However, very little is known regarding the regenerative capacity of resident lung MSCs (L-MSCs) during normal development and in BPD. In this study we aimed to characterize the L-MSC population in homeostasis and upon injury. We used single-cell RNA sequencing (scRNA-seq) to profile in situ Ly6a+ L-MSCs in the lungs of normal and O2-exposed neonatal mice (a well-established model to mimic BPD) at 3 developmental timepoints (postnatal days 3, 7, and 14). Hyperoxia exposure increased the number and altered the expression profile of L-MSCs, particularly by increasing the expression of multiple pro-inflammatory, pro-fibrotic, and anti-angiogenic genes. In order to identify potential changes induced in the L-MSCs transcriptome by storage and culture, we profiled 15 000 Ly6a+ L-MSCs after in vitro culture. We observed great differences in expression profiles of in situ and cultured L-MSCs, particularly those derived from healthy lungs. Additionally, we have identified the location of Ly6a+/Col14a1+ L-MSCs in the developing lung and propose Serpinf1 as a novel, culture-stable marker of L-MSCs. Finally, cell communication analysis suggests inflammatory signals from immune and endothelial cells as main drivers of hyperoxia-induced changes in L-MSCs transcriptome.

Pulmonary and Neurologic Effects of Mesenchymal Stromal Cell Extracellular Vesicles in a Multifactorial Lung Injury Model.

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.

Fully automated estimation of the mean linear intercept in histopathology images of mouse lung tissue.

Purpose: The mean linear intercept (MLI) score is a common metric for quantification of injury in lung histopathology images. The automated estimation of the MLI score is a challenging task because it requires accurate segmentation of different biological components of the lung tissue. Therefore, the most widely used approaches for MLI quantification are based on manual/semi-automated assessment of lung histopathology images, which can be expensive and time-consuming. We describe a fully automated pipeline for MLI estimation, which is capable of producing results comparable to human raters. Approach: We use a convolutional neural network based on U-Net architecture to segment the diagnostically relevant tissue segments in the whole slide images (WSI) of the mouse lung tissue. The proposed method extracts multiple field-of-view (FOV) images from the tissue segments and screen the FOV images, rejecting images based on presence of certain biological structures (i.e., blood vessels and bronchi). We used color slicing and region growing for segmentation of different biological structures in each FOV image. Results: The proposed method was tested on ten WSIs from mice and compared against the scores provided by three human raters. In segmenting the relevant tissue segments, our method obtained a mean accuracy, Dice coefficient, and Hausdorff distance of 98.34%, 98.22%, and 109.68 µ m , respectively. Our proposed method yields a mean precision, recall, and F 1 -score of 93.37%, 83.47%, and 87.87%, respectively, in screening of FOV images. There was substantial agreement found between the proposed method and the manual scores (Fleiss Kappa score of 0.76). The mean difference between the calculated MLI score between the automated method and average rater's score was 2.33 ± 4.13 ( 4.25 % ± 5.67 % ). Conclusion: The proposed pipeline for automated calculation of the MLI score demonstrates high consistency and accuracy with human raters and can be a potential replacement for manual/semi-automated approaches in the field.

Characterization of the innate immune response in a novel murine model mimicking bronchopulmonary dysplasia.

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% O2 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.

A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency.

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.

