Validating the Modified McGill Thyroid Nodule Score for Assessment of Preoperative Risk of Pediatric Thyroid Malignancy.

OBJECTIVE: The McGill Thyroid Nodule Score (MTNS) is a preoperative tool used to predict the risk for well-differentiated thyroid cancer in adults. It was developed by a multidisciplinary team using established evidence-based risk factors for thyroid cancer. The modified McGill Thyroid Nodule Score (mMTNS) was developed to predict malignancy risk in children. A pilot study suggested the mMTNS was able to assess malignancy risk in children with indeterminate cytology on fine needle aspiration (FNA). This study seeks to validate these findings. METHODS: Retrospective chart review identified subjects who underwent FNA biopsy and subsequent resection. Each patient was assigned a score to compare to final pathology. Statistical analysis was performed with SPSS. All tests were 2-tailed and statistical significance defined p < 0.05. Logistic regression used to determine predictive values of scores. RESULTS: 46 patients ≤21 years of age underwent resection of a thyroid nodule. Female predominance of 85% (n = 39). 78% (n = 36) of patients had palpable nodule. 65% (n = 30) found to have benign pathology and 35% (n = 16) found to have malignancy. Malignant nodules associated with greater mean mMTNS compared to benign [13.63 vs 7.23]. An mMTNS greater >12 had sensitivity of 86.7%, specificity of 90.3%, positive predictive value of 81.3%, and negative predictive value of 93.3%. CONCLUSION: Our data suggests the mMTNS continues to be a useful adjunct in predicting malignancy risk of pediatric thyroid nodules. An mMTNS >12 has a high risk for malignancy, which can aid in counseling and clinical decision making, particularly when there is indeterminate cytology on FNA. LEVEL OF EVIDENCE: IV.

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.

Development of a Wound-Healing Protocol for In Vitro Evaluation of Urothelial Cell Growth.

Urethral healing is plagued by strictures, impacting quality of life and medical costs. Various growth factors (GFs) have shown promise as therapeutic approaches to improve healing, but there is no protocol for in vitro comparison between GFs. This study focuses the development of a biomimetic in vitro urothelial healing assay designed to mimic early in vivo healing, followed by an evaluation of urothelial cell growth in response to GFs. METHODS: Wound-healing assays were developed with human urothelial cells and used to compared six GFs (EGF, FGF-2, IGF-1, PDGF, TGF-ß1, and VEGF) at three concentrations (1 ng/mL, 10 ng/mL, and 100 ng/mL) over a 48 h period. A commercial GF-containing medium (EGF, TGF-α, KGF, and Extract P) and a GF-free medium were used as controls. RESULTS: There was a statistically significant increase in cell growth for IGF-1 at 10 and 100 ng/mL compared to both controls (p < 0.05). There was a statistically significant increase in cell growth for EGF at all concentrations compared to the GF-free medium control (p < 0.05). CONCLUSION: This study shows the development of a clinically relevant wound-healing assay to evaluate urothelial cell growth. It is the first to compare GFs for future use in reconstructive techniques to improve urethral healing.

Esophageal Surveillance Practices in Esophageal Atresia Patients: A Survey by the Eastern Pediatric Surgery Network.

INTRODUCTION: Endoscopic surveillance guidelines for patients with repaired esophageal atresia (EA) rely primarily on expert opinion. Prior to embarking on a prospective EA surveillance registry, we sought to understand EA surveillance practices within the Eastern Pediatric Surgery Network (EPSN). METHODS: An anonymous, 23-question Qualtrics survey was emailed to 181 physicians (surgeons and gastroenterologists) at 19 member institutions. Likert scale questions gauged agreement with international EA surveillance guideline-derived statements. Multiple-choice questions assessed individual and institutional practices. RESULTS: The response rate was 77%. Most respondents (80%) strongly agree or agree that EA surveillance endoscopy should follow a set schedule, while only 36% claimed to perform routine upper GI endoscopy regardless of symptoms. Many institutions (77%) have an aerodigestive clinic, even if some lack a multi-disciplinary EA team. Most physicians (72%) expressed strong interest in helping develop evidence-based guidelines. CONCLUSIONS: Our survey reveals physician agreement with current guidelines but weak adherence. Surveillance methods vary greatly, underscoring the lack of evidence-based data to guide EA care. Aerodigestive clinics may help implement surveillance schedules. Respondents support evidence-based protocols, which bodes well for care standardization. Results will inform the first multi-institutional EA databases in the United States (US), which will be essential for evidence-based care. LEVEL OF EVIDENCE: This is a prognosis study with level 4 evidence.

