Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air‐liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic‐activated cell sorting system, and the generation of adult‐like airway epithelia through air‐liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air‐liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture

Human PSC (hPSC) differentiations can capture developmental phenotypes and processes and are useful for studying fundamental biological mechanisms driving tissue morphogenesis and cell lineage development. Here, we show for the first time the temporal development of lung cell lineages using hPSC that model developmental milestones observed in primary tissue, the generation of renewable fetal lung epithelial organoids, and the functional utility of the lung models at different differentiation stages for Cystic fibrosis disease modeling. We first show the presence of hPSC-derived lung progenitor cells reminiscent of early trimester lung development and can capture a population enriched with basal stem cells that generates renewable airway organoids. Maturation and polarization in air liquid interface (ALI) generates additional epithelial cell lineages found in adult lung tissues including pulmonary neuroendocrine, brush, mature basal, ciliated and secretory cell types. Finally, pseudotime analysis of the integrated datasets from the fetal and ALI stages reveal the developmental trajectories of the cells as they emerge during differentiation. Overall, hPSC differentiation can capture aspects of human lung development and potentially provide important insight into congenital causes of diseases.

Background: Physical exertion places a demand on cellular metabolic reserves. By providing key metabolic intermediates to mitochondria before, during and after training, optimal cellular homeostasis and metabolic replenishing can be augmented, enabling optimal maintenance and recovery of cellular reserves aimed at improving aerobic cellular function. The purpose of this pilot study was to evaluate if the exercise work capacity and recovery can be augmented through the administration of a synergistic blend of key metabolic intermediates which included: Citrate, Malate, Pyruvate and Vitamin C. Methods: A total of 14 participants volunteered to complete two identical training sessions; a Control session and a Supplemented session two weeks later. Three identical exercises, comprising 10 sets each, were included in the two sessions which included a 20 second work-set followed, by 90 seconds of rest. The number of repetitions, the repetition in which muscle fatigue and exercise induced muscle pain (burn) initiated, and peak muscle pain (burn) severity were recorded.Results: Nutritional intervention using tricarboxylic cycle intermediates provides a means for directly modulating mitochondrial performance expressed physiologically as improved work capacity and the ability to augment athletic performance. Concordantly, this study demonstrated improved recovery post-training minimizing delayed onset muscle soreness (DOMS).