ArticleLiterature Review

Molecular, cellular, and bioengineering approaches to stimulate lung regeneration after injury

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Abstract

The lung is susceptible to damage from a variety of sources throughout development and in adulthood. As a result, the lung has great capacities for repair and regeneration, directed by precisely controlled sequences of molecular and signaling pathways. Impairments or alterations in these signaling events can have deleterious effects on lung structure and function, ultimately leading to chronic lung disorders. When lung injury is too severe for the normal pathways to repair, or if those pathways do not function properly, lung regenerative medicine is needed to restore adequate structure and function. Great progress has been made in recent years in the number of regenerative techniques and their efficacy. This review will address recent progress in lung regenerative medicine focusing on pharmacotherapy including the expanding role of nanotechnology, stem cell-based therapies, and bioengineering techniques. The use of these techniques individually and collectively has the potential to significantly improve morbidity and mortality associated with congenital and acquired lung disorders.

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... Following injury, the lung has a robust ability to repair and regenerate through distinct cell types. "Repair" is the process of self-renewing and restoring the damaged cells and tissues in an organ, including regeneration or fibroplasia [29] and, if both can occur, then the structure and function of the lung can recover [30]. "Regeneration" refers to generation of new cells and organisation into tissues from progenitor cells derived from de-differentiated tissue-resident cells or cells recruited from the circulation [27][28][29]. ...
... "Repair" is the process of self-renewing and restoring the damaged cells and tissues in an organ, including regeneration or fibroplasia [29] and, if both can occur, then the structure and function of the lung can recover [30]. "Regeneration" refers to generation of new cells and organisation into tissues from progenitor cells derived from de-differentiated tissue-resident cells or cells recruited from the circulation [27][28][29]. Various cell types (e.g. epithelial, mesenchymal) [17,[31][32][33][34][35] participate in regeneration of the lung epithelium which is controlled by Wnt [11], Notch Fig. 2 Generation of lung organoids from hPSCs. ...
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Organoids are derived from stem cells or organ-specific progenitors. They display structures and functions consistent with organs in vivo. Multiple types of organoids, including lung organoids, can be generated. Organoids are applied widely in development, disease modelling, regenerative medicine, and other multiple aspects. Various human pulmonary diseases caused by several factors can be induced and lead to different degrees of lung epithelial injury. Epithelial repair involves the participation of multiple cells and signalling pathways. Lung organoids provide an excellent platform to model injury to and repair of lungs. Here, we review the recent methods of cultivating lung organoids, applications of lung organoids in epithelial repair after injury, and understanding the mechanisms of epithelial repair investigated using lung organoids. By using lung organoids, we can discover the regulatory mechanisms related to the repair of lung epithelia. This strategy could provide new insights for more effective management of lung diseases and the development of new drugs.
... Formation of alveolus, a gas-exchange unit mediating transport of gases between inhaled air and the blood, occurs prior to and after birth, when respiratory epithelial progenitors differentiate into alveolar type I (AT1) and AT2 cells which are located in close proximity to capillary endothelial cells 1,3 . Defects in formation of alveoli and pulmonary capillaries are associated with severe pediatric lung diseases, such as Bronchopulmonary Dysplasia (BPD) and Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) [4][5][6][7] . Recent single cell RNA sequencing of human and mouse neonatal lungs identified two types of alveolar endothelial cells: general capillary cells (gCAPs) and alveolar capillary cells (aCAPs or aerocytes) 8 . ...
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Pulmonary endothelial progenitor cells (EPCs) are critical for neonatal lung angiogenesis and represent a subset of general capillary cells (gCAPs). Molecular mechanisms through which EPCs stimulate lung angiogenesis are unknown. Herein, we used single-cell RNA sequencing to identify the BMP9/ACVRL1/SMAD1 pathway signature in pulmonary EPCs. BMP9 receptor, ACVRL1, and its downstream target genes were inhibited in EPCs from Foxf1WT/S52F mutant mice, a model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Expression of ACVRL1 and its targets were reduced in lungs of ACDMPV subjects. Inhibition of FOXF1 transcription factor reduced BMP9/ACVRL1 signaling and decreased angiogenesis in vitro. FOXF1 synergized with ETS transcription factor FLI1 to activate ACVRL1 promoter. Nanoparticle-mediated silencing of ACVRL1 in newborn mice decreased neonatal lung angiogenesis and alveolarization. Treatment with BMP9 restored lung angiogenesis and alveolarization in ACVRL1-deficient and Foxf1WT/S52F mice. Altogether, EPCs promote neonatal lung angiogenesis and alveolarization through FOXF1-mediated activation of BMP9/ACVRL1 signaling. The molecular mechanisms through which pulmonary endothelial progenitor cells stimulate lung angiogenesis are not clear. Here, authors show that these cells stimulate the growth of alveolar capillaries and alveoli of newborn mice through FOXF1 and FLI1 nuclear protein-activation of the BMP9/ACVRL1/SMAD1 signaling pathway.
