Effectiveness of extracellular vesicles derived from hiPSCs in repairing hyperoxia-induced injury in a fetal murine lung explant model.

BACKGROUND: Despite advances in neonatal care, the incidence of Bronchopulmonary Dysplasia (BPD) remains high among preterm infants. Human induced pluripotent stem cells (hiPSCs) have shown promise in repairing injury in animal BPD models. Evidence suggests they exert their effects via paracrine mechanisms. We aim herein to assess the effectiveness of extracellular vesicles (EVs) derived from hiPSCs and their alveolar progenies (diPSCs) in attenuating hyperoxic injury in a preterm lung explant model. METHODS: Murine lung lobes were harvested on embryonic day 17.5 and maintained in air-liquid interface. Following exposure to 95% O2 for 24 h, media was supplemented with 5 × 106 particles/mL of EVs isolated from hiPSCs or diPSCs by size-exclusion chromatography. On day 3, explants were assessed using Hematoxylin-Eosin staining with mean linear intercept (MLI) measurements, immunohistochemistry, VEGFa and antioxidant gene expression. Statistical analysis was conducted using one-way ANOVA and Multiple Comparison Test. EV proteomic profiling was performed, and annotations focused on alveolarization and angiogenesis signaling pathways, as well as anti-inflammatory, anti-oxidant, and regenerative pathways. RESULTS: Exposure of fetal lung explants to hyperoxia induced airspace enlargement, increased MLI, upregulation of anti-oxidants Prdx5 and Nfe2l2 with decreased VEGFa expression. Treatment with hiPSC-EVs improved parenchymal histologic changes. No overt changes in vasculature structure were observed on immunohistochemistry in our in vitro model. However, VEGFa and anti-oxidant genes were upregulated with diPSC-EVs, suggesting a pro-angiogenic and cytoprotective potential. EV proteomic analysis provided new insights in regard to potential pathways influencing lung regeneration. CONCLUSION: This proof-of-concept in vitro study reveals a potential role for hiPSC- and diPSC-EVs in attenuating lung changes associated with prematurity and oxygen exposure. Our findings pave the way for a novel cell free approach to prevent and/or treat BPD, and ultimately reduce the global burden of the disease.

Obesity and Adipose Tissue Dysfunction: From Pediatrics to Adults.

Obesity is a growing health problem that affects both children and adults. The increasing prevalence of childhood obesity is associated with comorbidities such as cardiovascular disease, type 2 diabetes and metabolic syndrome due to chronic low-grade inflammation present at early stages of the disease. In pediatric patients suffering from obesity, the role of epigenetics, the gut microbiome and intrauterine environment have emerged as causative factors Interestingly, pediatric obesity is strongly associated with low birth weight. Accelerated weight gain oftentimes occurs in these individuals during the post-natal period, which can lead to increased risk of adiposity and metabolic disease. The pathophysiology of obesity is complex and involves biological and physiological factors compounded by societal factors such as family and community. On a cellular level, adipocytes contained within adipose tissue become dysregulated and further contribute to development of comorbidities similar to those present in adults with obesity. This review provides an overview of the current understanding of adipose tissue immune, inflammatory and metabolic adaptation of the adipose tissue in obesity. Early cellular changes as well as the role of immune cells and inflammation on the progression of disease in pivotal pediatric clinical trials, adult studies and mouse models are emphasized. Understanding the initial molecular and cellular changes that occur during obesity can facilitate new and improved treatments aimed at early intervention and subsequent prevention of adulthood comorbidities.

Esophageal regeneration following surgical implantation of a tissue engineered esophageal implant in a pediatric model.

Diseases of the esophagus, damage of the esophagus due to injury or congenital defects during fetal esophageal development, i.e., esophageal atresia (EA), typically require surgical intervention to restore esophageal continuity. The development of tissue engineered tubular structures would improve the treatment options for these conditions by providing an alternative that is organ sparing and can be manufactured to fit the exact dimensions of the defect. An autologous tissue engineered Cellspan Esophageal ImplantTM (CEI) was surgically implanted into piglets that underwent surgical resection of the esophagus. Multiple survival time points, post-implantation, were analyzed histologically to understand the tissue architecture and time course of the regeneration process. In addition, we investigated CT imaging as an "in-life" monitoring protocol to assess tissue regeneration. We also utilized a clinically relevant animal management paradigm that was essential for long term survival. Following implantation, CT imaging revealed early tissue deposition and the formation of a contiguous tissue conduit. Endoscopic evaluation at multiple time points revealed complete epithelialization of the lumenal surface by day 90. Histologic evaluation at several necropsy time points, post-implantation, determined the time course of tissue regeneration and demonstrated that the tissue continues to remodel over the course of a 1-year survival time period, resulting in the development of esophageal structural features, including the mucosal epithelium, muscularis mucosae, lamina propria, as well as smooth muscle proliferation/migration initiating the formation of a laminated adventitia. Long term survival (1 year) demonstrated restoration of oral nutrition, normal animal growth and the overall safety of this treatment regimen.