Late Rescue Therapy with Cord-Derived Mesenchymal Stromal Cells for Established Lung Injury in Experimental Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), the main complication of extreme prematurity, has lifelong consequences for lung health. Mesenchymal stromal cells (MSCs) prevent lung injury in experimental BPD in newborn rodents when given in the immediate neonatal period. Whether MSC therapy can restore normal lung growth after established lung injury in adulthood is clinically relevant, but currently unknown. Experimental BPD was achieved by exposing newborn rats to 95% O2 from postnatal days 4-14. Human umbilical cord-derived MSCs were intratracheally administered to rats (1 × 106cells/kg body weight) as a single dose at 3 or 6 months of age followed by assessment at 5 or 8 months of age, respectively. Lung alveolar structure and vessel density were histologically analyzed. O2-exposed rats exhibited persistent lung injury characterized by arrested alveolar growth with airspace enlargement and a lower vessel density at both 5 and 8 months of age compared with controls. Single-dose MSC treatment at 3 months partially attenuated O2-induced alveolar injury and restored vessel density at 5 months. Treatment with a single dose at 6 months did not attenuate alveolar injury or vessel density at 8 months. However, treatment with multiple MSC doses at 6, 6.5, 7, and 7.5 months significantly attenuated alveolar injury and improved vessel density at 8 months of age. Treatment of the adult BPD lung with MSCs has the potential to improve lung injury if administered in multiple doses or at an early stage of adulthood.

Oxygen Disrupts Human Fetal Lung Mesenchymal Cells. Implications for Bronchopulmonary Dysplasia.

Exogenous mesenchymal stromal cells (MSCs) ameliorate experimental bronchopulmonary dysplasia. Moreover, data from term-born animal models and human tracheal aspirate-derived cells suggest altered mesenchymal signaling in the pathophysiology of neonatal lung disease. We hypothesized that hyperoxia, a factor contributing to the development of bronchopulmonary dysplasia, perturbs human lung-resident MSC function. Mesenchymal cells were isolated from human fetal lung tissue (16-18 wk of gestation), characterized and cultured in conditions resembling either intrauterine (5% O2) or extrauterine (21% and 60% O2) atmospheres. Secretome data were compared with MSCs obtained from term umbilical cord tissues. The human fetal lung mesenchyme almost exclusively contains CD146pos. MSCs expressing SOX-2 and OCT-4, which secrete elastin, fibroblast growth factors 7 and 10, vascular endothelial growth factor, angiogenin, and other lung cell-protecting/-maturing proteins. Exposure to extrauterine atmospheres in vitro leads to excessive proliferation, reduced colony-forming ability, alterations in the cell's surface marker profile, decreased elastin deposition, and impaired secretion of factors important for lung growth. Conversely, umbilical cord-derived MSCs abundantly secreted factors that impaired lung MSCs are unable to produce. Oxygen-impaired human fetal lung MSC function may contribute to disrupted repair capacity and arrested lung growth. Exogenous MSCs may act by triggering the signaling pathways lost by impaired endogenous lung mesenchymal cells.

Endothelial colony-forming cell therapy for heart morphological changes after neonatal high oxygen exposure in rats, a model of complications of prematurity.

Very preterm birth is associated with increased cardiovascular diseases and changes in myocardial structure. The current study aimed to investigate the impact of endothelial colony-forming cell (ECFC) treatment on heart morphological changes in the experimental model of neonatal high oxygen (O2 )-induced cardiomyopathy, mimicking prematurity-related conditions. Sprague-Dawley rat pups exposed to 95% O2 or room air (RA) from day 4 (P4) to day 14 (P14) were randomized to receive (jugular vein) exogenous human cord blood ECFC or vehicle at P14 (n = 5 RA-vehicle, n = 8 RA-ECFC, n = 8 O2 -vehicle and n = 7 O2 -ECFC) and the hearts collected at P28. Body and heart weights and heart to body weight ratio did not differ between groups. ECFC treatment prevented the increase in cardiomyocyte surface area in both the left (LV) and right (RV) ventricles of the O2 group (O2 -ECFC vs. O2 -vehicle LV: 121 ± 13 vs. 179 ± 21 µm2 , RV: 118 ± 12 vs. 169 ± 21 µm2 ). In O2 rats, ECFC treatment was also associated with a significant reduction in interstitial fibrosis in both ventricles (O2 -ECFC vs. O2 -vehicle LV: 1.07 ± 0.47 vs. 1.68 ± 0.41% of surface area, RV: 1.01 ± 0.74 vs. 1.77 ± 0.67%) and in perivascular fibrosis in the LV (2.29 ± 0.47 vs. 3.85 ± 1.23%) but in not the RV (1.95 ± 0.95 vs. 2.74 ± 1.14), and with increased expression of angiogenesis marker CD31. ECFC treatment had no effect on cardiomyocyte surface area or on tissue fibrosis of RA rats. Human cord blood ECFC treatment prevented cardiomyocyte hypertrophy and myocardial and perivascular fibrosis observed after neonatal high O2 exposure. ECFC could constitute a new regenerative therapy against cardiac sequelae caused by deleterious conditions of prematurity.