... FOXF1, a transcription factor from the Forkhead Box (FOX) family, is expressed in mesenchyme-derived cells such as endothelial cells, fibroblasts, pericytes, and visceral smooth muscle cells [32][33][34][35]. Published studies demonstrated that FOXF1 is critical for embryonic development [36][37][38][39], carcinogenesis [40,41], organ regeneration [36,42,43], and lung repair after various injuries [44][45][46][47]. The Foxf1 WT/S52F and Foxf1 +/mutant mice were recently used to develop two potential therapeutic approaches for ACDMPV patients. ...
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Compromised alveolar development and pulmonary vascular remodeling are hallmarks of pediatric lung diseases such as bronchopulmonary dysplasia (BPD) and alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Although advances in surfactant therapy, corticosteroids, and anti-inflammatory drugs have improved clinical management of preterm infants, still those who suffer with severe vascular complications lack viable treatment options. Paucity of the alveolar capillary network in ACDMPV causes respiratory distress and leads to mortality in a vast majority of ACDMPV infants. The discovery of endothelial progenitor cells (EPCs) in 1997 brought forth the paradigm of postnatal vasculogenesis and hope for promoting vascularization in fragile patient populations, such as those with BPD and ACDMPV. The identification of diverse EPC populations, both hematopoietic and nonhematopoietic in origin, provided a need to identify progenitor cell selective markers which are linked to progenitor properties needed to develop cell-based therapies. Focusing to the future potential of EPCs for regenerative medicine, this review will discuss various aspects of EPC biology, beginning with the identification of hematopoietic, nonhematopoietic, and tissue-resident EPC populations. We will review knowledge related to cell surface markers, signature gene expression, key transcriptional regulators, and will explore the translational potential of EPCs for cell-based therapy for BPD and ACDMPV. The ability to produce pulmonary EPCs from patient-derived induced pluripotent stem cells (iPSCs) in vitro, holds promise for restoring vascular growth and function in the lungs of patients with pediatric pulmonary disorders.
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One-way endobronchial valves (EBV) insertion to reduce pulmonary air trapping has been used as therapy for chronic obstructive pulmonary disease (COPD) patients. However, local inflammation may result and can contribute to worsening of clinical status in these patients. We hypothesized that combined EBV insertion and intrabronchial administration of mesenchymal stromal cells (MSCs) would decrease the inflammatory process, thus mitigating EBV complications in severe COPD patients. This initial study sought to investigate the safety of this approach. For this purpose, a phase I, prospective, patient-blinded, randomized, placebo-controlled design was used. Heterogeneous advanced emphysema (Global Initiative for Chronic Lung Disease [GOLD] III or IV) patients randomly received either allogeneic bone marrow-derived MSCs (10(8) cells, EBV+MSC) or 0.9% saline solution (EBV) (n = 5 per group), bronchoscopically, just before insertion of one-way EBVs. Patients were evaluated 1, 7, 30, and 90 days after therapy. All patients completed the study protocol and 90-day follow-up. MSC delivery did not result in acute administration-related toxicity, serious adverse events, or death. No significant between-group differences were observed in overall number of adverse events, frequency of COPD exacerbations, or worsening of disease. Additionally, there were no significant differences in blood tests, lung function, or radiological outcomes. However, quality-of-life indicators were higher in EBV + MSC compared with EBV. EBV + MSC patients presented decreased levels of circulating C-reactive protein at 30 and 90 days, as well as BODE (Body mass index, airway Obstruction, Dyspnea, and Exercise index) and MMRC (Modified Medical Research Council) scores. Thus, combined use of EBV and MSCs appears to be safe in patients with severe COPD, providing a basis for subsequent investigations using MSCs as concomitant therapy. Stem Cells Translational Medicine 2016.
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A current approach to obtain bioengineered lungs as a future alternative for transplantation is based on seeding stem cells on decellularized lung scaffolds. A fundamental question to be solved in this approach is how to drive stem cell differentiation onto the different lung cell phenotypes. Whereas the use of soluble factors as agents to modulate the fate of stem cells was established from an early stage of the research with this type of cells, it took longer to recognize that the physical microenvironment locally sensed by stem cells (e.g. substrate stiffness, 3D architecture, cyclic stretch, shear stress, air-liquid interface, oxygenation gradient) also contributes to their differentiation. The potential role played by physical stimuli would be particularly relevant in lung bioengineering since cells within the organ are physiologically subjected to two main stimuli required to facilitate efficient gas exchange: air ventilation and blood perfusion across the organ. The present review focuses on describing how the cell mechanical microenvironment can modulate stem cell differentiation and how these stimuli could be incorporated into lung bioreactors for optimizing organ bioengineering.