Advanced single-cell technologies to guide the development of bioengineered lungs.

Lung transplantation remains the only viable option for individuals suffering from end-stage lung failure. However, a number of current limitations exist including a continuing shortage of suitable donor lungs and immune rejection following transplantation. To address these concerns, engineering a decellularized biocompatible lung scaffold from cadavers reseeded with autologous lung cells to promote tissue regeneration is being explored. Proof-of-concept transplantation of these bioengineered lungs into animal models has been accomplished. However, these lungs were incompletely recellularized with resulting epithelial and endothelial leakage and insufficient basement membrane integrity. Failure to repopulate lung scaffolds with all of the distinct cell populations necessary for proper function remains a significant hurdle for the progression of current engineering approaches and precludes clinical translation. Advancements in 3D bioprinting, lung organoid models, and microfluidic device and bioreactor development have enhanced our knowledge of pulmonary lung development, as well as important cell-cell and cell-matrix interactions, all of which will help in the path to a bioengineered transplantable lung. However, a significant gap in knowledge of the spatiotemporal interactions between cell populations as well as relative quantities and localization within each compartment of the lung necessary for its proper growth and function remains. This review will provide an update on cells currently used for reseeding decellularized scaffolds with outcomes of recent lung engineering attempts. Focus will then be on how data obtained from advanced single-cell analyses, coupled with multiomics approaches and high-resolution 3D imaging, can guide current lung bioengineering efforts for the development of fully functional, transplantable lungs.

Hyperoxia-induced bronchopulmonary dysplasia: better models for better therapies.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease caused by exposure to high levels of oxygen (hyperoxia) and is the most common complication that affects preterm newborns. At present, there is no cure for BPD. Infants can recover from BPD; however, they will suffer from significant morbidity into adulthood in the form of neurodevelopmental impairment, asthma and emphysematous changes of the lung. The development of hyperoxia-induced lung injury models in small and large animals to test potential treatments for BPD has shown some success, yet a lack of standardization in approaches and methods makes clinical translation difficult. In vitro models have also been developed to investigate the molecular pathways altered during BPD and to address the pitfalls associated with animal models. Preclinical studies have investigated the efficacy of stem cell-based therapies to improve lung morphology after damage. However, variability regarding the type of animal model and duration of hyperoxia to elicit damage exists in the literature. These models should be further developed and standardized, to cover the degree and duration of hyperoxia, type of animal model, and lung injury endpoint, to improve their translational relevance. The purpose of this Review is to highlight concerns associated with current animal models of hyperoxia-induced BPD and to show the potential of in vitro models to complement in vivo studies in the significant improvement to our understanding of BPD pathogenesis and treatment. The status of current stem cell therapies for treatment of BPD is also discussed. We offer suggestions to optimize models and therapeutic modalities for treatment of hyperoxia-induced lung damage in order to advance the standardization of procedures for clinical translation.

Characterization of mesenchymal stem cells in patients with esophageal atresia.

BACKGROUND: Preclinical studies demonstrate that tissue engineering and patient-derived stem cells can regenerate tissue. The goal of this study was to determine whether stem cells from esophageal atresia patients (EA) could be utilized for this purpose. METHODS: Adipose tissue was obtained from control, esophageal atresia (EA) and long gap esophageal atresia (LGEA) patients. Mesenchymal stem cells (MSCs) were isolated, expanded, characterized and seeded onto tubular scaffolds for 6 days. Scaffolds were characterized for viability, gene expression and cytokine production. RESULTS: The average weight of tissue from the EA and LGEA patients was 145.8mg compared to 2981 mg in controls. Despite the small amount of tissue obtained from neonatal patients, cells were expanded to cover a scaffold. After incubating 6 days on the scaffold, cells were viable and proliferating with differences in gene expression between groups. VEGFA production in the supernatant was increased in EA and LGEA patients; while IL6 production was significantly increased in the control patients. CONCLUSIONS: This study demonstrates the ability to utilize small amounts of adipose tissue from esophageal atresia patients as a cell source for regenerative medicine. Future studies will focus on use of these cells for tissue regeneration in vivo.

Human induced pluripotent stem cells ameliorate hyperoxia-induced lung injury in a mouse model.