Impaired Angiogenic Supportive Capacity and Altered Gene Expression Profile of Resident CD146+ Mesenchymal Stromal Cells Isolated from Hyperoxia-Injured Neonatal Rat Lungs.

Bronchopulmonary dysplasia (BPD), the most common complication of extreme preterm birth, can be caused by oxygen-related lung injury and is characterized by impaired alveolar and vascular development. Mesenchymal stromal cells (MSCs) have lung protective effects. Conversely, BPD is associated with increased MSCs in tracheal aspirates. We hypothesized that endogenous lung (L-)MSCs are perturbed in a well-established oxygen-induced rat model mimicking BPD features. Rat pups were exposed to 21% or 95% oxygen from birth to postnatal day 10. On day 12, CD146+ L-MSCs were isolated and characterized according to the International Society for Cellular Therapy criteria. Epithelial and vascular repair potential were tested by scratch assay and endothelial network formation, respectively, immune function by mixed lymphocyte reaction assay. Microarray analysis was performed using the Affymetrix GeneChip and gene set enrichment analysis software. CD146+ L-MSCs isolated from rat pups exposed to hyperoxia had decreased CD73 expression and inhibited lung endothelial network formation. CD146+ L-MSCs indiscriminately promoted epithelial wound healing and limited T cell proliferation. Expression of potent antiangiogenic genes of the axonal guidance cue and CDC42 pathways was increased after in vivo hyperoxia, whereas genes of the anti-inflammatory Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and lung/vascular growth-promoting fibroblast growth factor (FGF) pathways were decreased. In conclusion, in vivo hyperoxia exposure alters the proangiogenic effects and FGF expression of L-MSCs. In addition, decreased CD73 and JAK/STAT expression suggests decreased immune function. L-MSC function may be perturbed and contribute to BPD pathogenesis. These findings may lead to improvements in manufacturing exogenous MSCs with superior repair capabilities.

Human induced pluripotent stem cell-derived lung progenitor and alveolar epithelial cells attenuate hyperoxia-induced lung injury.

BACKGROUND AIMS: Bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by disrupted lung growth, is the most common complication in extreme premature infants. BPD leads to persistent pulmonary disease later in life. Alveolar epithelial type 2 cells (AEC2s), a subset of which represent distal lung progenitor cells (LPCs), promote normal lung growth and repair. AEC2 depletion may contribute to persistent lung injury in BPD. We hypothesized that induced pluripotent stem cell (iPSC)-derived AECs prevent lung damage in experimental oxygen-induced BPD. METHODS: Mouse AECs (mAECs), miPSCs/mouse embryonic stem sells, human umbilical cord mesenchymal stromal cells (hUCMSCs), human (h)iPSCs, hiPSC-derived LPCs and hiPSC-derived AECs were delivered intratracheally to hyperoxia-exposed newborn mice. Cells were pre-labeled with a red fluorescent dye for in vivo tracking. RESULTS: Airway delivery of primary mAECs and undifferentiated murine pluripotent cells prevented hyperoxia-induced impairment in lung function and alveolar growth in neonatal mice. Similar to hUCMSC therapy, undifferentiated hiPSCs also preserved lung function and alveolar growth in hyperoxia-exposed neonatal NOD/SCID mice. Long-term assessment of hiPSC administration revealed local teratoma formation and cellular infiltration in various organs. To develop a clinically relevant cell therapy, we used a highly efficient method to differentiate hiPSCs into a homogenous population of AEC2s. Airway delivery of hiPSC-derived AEC2s and hiPSC-derived LPCs, improved lung function and structure and resulted in long-term engraftment without evidence of tumor formation. CONCLUSIONS: hiPSC-derived AEC2 therapy appears effective and safe in this model and warrants further exploration as a therapeutic option for BPD and other lung diseases characterized by AEC injury.

