Combined low-carbohydrate diet and long-term exercise in hypoxia in type 2 diabetes: A randomized controlled trial protocol to assess glycemic control, cardiovascular risk factors and body composition.

Background: Cardiovascular disease is the leading cause of mortality associated with diabetes, which is characterized by chronic hyperglycemia. Low-carbohydrate diet has gained popularity as an intervention in patients with type 2 diabetes mellitus, acting to improve glycemic profile and serum lipids. In its turn, exercise in hypoxia induces specific adaptations, mostly modulated via hypoxia-induced transcription factor signaling cascade, which increases with exposure to altitude, and promotes angiogenesis, glycogen supply, glucose tolerance, and raises GLUT-4 expression. Aim: Given that hyperglycemia decreases HIF-1α and it is better controlled when following a low-carbohydrate diet, this study aims to examine the hypothesis that a combination of both low-carbohydrate diet and chronic exercise in hypoxia in type 2 diabetes mellitus is associated with improved glycemic control and cardiovascular parameters, whose protocol is described. Methods: Patients with type 2 diabetes mellitus (n = 48) will be recruited and randomized into one of the three groups: (a) Control group: Control diet (low-fat and moderate-carbohydrate diet) + exercise in normoxia; (2) exercise in hypoxia group: Control diet + exercise in hypoxia; (3) intervention group: Low-carbohydrate diet (low-carbohydrate and high-fat diet) + exercise in hypoxia. Before and after 8 weeks of interventions, cardiopulmonary tests (Bruce protocol), body composition and blood pressure will be evaluated. Blood samples will be collected to measure hypoxia-induced transcription factor, C-reactive protein, glycemic and lipid profiles. Summary: This will be the first trial to examine the isolated and combined effect of chronic exercise in hypoxia and low-carbohydrate diet in type 2 diabetes mellitus. This trial will help to fill a significant research gap, guide future research and contribute to the combined nutrition and exercise approach to type 2 diabetes mellitus.

Early human lung immune cell development and its role in epithelial cell fate.

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1ß drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1ß-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.

The BREAK study protocol: Effects of intermittent energy restriction on adaptive thermogenesis during weight loss and its maintenance.

BACKGROUND: Adaptive thermogenesis, defined as the decrease in the energy expenditure components beyond what can be predicted by changes in body mass stores, has been studied as a possible barrier to weight loss and weight maintenance. Intermittent energy restriction (IER), using energy balance refeeds, has been pointed out as a viable strategy to reduce adaptive thermogenesis and improve weight loss efficiency (greater weight loss per unit of energy deficit), as an alternative to a continuous energy restriction (CER). Following a randomized clinical trial design, the BREAK Study aims to compare the effects of IER versus CER on body composition and in adaptive thermogenesis, and understand whether participants will successfully maintain their weight loss after 12 months. METHODS: Seventy-four women with obesity and inactive (20-45 y) will be randomized to 16 weeks of CER or IER (8x2 weeks of energy restriction interspersed with 7x1 week in energy balance). Both groups will start with 2 weeks in energy balance before energy restriction, followed by 16 weeks in energy restriction, then 8 weeks in energy balance and finally a 12-month weight maintenance phase. Primary outcomes are changes in fat-mass and adaptive thermogenesis after weight loss and weight maintenance. Secondary outcomes include weight loss, fat-free mass preservation, alterations in energy expenditure components, and changes in hormones (thyroid function, insulin, leptin, and cortisol). DISCUSSION: We anticipate that The BREAK Study will allow us to better understand adaptive thermogenesis during weight loss and weight maintenance, in women with obesity. These findings will enable evidence-based decisions for obesity treatment. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05184361.

Perspectives and practices of nutritionists on dietary supplements for elite soccer teams: a cross-sectional survey study.

Introduction and objectives: Dietary supplements are part of the nutritional strategies frequently applied in sports performance support. With growing research on this subject and high demand from athletes, nutritionists need to keep up to date with the latest evidence and utility of dietary supplements, particularly in real-world contexts. As information about the use of dietary supplements among elite soccer players is still scarce, this work aimed to know how nutritionists working with elite soccer teams perceive and use these substances in their daily practice. Methods: A questionnaire previously used to describe nutritionists' beliefs and attitudes regarding the use of dietary supplements in a clinical context was adapted for this study. The online questionnaire was addressed to nutritionists working with elite soccer teams from six European Leagues and Brazil, between November 2022 and February 2023. Results: Overall, the participants considered themselves well-trained (76.9%), knowledgeable (95.4%), and interested in dietary supplements (95.4%). The majority (70.8%) of the participants agreed or strongly agreed to recommend dietary supplements to soccer players. Personal usage of dietary supplements was associated with recommending supplements (p < 0.001), but no relationships were found with years of experience and academic level. Discussion: Nutritionists working with elite soccer players consider the use of dietary supplements for performance-enhancement purposes and not only to compensate for nutritional deficits, which might contribute to their higher interest, training and perceived knowledge about this topic. Participants recognize players' interest in dietary supplements, and are mindful of the safety and efficacy of these products. The present study suggests that nutritionists working with elite soccer teams are among the highest prescribers of dietary supplements, although personal usage is lower than that of nutritionists working in a clinical context.