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Background: Despite recent FDA approval of two new drugs for idiopathic pulmonary fibrosis (IPF), curative therapies remain elusive and mortality remains high. Pre-clinical and clinical data support the safety of human mesenchymal stem cells as a potential novel therapy for this fatal condition. The AETHER trial was the first study designed to evaluate the safety of a single infusion of bone marrow-derived mesenchymal stem cells in patients with idiopathic pulmonary fibrosis. Methods: Nine patients with mild to moderate IPF were sequentially assigned to one of three cohorts and dosed with a single intravenous infusion of 20, 100, or 200 x 10(6) human bone marrow-derived mesenchymal stem cells per infusion from young, unrelated, male donors. All baseline patient data were reviewed by a multidisciplinary study team to ensure accurate diagnosis. The primary endpoint was the incidence (at week four post infusion) of treatment emergent serious adverse events, defined as the composite of: death, non-fatal pulmonary embolism, stroke, hospitalization for worsening dyspnea, and clinically significant laboratory test abnormalities. Safety was assessed until week 60, and additionally 28 days thereafter. Secondary efficacy endpoints were exploratory and measured disease progression. Results: No treatment-emergent serious adverse events were reported. Two non-treatment related deaths occurred due to progression of IPF (disease worsening and/or acute exacerbation). By 60 weeks post-infusion, there was a 3.0% mean decline in % predicted FVC and 5.4% mean decline in % predicted DLCO. Conclusions: Data from this trial support the safety of a single infusion of hMSC in patients with mild-moderate IPF.
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Preterm birth occurs in approximately 11 % of all births worldwide. Advances in perinatal care have enabled the survival of preterm infants born as early as 23–24 weeks of gestation. However, many are affected by bronchopulmonary dysplasia (BPD)—a common respiratory complication of preterm birth, which has life-long consequences for lung health. Currently, there is no specific treatment for BPD. Recent advances in stem cell research have opened new therapeutic avenues for prevention/repair of lung damage. This review summarizes recent pre-clinical data and early clinical translation of cell-based therapies for BPD.
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FOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with a neonatally lethal lung developmental disorder, Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in human disease. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 E15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.
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Objectives: The aim of this study was to assess the feasibility of fetal tracheal injection in the late-gestational pig to target the airways. Methods: Following laparotomy and hysterotomy, fetoscopy was performed in pregnant sows to access the fetal trachea. Two volumes of fluospheres were injected (1 and 3 mL). Fluosphere distribution to the different lung lobes was investigated by microscopy. Possible fetal airway injury, caused by the surgical procedure or intratracheal injection, was investigated. Lung morphology and fetal lung volumes were calculated by micro computed tomography (μCT). Results: Intratracheal administration was successfully performed in 20/21 fetuses. Analysis by confocal microscopy demonstrated that 3 mL, and not 1 mL, most efficiently targeted all lung lobes. On high-resolution μCT, total airway volume was estimated at 2.9 mL; strengthening that 3 mL is appropriate to target all lung lobes. No procedural damage was evidenced in the lungs or trachea. Conclusions: Intratracheal injection of nanoparticles is feasible in the pregnant pig and does not cause procedural lung damage. Using an injection volume of 3 mL, all lung lobes were efficiently targeted. This nanoparticle delivery model to fetal airways opens perspectives for therapeutic interventions. © 2016 John Wiley & Sons, Ltd.
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Recent studies have shown that the respiratory system has an extensive ability to respond to injury and regenerate lost or damaged cells. The unperturbed adult lung is remarkably quiescent, but after insult or injury progenitor populations can be activated or remaining cells can re-enter the cell cycle. Techniques including cell-lineage tracing and transcriptome analysis have provided novel and exciting insights into how the lungs and trachea regenerate in response to injury and have allowed the identification of pathways important in lung development and regeneration. These studies are now informing approaches for modulating the pathways that may promote endogenous regeneration as well as the generation of exogenous lung cell lineages from pluripotent stem cells. The emerging advances, highlighted in this Review, are providing new techniques and assays for basic mechanistic studies as well as generating new model systems for human disease and strategies for cell replacement.
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Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital disorder causing respiratory failure and pulmonary hypertension shortly after birth. There are no effective treatments for ACDMPV other than lung transplant, and new therapeutic approaches are urgently needed. Although ACDMPV is linked to mutations in the FOXF1 gene, molecular mechanisms through which FOXF1 mutations cause ACDMPV are unknown.Objectives: To identify molecular mechanisms by which S52F FOXF1 mutations cause ACDMPV.Methods: We generated a clinically relevant mouse model of ACDMPV by introducing the S52F FOXF1 mutation into the mouse Foxf1 gene locus using CRISPR/Cas9 technology. Immunohistochemistry, whole-lung imaging, and biochemical methods were used to examine vasculature in Foxf1 WT/S52F lungs and identify molecular mechanisms regulated by FOXF1.Measurements and Main Results: FOXF1 mutations were identified in 28 subjects with ACDMPV. Foxf1 WT/S52F knock-in mice recapitulated histopathologic findings in ACDMPV infants. The S52F FOXF1 mutation disrupted STAT3-FOXF1 protein-protein interactions and inhibited transcription of Stat3, a critical transcriptional regulator of angiogenesis. STAT3 signaling and endothelial proliferation were reduced in Foxf1 WT/S52F mice and human ACDMPV lungs. S52F FOXF1 mutant protein did not bind chromatin and was transcriptionally inactive. Furthermore, we have developed a novel formulation of highly efficient nanoparticles and demonstrated that nanoparticle delivery of STAT3 cDNA into the neonatal circulation restored endothelial proliferation and stimulated lung angiogenesis in Foxf1 WT/S52F mice.Conclusions: FOXF1 acts through STAT3 to stimulate neonatal lung angiogenesis. Nanoparticle delivery of STAT3 is a promising strategy to treat ACDMPV associated with decreased STAT3 signaling.