Hyperoxia-induced lung injury occurs in neonates on oxygen support due to premature birth, often leading to the development of bronchopulmonary dysplasia. Current treatment options have limited effect. The aim of this study was to determine if human induced pluripotent stem cells (iPSCs) and those differentiated to an alveolar-like phenotype (diPSCs) could repair hyperoxia-induced lung damage in a mouse model. Neonatal C57BL6/J mice were separated into two groups and exposed to 75% oxygen over 6 or 14 days. Cell treatments were instilled intra-orally following removal. Controls included hyperoxia, normoxia, and a vehicle. 7 and 14 days post treatment, lungs were extracted and histomorphometric analysis performed. Gene expression of markers mediating inflammation (Tgfß1, Nfkb1, and Il-6) were investigated. In addition, exosomes from each cell type were isolated and administered as a cell free alternative. There was a significant difference between the mean linear intercept (MLI) in hyperoxic vs. normoxic lungs prior to treatment. No difference existed between the MLI in iPSC-treated lungs vs. normoxic lungs after 6 and 14 days of hyperoxia. For mice exposed to 6 days of hyperoxia, gene expression in iPSC-treated lungs returned to normal 14 days later. At the same time points, diPSCs were not as effective. Exosomes were also not as effective in reversing hyperoxic lung damage as their cellular counterparts. This study highlights the potential benefit of using iPSCs to repair damaged lung tissue through possible modulation of the inflammatory response, leading to novel therapies for acute hyperoxia-induced lung injury and the prevention of bronchopulmonary dysplasia.

Assessment of iPSC teratogenicity throughout directed differentiation toward an alveolar-like phenotype.

Considerable work has gone into creating cell therapies from induced pluripotent stem cells (iPSCs) since their discovery just over a decade ago. However, comparatively little research has been done concerning the safety of iPSCs and their progeny and specifically the mechanisms governing teratogenicity. The aim of this study was to ascertain at what developmental phase iPSCs undergoing differentiation to an alveolar-like phenotype lose their capacity to form a teratoma and uncover potential mechanisms responsible. iPSCs were differentiated using a previously published directed differentiation protocol mirroring alveolar embryogenesis. At each developmental phase cell phenotype was assessed and cells mixed with Matrigel and injected subcutaneously above the hind limbs of NSG mice to determine teratogenicity. A genetic screen of 42 genes commonly associated with teratoma formation was conducted on all the cells and any resulting teratoma. It was found that neither NKX2-1 lung progenitors nor terminally differentiated alveolar-like cells formed teratomas. As expected the expression of pluripotency markers was diminished over differentiation. However, the expression of two proteoglycans, decorin and lumican, was increased more than 3000x during differentiation. Both decorin and lumican are putative tumor suppressors with additional functions in angiogenesis, fibrosis, inflammation and autophagy. We hypothesize that the increasing expression of these proteoglycans by iPSCs as they differentiate may act to inhibit host endothelial cell recruitment when implanted resulting in the inhibition of any teratoma formation by any remaining undifferentiated iPSCs.

Polyurethane scaffolds seeded with autologous cells can regenerate long esophageal gaps: An esophageal atresia treatment model.

BACKGROUND: Pediatric patients suffering from long gap esophageal defects or injuries are in desperate need of innovative treatment options. Our study demonstrates that two different cell sources can adhere to and proliferate on a retrievable synthetic scaffold. In feasibility testing of translational applicability, these cell seeded scaffolds were implanted into piglets and demonstrated esophageal regeneration. METHODS: Either porcine esophageal epithelial cells or porcine amniotic fluid was obtained and cultured in 3 dimensions on a polyurethane scaffold (Biostage). The amniotic fluid was obtained prior to birth of the piglet and was a source of mesenchymal stem cells (AF-MSC). Scaffolds that had been seeded were implanted into their respective Yucatan mini-swine. The cell seeded scaffolds in the bioreactor were evaluated for cell viability, proliferation, genotypic expression, and metabolism. Feasibility studies with implantation evaluated tissue regeneration and functional recovery of the esophagus. RESULTS: Both cell types seeded onto scaffolds in the bioreactor demonstrated viability, adherence and metabolism over time. The seeded scaffolds demonstrated increased expression of VEGF after 6 days in culture. Once implanted, endoscopy 3 weeks after surgery revealed an extruded scaffold with newly regenerated tissue. Both cell seeded scaffolds demonstrated epithelial and muscle regeneration and the piglets were able to eat and grow over time. CONCLUSIONS: Autologous esophageal epithelial cells or maternal AF-MSC can be cultured on a 3D scaffold in a bioreactor. These cells maintain viability, proliferation, and adherence over time. Implantation into piglets demonstrated esophageal regeneration with extrusion of the scaffold. This sets the stage for translational application in a neonatal model of esophageal atresia.

Cloning and characterization of a hybridoma secreting a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-specific monoclonal antibody and recombinant F(ab).

Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.