Effects of dietary supplements on athletic performance in elite soccer players: a systematic review.

Dietary supplements are widely used among athletes, and soccer players are no exception. Nevertheless, evidence supporting the use of dietary supplements aiming to enhance performance in soccer is somewhat contradictory, scarce, or even nonexistent. Thus, the present study aimed to systematically review and synthesize the effects of dietary supplements on athletic performance (e.g. distance covered, sprinting, jump performance) in elite soccer players. Studies enrolling highly trained, elite, and world-class soccer players using dietary supplements were searched in MEDLINE/PubMed, Web of Science, Scopus, and EBSCO databases in June 2022. In total, 1043 studies were identified, and 18 met the eligibility criteria. The studies evaluated the impacts on athletic performance of several dietary supplements, including caffeine, creatine, protein, beverages with carbohydrates and electrolytes, tart cherry juice, nitrate-rich beetroot juice, sodium bicarbonate with minerals, yohimbine, and a proprietary nutraceutical blend. Caffeine supplementation in doses between 3 and 6 mg/kg of body mass may improve jump height and sprint ability, particularly in female players, but individual response to caffeine must be considered. Creatine may improve sprint, agility, and in female players, jump performance. Protein supplementation can improve sprint and jump performance between matches, especially if protein ingested from food is not up to recommendations. Beverages containing carbohydrates and electrolytes can be used as part of the strategies to achieve carbohydrate intake during training and match-days but used alone do not benefit athletic performance. Tart cherry juice might be useful for maintaining athletic performance after matches that produce higher force loss and exercise-induced muscle damage, although polyphenols from the diet might attenuate the effects of tart cherry supplementation. Nitrate-rich beetroot concentrate can attenuate performance decrease in the days following matches. Further investigation with sodium bicarbonate alone is necessary, as supplementation protocols with elite players included other substances. Finally, the available data does not support yohimbine supplementation or the use of Resurgex Plus® to improve athletic performance in elite soccer players. Still, more well-designed research with elite soccer players is needed to improve support and advice regarding the use of dietary supplements for athletic performance enhancement.

Adding protein to a carbohydrate pre-exercise beverage does not influence running performance and metabolism.

BACKGROUND: To analyze whether pre-exercise CHO+PRO vs. CHO intake distinctly influences running performance and metabolic biomarkers along a various of exercise intensities. METHODS: In a randomized, double blind, counterbalanced, crossover and placebo control design, 10 middle distance runners were tested in 3 occasions. After 10 h of fasting, participants ingested isovolumic beverages (0.75+0.25g·BW-1 of CHO+PRO, 1.0g·BW-1 of CHO and placebo control) 30 min before a treadmill running incremental protocol of 4 min steps until exhaustion. Venous blood was collected at fasting, 30 min after beverage ingestion and after the 3rd and 7th running steps. Oxygen uptake-related variables, including respiratory exchange ratio, heart rate, plasma glucose, insulin, glucagon, free fatty acids, blood lactate concentrations, gastrointestinal discomfort and rate of perceived exertion were measured. RESULTS: The addition of PRO to CHO had no influence on the measured variables, which did not differ between conditions along all incremental protocol intensities. The intake of CHO+PRO (compared to CHO) tended to decrease glycemia (106.5±21.3 vs. 113.6±26.5) and to increase insulinemia (14.4±15.1 vs. 12.7±10.8) at intensities close to maximum oxygen uptake. CONCLUSIONS: The addition of PRO to a pre-exercise CHO beverage had no impact on running performance and related metabolic variables at a wide spectrum of exercise intensities.

Toll-like receptor 2 orchestrates a tumor suppressor response in non-small cell lung cancer.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

Cell-intrinsic differences between human airway epithelial cells from children and adults.

The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells, and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium was similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony formation ability, sustained in vitro growth, and outcompeted adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states.

Between-devices agreement in obtaining raw bioelectrical parameters after a lifestyle intervention targeting weight loss in former athletes.