The First Two Years of the Association of Pediatric Surgery Training Program Directors (APSTPD) Transition to Fellowship Course: Lessons Learned and Future Directions.

OBJECTIVE: The first transition to fellowship course for incoming pediatric surgery fellows was held in the US in 2018 and the second in 2019. The course aimed to facilitate a successful transition in to fellowship by introduction of the professional, patient care, and technical aspects unique to pediatric surgery training. The purpose of this study was to evaluate the feasibility and effectiveness of the first two years of this course in the US and discuss subsequent evolution of this endeavor. DESIGN: This is a descriptive and qualitative analysis of two years' experience with the Association of Pediatric Surgery Training Program Directors' (APSTPD) Transition to Fellowship course. Course development and curriculum, including clinical knowledge, soft skills, and hands-on skills labs, are presented. Participating incoming fellows completed multiple choice, boards-style pre- and post-tests. Scores were compared to determine if knowledge was effectively transferred. Participants also completed post-course evaluations and subsequent 3- or 12-month surveys inquiring on the lasting impact of the course on their transition into fellowship. Standard univariate statistics were used to present results. SETTING: The first APSTPD Transition to Fellowship course was held at the Johns Hopkins Hospital in Baltimore, Maryland in 2018, and the second course was held at the Oregon Health and Science University in Portland, Oregon in 2019. PARTICIPANTS: All fellows entering ACGME-certified Pediatric Surgery fellowships in the United States were invited to participate. Twenty fellows accepted and attended in 2018, and fourteen fellows participated in 2019. RESULTS: There were 34 incoming pediatric surgery fellow participants over 2 years. Faculty represented more than 10 institutions each year. Pre- and post-test scores were similar between years, with a significant improvement of scores after completion of the course (67±10% vs 79±8%, p < 0.001). Feedback from participants was overwhelmingly positive, with skills labs being attendees' favorite component. When asked about usefulness of individual course sessions, more attendees found clinical sessions more useful than soft skills (93% vs 73%, p = 0.011). Almost all (90%) of participants reported the course met its stated purpose and would recommend the course to future fellows. This was further reflected on 3 and 12 month follow up surveys wherein 85% stated they found the course helpful during the first few months of fellowship and 90% would still recommend it. CONCLUSIONS: A transition to fellowship course in the US for incoming pediatric surgery fellows is logistically feasible, effective in transfer of knowledge, and highly regarded among attendees. Feedback from each course has been used to improve the subsequent courses, ensuring that it remains a valuable addition to pediatric surgical training in the US.

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.

Development of alginate and gelatin-based pleural and tracheal sealants.

Pleural and tracheal injuries remain significant problems, and an easy to use, effective pleural or tracheal sealant would be a significant advance. The major challenges are requirements for adherence, high strength and elasticity, dynamic durability, appropriate biodegradability, and lack of cell or systemic toxicity. We designed and evaluated two sealant materials comprised respectively of alginate methacrylate and of gelatin methacryloyl, each functionalized by conjugation with dopamine HCl. Both compounds are cross-linked into easily applied as pre-formed hydrogel patches or as in situ hydrogels formed at the wound site utilizing FDA-approved photo-initiators and oxidants. Material testing demonstrates appropriate adhesiveness, tensile strength, burst pressure, and elasticity with no significant cell toxicity in vitro assessments. Air-leak was absent after sealant application to experimentally-induced injuries in ex-vivo rat lung and tracheal models and in ex vivo pig lungs. Sustained repair of experimentally-induced pleural injury was observed for up to one month in vivo rat models and for up to 2 weeks in vivo rat tracheal injury models without obvious air leak or obvious toxicities. The alginate-based sealant worked best in a pre-formed hydrogel patch whereas the gelatin-based sealant worked best in an in situ formed hydrogel at the wound site thus providing two potential approaches. These studies provide a platform for further pre-clinical and potential clinical investigations. STATEMENT OF SIGNIFICANCE: Pneumothorax and pleural effusions resulting from trauma and a range of lung diseases and critical illnesses can result in lung collapse that can be immediately life-threatening or result in chronic leaking (bronchopleural fistula) that is currently difficult to manage. This leads to significantly increased morbidity, mortality, hospital stays, health care costs, and other complications. We have developed sealants originating from alginate and gelatin biomaterials, each functionalized by methacryloylation and by dopamine conjugation to have desired mechanical characteristics for use in pleural and tracheal injuries. The sealants are easily applied, non-cytotoxic, and perform well in vitro and in vivo model systems of lung and tracheal injuries. These initial proof of concept investigations provide a platform for further studies.