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The unique architecture of the mammalian lung is required for adaptation to air breathing at birth and thereafter. Understanding the cellular and molecular mechanisms controlling its morphogenesis provides the framework for understanding the pathogenesis of acute and chronic lung diseases. Recent single-cell RNA sequencing data and high-resolution imaging identify the remarkable heterogeneity of pulmonary cell types and provides cell selective gene expression underlying lung development. We will address fundamental issues related to the diversity of pulmonary cells, to the formation and function of the mammalian lung, and will review recent advances regarding the cellular and molecular pathways involved in lung organogenesis. What cells form the lung in the early embryo? How are cell proliferation, migration, and differentiation regulated during lung morphogenesis? How do cells interact during lung formation and repair? How do signaling and transcriptional programs determine cell-cell interactions necessary for lung morphogenesis and function?
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Pulmonary vascular disease encompasses a wide range of serious afflictions with important clinical implications. There is critical need for the development of efficient, nonviral gene therapy delivery systems. Here, a promising avenue to overcome critical issues in efficient cell targeting within the lung via a uniquely designed nanosystem is reported. Polyplexes are created by functionalizing hyperbranched polyethylenimine (PEI) with biological fatty acids and carboxylate‐terminated poly(ethylene glycol) (PEG) through a one‐pot 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride/N‐hydroxysuccinimide reaction. Following intravenous injection, polyplexes show an exceptionally high specificity to the pulmonary microvascular endothelium, allowing for the successful delivery of stabilized enhanced green fluorescent protein (eGFP) expressing messenger ribonucleic acid (mRNA). It is further shown, quantitatively, that positive surface charge is the main mechanism behind such high targeting efficiency for these polyplexes. Live in vivo imaging, flow cytometry of single cell suspensions, and confocal microscopy are used to demonstrate that positive polyplexes are enriched in the lung tissue and disseminated in 85–90% of the alveolar capillary endothelium, whilst being sparse in large vessels. Charge modification, achieved through poly(acrylic acid) or heparin coating, drives a highly significant reduction in both targeting percentage and targeting strength, highlighting the importance of specific surface charge, derived from chemical formulation, for efficient targeting of the pulmonary microvascular endothelium.
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Basic fibroblast growth factor (bFGF) can protect the lung against radiation-induced pulmonary vascular endothelial apoptosis and subsequent radiation-induced lung injury (RILI). However, guiding bFGF to pulmonary vascular endothelial cells is a key determinant for the success of bFGF therapy. To improve the lung-targeting ability of bFGF, a lung endothelial cell-targeting peptide was fused to bFGF (LET-bFGF). An in vitro biological activity assay indicated that fusion of LET did not affect the bioactivity of bFGF. In addition, the fused protein showed superior lung-targeting ability following intravenous injection. Upon injecting LET-bFGF intravenously after thorax radiation, LET-bFGF could better protect against pulmonary vascular endothelial cell apoptosis as early as 4 h post-radiation. Compared with native bFGF, enhanced therapeutic effects of LET-bFGF were also observed in terms of decreased vascular abnormalities, disorganized lung structure, inflammatory cell migration, and lung density at 2 months post-radiation. Therefore, lung endothelial cell-targeted bFGF may represent a promising remedy for RILI.
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Organogenesis is regulated by mesenchymal-epithelial signaling events that induce expression of cell-type specific transcription factors critical for cellular proliferation, differentiation and appropriate tissue patterning. While mesenchymal transcription factors play a key role in mesenchymal-epithelial interactions, transcriptional networks in septum transversum and splanchnic mesenchyme remain poorly characterized. Forkhead Box F1 (FOXF1) transcription factor is expressed in mesenchymal cell lineages; however, its role in organogenesis remains uncharacterized due to early embryonic lethality of Foxf1-/- mice. In the present study, we generated mesenchyme-specific Foxf1 knockout mice (Dermo1-Cre Foxf1-/-) and demonstrated that FOXF1 is required for development of respiratory, cardiovascular and gastrointestinal organ systems. Deletion of Foxf1 from mesenchyme caused embryonic lethality in the middle of gestation due to multiple developmental defects in the heart, lung, liver and esophagus. Deletion of Foxf1 inhibited mesenchyme proliferation and delayed branching lung morphogenesis. Gene expression profiling of micro-dissected distal lung mesenchyme and ChIP sequencing of fetal lung tissue identified multiple target genes activated by FOXF1, including Wnt2, Wnt11, Wnt5A and Hoxb7. FOXF1 decreased expression of the Wnt inhibitor Wif1 through direct transcriptional repression. Furthermore, using a global Foxf1 knockout mouse line (Foxf1-/-) we demonstrated that FOXF1-deficiency disrupts the formation of the lung bud in foregut tissue explants. Finally, deletion of Foxf1 from smooth muscle cell lineage (smMHC-Cre Foxf1-/-) caused hyper-extension of esophagus and trachea, loss of tracheal and esophageal muscle, mispatterning of esophageal epithelium and decreased proliferation of smooth muscle cells. Altogether, FOXF1 promotes lung morphogenesis by regulating mesenchymal-epithelial signaling and stimulating cellular proliferation in fetal lung mesenchyme.