Human Umbilical Cord Mesenchymal Stromal Cells Improve Survival and Bacterial Clearance in Neonatal Sepsis in Rats.

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 × 105/rat). One hour after infection, rats were treated intravenously with normal saline, hUC-MSCs, or with interferon-γ preconditioned hUC-MSCs (107 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.

Impact of bronchopulmonary dysplasia on brain and retina.

Many premature newborns develop bronchopulmonary dysplasia (BPD), a chronic lung disease resulting from prolonged mechanical ventilation and hyperoxia. BPD survivors typically suffer long-term injuries not only to the lungs, but also to the brain and retina. However, currently it is not clear whether the brain and retinal injuries in these newborns are related only to their prematurity, or also to BPD. We investigated whether the hyperoxia known to cause histologic changes in the lungs similar to BPD in an animal model also causes brain and retinal injuries. Sprague Dawley rat pups were exposed to hyperoxia (95% O2, 'BPD' group) or room air (21% O2, 'control' group) from postnatal day 4-14 (P4-14); the rat pups were housed in room air between P14 and P28. At P28, they were sacrificed, and their lungs, brain, and eyes were extracted. Hematoxylin and eosin staining was performed on lung and brain sections; retinas were stained with Toluidine Blue. Hyperoxia exposure resulted in an increased mean linear intercept in the lungs (P<0.0001). This increase was associated with a decrease in some brain structures [especially the whole-brain surface (P=0.02)], as well as a decrease in the thickness of the retinal layers [especially the total retina (P=0.0008)], compared to the room air control group. In addition, a significant negative relationship was observed between the lung structures and the brain (r=-0.49,P=0.02) and retina (r=-0.70,P=0.0008) structures. In conclusion, hyperoxia exposure impaired lung, brain, and retina structures. More severe lung injuries correlated with more severe brain and retinal injuries. This result suggests that the same animal model of chronic neonatal hyperoxia can be used to simultaneously study lung, brain and retinal injuries related to hyperoxia.

The isolation and culture of endothelial colony-forming cells from human and rat lungs.

Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell-driven 'postnatal vasculogenesis' has been suggested as an important mechanism that contributes to new blood vessel formation and organ repair. Among several described progenitor cell types that contribute to blood vessel formation, endothelial colony-forming cells (ECFCs) have received widespread attention as lineage-specific 'true' vascular progenitors. Here we describe a protocol for the isolation of pulmonary microvascular ECFCs from human and rat lung tissue. Our technique takes advantage of an earlier protocol for the isolation of circulating ECFCs from the mononuclear cellular fraction of peripheral blood. We adapted the earlier protocol to isolate resident ECFCs from the distal lung tissue. After enzymatic dispersion of rat or human lung samples into a cellular suspension, CD31-expressing cells are positively selected using magnetic-activated cell sorting and plated in endothelial-specific growth conditions. The colonies arising after 1-2 weeks in culture are carefully separated and expanded to yield pure ECFC cultures after a further 2-3 weeks. The resulting cells demonstrate the defining characteristics of ECFCs such as (i) 'cobblestone' morphology of cultured cell monolayers; (ii) acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding; (iii) tube-like network formation in Matrigel; (iv) expression of endothelial cell-specific surface markers and the absence of hematopoietic or myeloid surface antigens; (v) self-renewal potential displayed by the most proliferative cells; and (vi) contribution to de novo vessel formation in an in vivo mouse implant model. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 4 weeks. Isolation and culture of lung vascular ECFCs will allow assessment of the functional state of these cells in experimental and human lung diseases, providing newer insights into their pathophysiological mechanisms.