It is unclear if different bioelectrical impedance (BI) devices provide similar results regarding raw parameters [Resistance (R), Reactance (Xc), Phase Angle (PhA), and Impedance (Z)] for the same population/individual undergoing a weight loss intervention. The aim was to evaluate the cross-sectional and longitudinal agreement of raw data obtained by two BI devices in former athletes with overweight/obesity. Fifty-nine participants [mean (SD): 43.5 (9.2) years, 30.5 (4.0) kg/m2, 42% females] were included. All the assessments were performed before and after a 4-months lifestyle intervention targeting weight loss (WL). BI parameters were assessed at 50 kHz by two devices: a BI spectroscopy (Xitron Technologies, 4200B, San Diego, USA) and a phase-sensitive single-frequency device (BIA 101 AKERN, Florence, Italy). Cross-sectionally, BIS provided lower mean values for all parameters (0.4% for R, 1.6% for Xc, 1.0% for PhA and 0.4% for Z, p <0.001) compared to SF-BIA. In individuals with a WL≥2.5% (n =18), no longitudinal differences were found in any of the raw parameters between devices (p≥0.128) and there was no proportional bias (p≥0.408). Despite small baseline differences in raw BI parameters, both devices agreed in tracking changes over time at the group level but interpretation should be careful at the individual level.

A Novel Plant-Based Protein Has Similar Effects Compared to Whey Protein on Body Composition, Strength, Power, and Aerobic Performance in Professional and Semi-Professional Futsal Players.

Introduction: The effects of dietary protein on body composition and physical performance seemingly depend on the essential amino acid profile of the given protein source, although controversy exists about whether animal protein sources may possess additional anabolic properties to plant-based protein sources. Purpose: To compare the effects of a novel plant-based protein matrix and whey protein supplementation on body composition, strength, power, and endurance performance of trained futsal players. Methods: Fifty male futsal players were followed during 8 weeks of supplementation, with 40 completing the study either with plant-based protein (N = 20) or whey protein (N = 20). The following measures were assessed: bone mineral content, lean body mass, and fat mass; muscle thickness of the rectus femoris; total body water; blood glucose, hematocrit, C-reactive protein, aspartate aminotransferase, alanine aminotransferase, creatine kinase, creatinine, and estimated glomerular filtration rate; salivary cortisol; maximal strength and 1-RM testing of the back squat and bench press exercises; muscle power and countermovement jump; VO2max and maximal aerobic speed. Subjects were asked to maintain regular dietary habits and record dietary intake every 4 weeks through 3-day food records. Results: No differences in any variable were observed between groups at baseline or pre- to post-intervention. Moreover, no time*group interaction was observed in any of the studied variables, and a time effect was only observed regarding fat mass reduction. Conclusions: Supplementing with either a novel plant-based protein matrix or whey protein did not affect any of the variables assessed in high-level futsal players over 8 wks. These results suggest that whey protein does not possess any unique anabolic properties over and above those of plant-based proteins when equated to an essential amino acid profile in the population studied. Furthermore, when consuming a daily protein intake >1.6 g/kg BW.day-1, additional protein supplementation does not affect body composition or performance in trained futsal players, regardless of protein type/source.

Portuguese Football Federation consensus statement 2020: nutrition and performance in football.

Nutrition is an undeniable part of promoting health and performance among football (soccer) players. Nevertheless, nutritional strategies adopted in elite football can vary significantly depending on culture, habit and practical constraints and might not always be supported by scientific evidence. Therefore, a group of 28 Portuguese experts on sports nutrition, sports science and sports medicine sought to discuss current practices in the elite football landscape and review the existing evidence on nutritional strategies to be applied when supporting football players. Starting from understanding football's physical and physiological demands, five different moments were identified: preparing to play, match-day, recovery after matches, between matches and during injury or rehabilitation periods. When applicable, specificities of nutritional support to young athletes and female players were also addressed. The result is a set of practical recommendations that gathered consensus among involved experts, highlighting carbohydrates periodisation, hydration and conscious use of dietary supplements.

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non-small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. SIGNIFICANCE: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355.

Immune Surveillance in Clinical Regression of Preinvasive Squamous Cell Lung Cancer.

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.

Sestrins induce natural killer function in senescent-like CD8+ T cells.

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8+ T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27-CD28-CD8+ T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27-CD28-CD8+ T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27-CD28-CD8+ T cells to acquire a broad-spectrum, innate-like killing activity.

Behavioural and psychological pretreatment predictors of short- and long-term weight loss among women with overweight and obesity.

PURPOSE: This study aims at identifying behavioural and psychological pretreatment predictors of 12- and 36-month weight loss in women with overweight/obesity enrolled in a behavioural weight management intervention. METHODS: A sample of 221 women participated in a randomized controlled trial on weight management (n12 month = 184; n36 month = 156). Multiple linear regressions were used to identify pretreatment predictors of successful weight loss, separately for intervention and control groups. Completers-only and baseline observation carried forward analyses were performed. This study is a secondary analysis of data from the 'Promotion of Exercise and Health in Obesity' randomized controlled trial. RESULTS: Fewer weight loss attempts in the last year positively predicted weight loss at 12 months in the intervention group, explaining 6% of the variance. At 36 months, in the intervention group, 20.2% of the variance in weight change was explained by lower eating disinhibition and higher weight-related quality of life in completers-only analyses, while baseline observation carried forward analyses explained only 9.8% of the variance in weight change via higher self-esteem and lower weight loss expectations. In the control group, higher exercise self-efficacy and a more internal weight locus of control predicted weight loss at 36 months, explaining 13.9% of the variance (completers-only analyses). CONCLUSIONS: Previous weight loss attempts were identified as the most efficient pretreatment predictor of 12-month weight loss. Eating disinhibition, weight-related quality of life, self-esteem, weight loss expectations, exercise self-efficacy, and weight locus of control seem to be key factors for long-term success. LEVEL OF EVIDENCE: Level I, randomized controlled trial. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00513084.

Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution.

The human bronchial epithelium is composed of multiple distinct cell types that cooperate to defend against environmental insults. While studies have shown that smoking alters bronchial epithelial function and morphology, its precise effects on specific cell types and overall tissue composition are unclear. We used single-cell RNA sequencing to profile bronchial epithelial cells from six never and six current smokers. Unsupervised analyses led to the characterization of a set of toxin metabolism genes that localized to smoker ciliated cells, tissue remodeling associated with a loss of club cells and extensive goblet cell hyperplasia, and a previously unidentified peri-goblet epithelial subpopulation in smokers who expressed a marker of bronchial premalignant lesions. Our data demonstrate that smoke exposure drives a complex landscape of cellular alterations that may prime the human bronchial epithelium for disease.

Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions.

The molecular alterations that occur in cells before cancer is manifest are largely uncharted. Lung carcinoma in situ (CIS) lesions are the pre-invasive precursor to squamous cell carcinoma. Although microscopically identical, their future is in equipoise, with half progressing to invasive cancer and half regressing or remaining static. The cellular basis of this clinical observation is unknown. Here, we profile the genomic, transcriptomic, and epigenomic landscape of CIS in a unique patient cohort with longitudinally monitored pre-invasive disease. Predictive modeling identifies which lesions will progress with remarkable accuracy. We identify progression-specific methylation changes on a background of widespread heterogeneity, alongside a strong chromosomal instability signature. We observed mutations and copy number changes characteristic of cancer and chart their emergence, offering a window into early carcinogenesis. We anticipate that this new understanding of cancer precursor biology will improve early detection, reduce overtreatment, and foster preventative therapies targeting early clonal events in lung cancer.

The role of electrostatics in TrxR electron transfer mechanism: A computational approach.

Thioredoxin reductase (TrxR) is an important enzyme in the control of the intracellular reduced redox environment. It transfers electrons from NADPH to several molecules, including its natural partner, thioredoxin. Although there is a generally accepted model describing how the electrons are transferred along TrxR, which involves a flexible arm working as a "shuttle," the molecular details of such mechanism are not completely understood. In this work, we use molecular dynamics simulations with Poisson-Boltzmann/Monte Carlo pKa calculations to investigate the role of electrostatics in the electron transfer mechanism. We observed that the combination of redox/protonation states of the N-terminal (FAD and Cys59/64) and C-terminal (Cys497/Selenocysteine498) redox centers defines the preferred relative positions and allows for the flexible arm to work as the desired "shuttle." Changing the redox/ionization states of those key players, leads to electrostatic triggers pushing the arm into the pocket when oxidized, and pulling it out, once it has been reduced. The calculated pKa values for Cys497 and Selenocysteine498 are 9.7 and 5.8, respectively, confirming that the selenocysteine is indeed deprotonated at physiological pH. This can be an important advantage in terms of reactivity (thiolate/selenolate are more nucleophilic than thiol/selenol) and ability to work as an electrostatic trigger (the "shuttle" mechanism) and may be the reason why TrxR uses selenium instead of sulfur. Proteins 2016; 84:1836-1843. © 2016 Wiley Periodicals, Inc.

pK(a) Values of Titrable Amino Acids at the Water/Membrane Interface.

Peptides and proteins protonation equilibrium is strongly influenced by its surrounding media. Remarkably, until now, there have been no quantitative and systematic studies reporting the pK(a) shifts in the common titrable amino acids upon lipid membrane insertion. Here, we applied our recently developed CpHMD-L method to calculate the pK(a) values of titrable amino acid residues incorporated in Ala-based pentapeptides at the water/membrane interface. We observed that membrane insertion leads to desolvation and a clear stabilization of the neutral forms, and we quantified the increases/decreases of the pK(a) values in the anionic/cationic residues along the membrane normal. This work highlights the importance of properly modeling the protonation equilibrium in peptides and proteins interacting with membranes using molecular dynamics simulations.

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering.

RATIONALE: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. OBJECTIVES: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. METHODS: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. MEASUREMENTS AND MAIN RESULTS: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. CONCLUSIONS: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.