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Importance Airway transplantation could be an option for patients with proximal lung tumor or with end-stage tracheobronchial disease. New methods for airway transplantation remain highly controversial. Objective To establish the feasibility of airway bioengineering using a technique based on the implantation of stented aortic matrices. Design, Setting, and Participants Uncontrolled single-center cohort study including 20 patients with end-stage tracheal lesions or with proximal lung tumors requiring a pneumonectomy. The study was conducted in Paris, France, from October 2009 through February 2017; final follow-up for all patients occurred on November 2, 2017. Exposures Radical resection of the lesions was performed using standard surgical techniques. After resection, airway reconstruction was performed using a human cryopreserved (−80°C) aortic allograft, which was not matched by the ABO and leukocyte antigen systems. To prevent airway collapse, a custom-made stent was inserted into the allograft. In patients with proximal lung tumors, the lung-sparing intervention of bronchial transplantation was used. Main Outcomes and Measures The primary outcome was 90-day mortality. The secondary outcome was 90-day morbidity. Results Twenty patients were included in the study (mean age, 54.9 years; age range, 24-79 years; 13 men [65%]). Thirteen patients underwent tracheal (n = 5), bronchial (n = 7), or carinal (n = 1) transplantation. Airway transplantation was not performed in 7 patients for the following reasons: medical contraindication (n = 1), unavoidable pneumonectomy (n = 1), exploratory thoracotomy only (n = 2), and a lobectomy or bilobectomy was possible (n = 3). Among the 20 patients initially included, the overall 90-day mortality rate was 5% (1 patient underwent a carinal transplantation and died). No mortality at 90 days was observed among patients who underwent tracheal or bronchial reconstruction. Among the 13 patients who underwent airway transplantation, major 90-day morbidity events occurred in 4 (30.8%) and included laryngeal edema, acute lung edema, acute respiratory distress syndrome, and atrial fibrillation. There was no adverse event directly related to the surgical technique. Stent removal was performed at a postoperative mean of 18.2 months. At a median follow-up of 3 years 11 months, 10 of the 13 patients (76.9%) were alive. Of these 10 patients, 8 (80%) breathed normally through newly formed airways after stent removal. Regeneration of epithelium and de novo generation of cartilage were observed within aortic matrices from recipient cells. Conclusions and Relevance In this uncontrolled study, airway bioengineering using stented aortic matrices demonstrated feasibility for complex tracheal and bronchial reconstruction. Further research is needed to assess efficacy and safety. Trial Registration clinicaltrials.gov Identifier: NCT01331863
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New protocols to efficiently generate functional airway epithelial organoids from human pluripotent stem cells (PSCs) would represent a major advance towards effective disease modeling, drug screening and cell based therapies for lung disorders. This unit describes an approach using stage‐specific signaling pathway manipulation to differentiate cells to proximal airway epithelium via key developmental intermediates. Cells are directed via definitive endoderm (DE) to anterior foregut, and then specified to NKX2‐1+ lung epithelial progenitors. These lung progenitors are purified using cell surface marker sorting and replated in defined culture conditions to form three‐dimensional, epithelial‐only airway organoids. This directed differentiation approach using serum‐free, defined media also includes protocols for evaluation of DE induction, intracellular FACS analysis of NKX2‐1 specification efficiency and enrichment, and approaches for characterization and expansion of airway organoids. Taken together, this represents an efficient and reproducible approach to generate expandable airway organoids from human PSCs for use in numerous downstream applications.
Chapter
Lung morphogenesis is a highly orchestrated process beginning with the appearance of lung buds on approximately embryonic day 9.5 in the mouse. Endodermally derived epithelial cells of the primitive lung buds undergo branching morphogenesis to generate the tree-like network of epithelial-lined tubules. The pulmonary vasculature develops in close proximity to epithelial progenitor cells in a process that is regulated by interactions between the developing epithelium and underlying mesenchyme. Studies in transgenic and knockout mouse models demonstrate that normal lung morphogenesis requires coordinated interactions between cells lining the tubules, which end in peripheral saccules, juxtaposed to an extensive network of capillaries. Multiple growth factors, microRNAs, transcription factors, and their associated signaling cascades regulate cellular proliferation, migration, survival, and differentiation during formation of the peripheral lung. Dysregulation of signaling events caused by gene mutations, teratogens, or premature birth causes severe congenital and acquired lung diseases in which normal alveolar architecture and the pulmonary capillary network are disrupted. Herein, we review scientific progress regarding signaling and transcriptional mechanisms regulating the development of pulmonary vasculature during lung morphogenesis.
