Respir. (scRNA-seq) and computational pipelines are designed to compare samples both globally and at the population level. A sample of native rat tracheal epithelium is also evaluated by scRNA-seq like a control for designed epithelium. Overall, this work identifies platform-specific effects that support the use of designed models to achieve the most physiologic differential results Kynurenic acid in pulmonary epithelial regenerative applications. Graphical Abstract In Brief Greaney et al. compare pulmonary epithelial regeneration across multiple modalities (Rock et al., 2009). Recently, studies of severe lung injury possess exposed a basal-like, Tp63-lineage, bipotent progenitor cell that may migrate to alveoli and participate in the regeneration of distal-like epithelium unique from proximal phenotypes (Vaughan et al., 2015; Xi et al., Kynurenic acid 2017; Kumar et al., 2011; Ray et al., 2016), as well as other epithelial progenitors with related regenerative potential (Zacharias et al., 2018; Kim et al., 2005; Liu et al., 2019). This observed phenomenon suggests that environmental cues throughout the airway tree, such as localized matrix bioactivity, air flow exposure, or mechanical cues, may influence the fate of such basal-like cells. For that reason, a better understanding of these extracellular cues could be exploited to drive the differentiation of pulmonary epithelial progenitors in Kynurenic acid regenerative medicine. The potential of main BCs for such regenerative applications has been enhanced from the development of pharmacological growth techniques for epithelial cells. While pluripotent stem cell-derived epithelial progenitors find great power in developmental modeling (Huang et al., 2015; McCauley et al., 2018), pharmacologic growth facilitates the use of main adult BCs for regenerative medicine by enabling growth to higher cell numbers while maintaining plasticity, without the use of exogenous genetic reprogramming factors or irradiated feeder cells. By this method, the addition of small molecule inhibitors of PAK1-ROCK-myosin II and transforming growth element (TGF-) signaling results in a >1 trillion-fold growth of human being epithelial stem and progenitor cells from pores and skin, airway, mammary, and prostate glands (Zhang et al., 2018). The regenerative RGS capacity of various pulmonary epithelial cell populations is definitely often evaluated in any quantity of tradition platforms. One common method is growing solitary cells suspended in Matrigel, therefore permitting cells to clonally increase and organize into spheroids or organoids (Rock et al., 2009; Barkauskas et al., 2013; Lee et al., 2014; Nichane et al., 2017). Proximal airway epithelial cells produced at an air-liquid interface (ALI) on a filter insert tend to display strong ciliation and self-organize into a pseudostratified epithelial coating (Nichane et al., 2017; Whitcutt et al., 1988; Schoch et al., 2004; Randell et al., 2011). Culturing rat tracheal epithelial cells in the lumens of denuded rat tracheas offers demonstrated the inherent regional regenerative capacity of proximal epithelium (Liu et al., 1994). This regional specificity of proximal epithelial populations has been supported by two recent evaluations in designed lung constructs, which produced little evidence of functional alveolar repair (LaRanger et Kynurenic acid al., 2018; Gilpin et al., 2016). While it is well known the physical and bioactive environments of differentiation have a huge influence on stem cell fate (McBeath et al., 2004; Bissell et al., 1982), this concept has been understudied in the pulmonary Kynurenic acid field. With this statement, we demonstrate strong growth and differentiation of rat pharmacologically expanded basal cells (peBCs) and the evaluation of platform-specific effects on regenerative results. Using a solitary line of peBCs, we conduct a focused evaluation of differentiation across four tradition platforms, including organoids, ALIs, and tracheal and lung regeneration models. Furthermore, we use 10 Drop-Seq single-cell RNA sequencing (scRNA-seq) to evaluate peBC heterogeneity and differential results like a function of tradition platform, including global platform-based variability, as well as population-level comparisons. For this analysis, we developed computational pipelines for transcriptomic assessment between designed samples, relating these results to a native rat tracheal epithelium control. We conclude that peBC differentiation in designed tissue constructs results in more mature epithelial populations tradition protocol for peBCs on a decellularized trachea scaffold. Native rat tracheas were decellularized using a detergent-enzymatic method adapted from a whole-lung decellularization protocol (Balestrini et al., 2015). peBCs were cultured in the lumen of the designed trachea for 2 days to allow for cell adhesion, followed by luminal perfusion inside a custom bioreactor. After 4 weeks, the peBC created a pseudostratified epithelial coating and achieved full mucociliary differentiation resembling native tracheal epithelium (Number 3C). When cultured in these three platforms, peBC consistently created pseudostratified epithelium and mucociliary differentiation of varying examples of business and maturity. The expressions of Foxj1 (ciliated marker) and Muc5ac following a differentiation of multiple peBC lines were both significantly higher in designed tracheas than in organoid or ALI (Number S3)..