Existence, functional impairment, and lung repair potential of endothelial colony-forming cells in oxygen-induced arrested alveolar growth.

BACKGROUND: Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth. METHODS AND RESULTS: Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth-arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood-derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension. CONCLUSIONS: Impaired ECFC function may contribute to arrested alveolar growth. Cord blood-derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage.

Exogenous hydrogen sulfide (H2S) protects alveolar growth in experimental O2-induced neonatal lung injury.

BACKGROUND: Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, remains a major health problem. BPD is characterized by impaired alveolar development and complicated by pulmonary hypertension (PHT). Currently there is no specific treatment for BPD. Hydrogen sulfide (H2S), carbon monoxide and nitric oxide (NO), belong to a class of endogenously synthesized gaseous molecules referred to as gasotransmitters. While inhaled NO is already used for the treatment of neonatal PHT and currently tested for the prevention of BPD, H2S has until recently been regarded exclusively as a toxic gas. Recent evidence suggests that endogenous H2S exerts beneficial biological effects, including cytoprotection and vasodilatation. We hypothesized that H2S preserves normal alveolar development and prevents PHT in experimental BPD. METHODS: We took advantage of a recently described slow-releasing H2S donor, GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate) to study its lung protective potential in vitro and in vivo. RESULTS: In vitro, GYY4137 promoted capillary-like network formation, viability and reduced reactive oxygen species in hyperoxia-exposed human pulmonary artery endothelial cells. GYY4137 also protected mitochondrial function in alveolar epithelial cells. In vivo, GYY4137 preserved and restored normal alveolar growth in rat pups exposed from birth for 2 weeks to hyperoxia. GYY4137 also attenuated PHT as determined by improved pulmonary arterial acceleration time on echo-Doppler, pulmonary artery remodeling and right ventricular hypertrophy. GYY4137 also prevented pulmonary artery smooth muscle cell proliferation. CONCLUSIONS: H2S protects from impaired alveolar growth and PHT in experimental O2-induced lung injury. H2S warrants further investigation as a new therapeutic target for alveolar damage and PHT.

Hypoxia-inducible factors promote alveolar development and regeneration.

Understanding how alveoli and the underlying capillary network develop and how these mechanisms are disrupted in disease states is critical for developing effective therapies for lung regeneration. Recent evidence suggests that lung angiogenesis promotes lung development and repair. Vascular endothelial growth factor (VEGF) preserves lung angiogenesis and alveolarization in experimental O2-induced arrested alveolar growth in newborn rats, but combined VEGF+angiopoietin 1 treatment is necessary to correct VEGF-induced vessel leakiness. Hypoxia-inducible factors (HIFs) are transcription factors that activate multiple O2-sensitive genes, including those encoding for angiogenic growth factors, but their role during postnatal lung growth is incompletely understood. By inducing the expression of a range of angiogenic factors in a coordinated fashion, HIF may orchestrate efficient and safe angiogenesis superior to VEGF. We hypothesized that HIF inhibition impairs alveolarization and that HIF activation regenerates irreversible O2-induced arrested alveolar growth. HIF inhibition by intratracheal dominant-negative adenovirus (dnHIF-1α)-mediated gene transfer or chetomin decreased lung HIF-1α, HIF-2α, and VEGF expression and led to air space enlargement and arrested lung vascular growth. In experimental O2-induced arrested alveolar growth in newborn rats, the characteristic features of air space enlargement and loss of lung capillaries were associated with decreased lung HIF-1α and HIF-2α expression. Intratracheal administration of Ad.HIF-1α restored HIF-1α, endothelial nitric oxide synthase, VEGF, VEGFR2, and Tie2 expression and preserved and rescued alveolar growth and lung capillary formation in this model. HIFs promote normal alveolar development and may be useful targets for alveolar regeneration.

The axonal guidance cue semaphorin 3C contributes to alveolar growth and repair.

Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD) in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C), contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O2-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.