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Advances in neonatal medicine have led to increased survival of infants born at the limits of viability resulting in an increased incidence of bronchopulmonary dysplasia (BPD). BPD is a chronic lung disease of premature infants characterized by arrest of alveolarization, fibroblast activation, and inflammation. BPD leads to significant morbidity and mortality in the neonatal period and is one of the leading causes of chronic lung disease in children. The past decade has brought a surge of trials investigating cellular therapies for the treatment of pulmonary diseases. Mesenchymal stem cells (MSCs) are of particular interest because of their ease of isolation, low immunogenicity, and anti-inflammatory and reparative properties. Clinical trials of MSCs have demonstrated short-term safety and tolerability, however, studies have also shown populations of MSCs with adverse pro-inflammatory and myofibroblastic characteristics. Cell-based therapies may represent the next breakthrough therapy for the treatment of BPD, however there remain barriers to implementation as well as gaps in knowledge of the role of endogenous MSCs in the pathogenesis of BPD. Concurrent high quality basic science, translational, and clinical studies investigating the fundamental pathophysiology underlying BPD, therapeutic mechanisms of exogenous MSCs, and logistics of translating cellular therapies will be important areas of future research.Pediatric Research accepted article preview online, 25 September 2017. doi:10.1038/pr.2017.237.
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The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6⁺ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6⁺ cells impairs airway injury repair in vivo. Distinct Lgr5⁺ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.
Article
Rationale: Mesenchymal stem/stromal cell (MSC) therapies have shown promise in preclinical models of pathologies relevant to newborn medicine, such as bronchopulmonary dysplasia (BPD). We have reported that the therapeutic capacity of MSCs is comprised in their secretome, and demonstrated that the therapeutic vectors are exosomes produced by MSCs (MSC-exos). Objective: To assess efficacy of MSC-exo treatment in a pre-clinical model of BPD and to investigate mechanisms underlying MSC-exo therapeutic action. Methods: Exosomes were isolated from media conditioned by human MSC cultures. Newborn mice were exposed to hyperoxia (HYRX, 75% O2), treated with exosomes on postnatal day 4 (PN4) and returned to room air on PN7. Treated animals and appropriate controls were harvested on PN7, 14 or 42 for assessment of pulmonary parameters. Measurements and main results: HYRX-exposed mice presented with pronounced alveolar simplification, fibrosis, and pulmonary vascular remodeling, which was effectively ameliorated by MSC-exo treatment. Pulmonary function tests and assessment of pulmonary hypertension showed functional improvements following MSC-exo treatment. Lung mRNA sequencing demonstrated that MSC-exo treatment induced pleiotropic effects on gene expression associated with HYRX-induced inflammation and immune responses. MSC-exos modulate the macrophage phenotype fulcrum, suppressing the proinflammatory M1 state and augmenting an anti-inflammatory M2-like state, both in vitro and in vivo. Conclusion: MSC-exo treatment blunts hyperoxia-associated inflammation and alters the hyperoxic lung transcriptome. This results in alleviation of HYRX-induced BPD, improvement of lung function, decrease in fibrosis and pulmonary vascular remodeling, and amelioration of pulmonary hypertension. The MSC exosome mechanism-of-action is associated with modulation of lung macrophage phenotype.
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Pulmonary immune homeostasis is maintained by a network of tissue-resident cells that continually monitor the external environment, and in health, instruct tolerance to innocuous inhaled particles while ensuring that efficient and rapid immune responses can be mounted against invading pathogens. Here we review the multiple pathways that underlie effective lung immunity in health, and discuss how these may be affected by external environmental factors and contribute to chronic inflammation during disease. In this context, we examine the current understanding of the impact of the microbiota in immune development and function and in the setting of the threshold for immune responses that maintains the balance between tolerance and chronic inflammation in the lung. We propose that host interactions with microbes are critical for establishing the immune landscape of the lungs.
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Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders such as cystic fibrosis. However, limited understanding of human airway patterning has made this goal challenging. Here, we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+ progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+ progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach, we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases.