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.

Pediatric Patient With Concurrent Eosinophilic Esophagitis, Erosive Reflux Esophagitis, and Barrett's Esophagus.

Eosinophilic esophagitis and Barrett's esophagus are believed to be separate disease processes, with erosive esophagitis leading to Barrett's esophagus. We report a rare case of concurrent diagnoses in a pediatric patient and examine the relevant genetic profiles in the esophagus.

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.

Reagent-Free and Rapid Assessment of T Cell Activation State Using Diffraction Phase Microscopy and Deep Learning.

CD8+ T cells constitute an essential compartment of the adaptive immune system. During immune responses, naïve T cells become functional, as they are primed with their cognate determinants by the antigen presenting cells. Current methods of identifying activated CD8+ T cells are laborious, time-consuming and expensive due to the extensive list of required reagents. Here, we demonstrate an optical imaging approach featuring quantitative phase imaging to distinguish activated CD8+ T cells from naïve CD8+ T cells in a rapid and reagent-free manner. We measured the dry mass of live cells and employed transport-based morphometry to better understand their differential morphological attributes. Our results reveal that, upon activation, the dry cell mass of T cells increases significantly in comparison to that of unstimulated cells. By employing deep learning formalism, we are able to accurately predict the population ratios of unknown mixed population based on the acquired quantitative phase images. We envision that, with further refinement, this label-free method of T cell phenotyping will lead to a rapid and cost-effective platform for assaying T cell responses to candidate antigens in the near future.

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.

Integration of diffraction phase microscopy and Raman imaging for label-free morpho-molecular assessment of live cells.

Label-free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label-free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho-molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr-path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi-wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label-free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.

Expanding and characterizing esophageal epithelial cells obtained from children with eosinophilic esophagitis.

BACKGROUND: The role of epithelial cells in eosinophilic esophagitis (EoE) is not well understood. In this study, our aim was to isolate, culture, and expand esophageal epithelial cells obtained from patients with or without EoE and characterize differences observed over time in culture. METHODS: Biopsies were obtained at the time of endoscopy from children with EoE or suspected to have EoE. We established patient-derived esophageal epithelial cell (PDEEC) lines utilizing conditional reprogramming methods. We determined integrin profiles, gene expression, MHC class II expression, and reactivity to antigen stimulation. RESULTS: The PDEECs were found to maintain their phenotype over several passages. There were differences in integrin profiles and gene expression levels in EoE-Active compared to normal controls and EoE-Remission patients. Once stimulated with antigens, PDEECs express MHC class II molecules on their surface, and when co-cultured with autologous T-cells, there is increased IL-6 and TNF-α secretion in EoE-Active patients vs. controls. CONCLUSION: We are able to isolate, culture, and expand esophageal epithelial cells from pediatric patients with and without EoE. Once stimulated with antigens, these cells express MHC class II molecules and behave as non-professional antigen-presenting cells. This method will help us in developing an ex vivo, individualized, patient-specific model for diagnostic testing for causative antigens.

Production of high purity alveolar-like cells from iPSCs through depletion of uncommitted cells after AFE induction.

Protocols to differentiate induced pluripotent stem cells (iPSCs) into specialized cells are continually evolving. iPSCs can be differentiated to alveolar cells with protocols that focus on development, specifically by inducing differentiation into definitive endoderm (DE), anterior foregut endoderm (AFE) and then lung bud progenitor intermediaries. However, current protocols result in a relatively low yield of the desired alveolar cells. The aim of this study was to evaluate whether depleting uncommitted cells after AFE induction would have a beneficial effect on alveolar cell yield. iPSCs were differentiated on Matrigel-coated plates for 25days. At each stage, phenotype was assessed using flow cytometry, immunofluorescence and qRT-PCR. Additionally, samples were dissociated in trypsin following AFE induction to improve the purity of the cells for the subsequent lung differentiation phase. Finally, the efficacy of dissociating the samples was confirmed comparing the expression of markers indicative of pluripotency and apoptosis. The ability to differentiate iPSCs to DE was 96% and to AFE was 97% utilizing our current protocol. After depletion of uncommitted cells and 12 days in culture, the purity of lung bud progenitors was 99%. Finally, the percentage of alveolar types I and II at the end of differentiation was 74% as compared to 31% in control cultures that had not been depleted of uncommitted cells after AFE induction. In conclusion, depletion of uncommitted cells after AFE induction, improves terminal differentiation of alveolar cells from 31% to 74%.