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Very small embryonic-like stem cells (VSELs) are major pluripotent stem cells involved in vascular and tissue regeneration and constitute a recruitable pool of stem/progenitor cells with putative instrumental role in organ repair. Here, we hypothesized that VSELs might be mobilized from the bone marrow (BM) to peripheral blood (PB) in patients with hypoxic lung disease or pulmonary hypertension (PH). The objective of the present study was then to investigate the changes in VSELs number in peripheral blood of patients with hypoxic lung disease and PH. We enrolled 26 patients with Chronic Obstructive Pulmonary Disease (COPD) with or without hypoxemia, 13 patients with PH and 20 controls without any respiratory or cardiovascular diseases. In PH patients, VSELs levels have been determined during right heart catheterization in pulmonary blood and PB. For this purpose, mononuclear cells were separated by density gradient and VSELs have been quantified by using a multiparametric flow cytometry approach. The number of PB-VSELs in hypoxic COPD patients was significantly increased compared with non-hypoxic COPD patients or controls (p = 0.0055). In patients with PH, we did not find any difference in VSELs numbers between arterial pulmonary blood and venous PB (p = 0.93). However, we found an increase in VSELs in the peripheral blood of patients with PH (p = 0.03). In conclusion, we unraveled that circulating VSELs were increased in peripheral blood of patients with hypoxic COPD or with PH. Thus, VSELs may serve as a reservoir of pluripotent stem cells that can be recruited into PB and may play an important role in promoting lung repair.
Article
Objective: Bioengineering of viable, functional, and implantable human lung grafts on porcine matrix. Summary background data: Implantable bioartificial organ grafts could revolutionize transplant surgery. To date, several milestones toward that goal have been achieved in rodent models. To make bioengineered organ grafts clinically relevant, scaling to human cells and graft size are the next steps. Methods: We seeded porcine decellularized lung scaffolds with human airway epithelial progenitor cells derived from rejected donor lungs, and banked human umbilical vein endothelial cells. We subsequently enabled tissue formation in whole organ culture. The resulting grafts were then either analyzed in vitro (n = 15) or transplanted into porcine recipients in vivo (n = 3). Results: By repopulating porcine extracellular matrix scaffolds with human endothelial cells, we generated pulmonary vasculature with mature endothelial lining and sufficient anti-thrombotic function to enable blood perfusion. By repopulating the epithelial surface with human epithelial progenitor cells, we created a living, functioning gas exchange graft. After surgical implantation, the bioengineered lung grafts were able to withstand physiological blood flow from the recipient's pulmonary circulation, and exchanged gases upon ventilation during the 1-hour observation. Conclusions: Engineering and transplantation of viable lung grafts based on decellularized porcine lung scaffolds and human endothelial and epithelial cells is technically feasible. Further graft maturation will be necessary to enable higher-level functions such as mucociliary clearance, and ventilation-perfusion matching.
Article
Background: Lung morphogenesis is regulated by interactions between the canonical Wnt/β-catenin and Kras/ERK/Foxm1 signaling pathways that establish proximal-peripheral patterning of lung tubules. How these interactions influence the development of respiratory epithelial progenitors to acquire airway as compared to alveolar epithelial cell fate is unknown. During branching morphogenesis, SOX9 transcription factor is normally restricted from conducting airway epithelial cells and is highly expressed in peripheral, acinar progenitor cells that serve as precursors of alveolar type 2 (AT2) and AT1 cells as the lung matures. Results: To identify signaling pathways that determine proximal-peripheral cell fate decisions, we used the SFTPC gene promoter to delete or overexpress key members of Wnt/β-catenin and Kras/ERK/Foxm1 pathways in fetal respiratory epithelial progenitor cells. Activation of β-catenin enhanced SOX9 expression in peripheral epithelial progenitors, whereas deletion of β-catenin inhibited SOX9. Surprisingly, deletion of β-catenin caused accumulation of atypical SOX9-positive basal cells in conducting airways. Inhibition of Wnt/β-catenin signaling by Kras(G12D) or its downstream target Foxm1 stimulated SOX9 expression in basal cells. Genetic inactivation of Foxm1 from Kras(G12D) -expressing epithelial cells prevented the accumulation of SOX9-positive basal cells in developing airways. Conclusions: Interactions between the Wnt/β-catenin and the Kras/ERK/Foxm1 pathways are essential to restrict SOX9 expression in basal cells. This article is protected by copyright. All rights reserved.
Article
In this manuscript we will review the biology and physiological importance of transforming growth factor-β (TGF-β) to homeostasis in the respiratory system, its importance to innate and adaptive immune responses in the lung, and its pathophysiological role in various chronic pulmonary diseases including pulmonary arterial hypertension, COPD, asthma, and pulmonary fibrosis. The TGF-β family is responsible for initiation of intracellular signaling pathways that direct numerous cellular activities, including proliferation, differentiation, extracellular matrix synthesis and apoptosis. When TGF-β signaling is dysregulated or essential control mechanisms are unbalanced, the consequences of organ and tissue dysfunction can be profound. The complexities and myriad checkpoints built into TGF-β signaling pathways provide attractive targets for treatment of these disease states, many of which are currently being studied. This review will focus on those aspects of TGF-β biology that are most relevant to pulmonary diseases and hold promise as novel therapeutic targets.
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Hippo pathway is comprised of a kinase cascade that involves MST1/2 and LATS1/2, and leads to inactivation of the transcriptional coactivators, TAZ and YAP. Protein stability and subcellular localization of TAZ/YAP determine its ability to regulate a diverse array of biological processes, including proliferation, apoptosis, differentiation, stem/progenitor cell properties, organ size control, and tumorigenesis. These actions are enabled by interactions with various transcription factors or through crosstalk with other signaling pathways. Interestingly, mechanical stress has been shown to be an upstream regulator of TAZ/YAP activity, and this finding provides a novel clue for understanding how mechanical forces influence a broad spectrum of biological processes, which involve cytoskeletal structure, cell adhesion, and extracellular matrix (ECM) organization. TGF-β pathway is a critical component of lung development and the progression of lung diseases including emphysema, fibrosis, and cancer. In addition, TGF-β is a key regulator of ECM remodeling and cell differentiation processes such as epithelial-mesenchymal transition (EMT). In this review, we summarize the current knowledge of Hippo pathway regarding lung development and diseases, with an emphasis on its interplay with TGF-β signaling. Copyright © 2015, American Journal of Physiology - Lung Cellular and Molecular Physiology.
Article
No effective pharmacotherapy for acute respiratory distress syndrome (ARDS) exists, and mortality remains high. Preclinical studies support the efficacy of mesenchymal stem (stromal) cells (MSCs) in the treatment of lung injury. We aimed to test the safety of a single dose of allogeneic bone marrow-derived MSCs in patients with moderate-to-severe ARDS. The STem cells for ARDS Treatment (START) trial was a multicentre, open-label, dose-escalation, phase 1 clinical trial. Patients were enrolled in the intensive care units at University of California, San Francisco, CA, USA, Stanford University, Stanford, CA, USA, and Massachusetts General Hospital, Boston, MA, USA, between July 8, 2013, and Jan 13, 2014. Patients were included if they had moderate-to-severe ARDS as defined by the acute onset of the need for positive pressure ventilation by an endotracheal or tracheal tube, a PaO2:FiO2 less than 200 mm Hg with at least 8 cm H2O positive end-expiratory airway pressure (PEEP), and bilateral infiltrates consistent with pulmonary oedema on frontal chest radiograph. The first three patients were treated with low dose MSCs (1 million cells/kg predicted bodyweight [PBW]), the next three patients received intermediate dose MSCs (5 million cells/kg PBW), and the final three patients received high dose MSCs (10 million cells/kg PBW). Primary outcomes included the incidence of prespecified infusion-associated events and serious adverse events. The trial is registered with ClinicalTrials.gov, number NCT01775774. No prespecified infusion-associated events or treatment-related adverse events were reported in any of the nine patients. Serious adverse events were subsequently noted in three patients during the weeks after the infusion: one patient died on study day 9, one patient died on study day 31, and one patient was discovered to have multiple embolic infarcts of the spleen, kidneys, and brain that were age-indeterminate, but thought to have occurred before the MSC infusion based on MRI results. None of these severe adverse events were thought to be MSC-related. A single intravenous infusion of allogeneic, bone marrow-derived human MSCs was well tolerated in nine patients with moderate to severe ARDS. Based on this phase 1 experience, we have proceeded to phase 2 testing of MSCs for moderate to severe ARDS with a primary focus on safety and secondary outcomes including respiratory, systemic, and biological endpoints. The National Heart, Lung, and Blood Institute. Copyright © 2014 Elsevier Ltd. All rights reserved.
Article
Current treatments for inflammation associated with bronchopulmonary dysplasia fail to show clinical efficacy. Foxm1, a transcription factor of the Forkhead box family, is a critical mediator of lung development and carcinogenesis, but its role in bronchopulmonary dysplasia-associated pulmonary inflammation is unknown. Immunohistochemistry and RNA analysis were used to assess Foxm1 in lung tissue from hyperoxia-treated mice and patients with bronchopulmonary dysplasia. LysM-Cre/Foxm1-/- mice, in which Foxm1 was deleted from myeloid-derived inflammatory cells, including macrophages, monocytes and neutrophils, were exposed to neonatal hyperoxia causing lung injury and remodeling. Measurements of lung function and flow cytometry were used to evaluate effects of Foxm1 deletion on pulmonary inflammation and repair. Increased Foxm1 expression was observed in pulmonary macrophages of hyperoxia-exposed mice and lung tissue from patients with bronchopulmonary dysplasia. After hyperoxia, deletion of Foxm1 from the myeloid cell lineage decreased numbers of interstitial macrophages (CD45+CD11b+Ly6C-Ly6G-F4/80+CD68-) and impaired alveologenesis and lung function. The exaggerated bronchopulmonary dysplasia-like phenotype observed in hyperoxia-exposed LysM-Cre/Foxm1-/- mice was associated with increased expression of neutrophil-derived myeloperoxidase, proteinase 3 and cathepsin-g, all of which are critical for lung remodeling and inflammation. Our data demonstrate that Foxm1 influences pulmonary inflammatory responses to hyperoxia, inhibiting neutrophil-derived enzymes and enhancing monocytic responses that limit alveolar injury and remodeling in neonatal lungs.