Defining the contribution of Troy-positive progenitor cells to the mouse esophageal epithelium.

Progenitor cells adapt their behavior in response to tissue demands. However, the molecular mechanisms controlling esophageal progenitor decisions remain largely unknown. Here, we demonstrate the presence of a Troy (Tnfrsf19)-expressing progenitor subpopulation localized to defined regions along the mouse esophageal axis. Lineage tracing and mathematical modeling demonstrate that Troy-positive progenitor cells are prone to undergoing symmetrical fate choices and contribute to esophageal tissue homeostasis long term. Functionally, TROY inhibits progenitor proliferation and enables commitment to differentiation without affecting fate symmetry. Whereas Troy expression is stable during esophageal homeostasis, progenitor cells downregulate Troy in response to tissue stress, enabling proliferative expansion of basal cells refractory to differentiation and reestablishment of tissue homeostasis. Our results demonstrate functional, spatially restricted progenitor heterogeneity in the esophageal epithelium and identify how dynamic regulation of Troy coordinates tissue generation.

Distinct roles for SOX2 and SOX21 in differentiation, distribution and maturation of pulmonary neuroendocrine cells.

Pulmonary neuroendocrine (NE) cells represent a small population in the airway epithelium, but despite this, hyperplasia of NE cells is associated with several lung diseases, such as congenital diaphragmatic hernia and bronchopulmonary dysplasia. The molecular mechanisms causing the development of NE cell hyperplasia remains poorly understood. Previously, we showed that the SOX21 modulates the SOX2-initiated differentiation of epithelial cells in the airways. Here, we show that precursor NE cells start to develop in the SOX2 + SOX21 + airway region and that SOX21 suppresses the differentiation of airway progenitors to precursor NE cells. During development, clusters of NE cells start to form and NE cells mature by expressing neuropeptide proteins, such as CGRP. Deficiency in SOX2 resulted in decreased clustering, while deficiency in SOX21 increased both the numbers of NE ASCL1 + precursor cells early in development, and the number of mature cell clusters at E18.5. In addition, at the end of gestation (E18.5), a number of NE cells in Sox2 heterozygous mice, did not yet express CGRP suggesting a delay in maturation. In conclusion, SOX2 and SOX21 function in the initiation, migration and maturation of NE cells.

SOX2 and SOX21 in Lung Epithelial Differentiation and Repair.

The lung originates from the ventral foregut and develops into an intricate branched structure of airways, alveoli, vessels and support tissue. As the lung develops, cells become specified and differentiate into the various cell lineages. This process is controlled by specific transcription factors, such as the SRY-related HMG-box genes SOX2 and SOX21, that are activated or repressed through intrinsic and extrinsic signals. Disturbances in any of these processes during the development of the lung may lead to various pediatric lung disorders, such as Congenital Diaphragmatic Hernia (CDH), Congenital Pulmonary Airway Malformation (CPAM) and Broncho-Pulmonary Dysplasia (BPD). Changes in the composition of the airways and the alveoli may result in reduced respiratory function and eventually lead to chronic lung disorders. In this concise review, we describe different intrinsic and extrinsic cellular processes required for proper differentiation of the epithelium during development and regeneration, and the influence of the microenvironment on this process with special focus on SOX2 and SOX21.

Lung epithelium development and airway regeneration.

The lung is composed of a highly branched airway structure, which humidifies and warms the inhaled air before entering the alveolar compartment. In the alveoli, a thin layer of epithelium is in close proximity with the capillary endothelium, allowing for an efficient exchange of oxygen and carbon dioxide. During development proliferation and differentiation of progenitor cells generates the lung architecture, and in the adult lung a proper function of progenitor cells is needed to regenerate after injury. Malfunctioning of progenitors during development results in various congenital lung disorders, such as Congenital Diaphragmatic Hernia (CDH) and Congenital Pulmonary Adenomatoid Malformation (CPAM). In addition, many premature neonates experience continuous insults on the lung caused by artificial ventilation and supplemental oxygen, which requires a highly controlled mechanism of airway repair. Malfunctioning of airway progenitors during regeneration can result in reduction of respiratory function or (chronic) airway diseases. Pathways that are active during development are frequently re-activated upon damage. Understanding the basic mechanisms of lung development and the behavior of progenitor cell in the ontogeny and regeneration of the lung may help to better understand the underlying cause of lung diseases, especially those occurring in prenatal development or in the immediate postnatal period of life. This review provides an overview of lung development and the cell types involved in repair of lung damage with a focus on the airway.

Disease modeling following organoid-based expansion of airway epithelial cells.

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion [three-dimensional (3D)] can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to 1) cellular composition, 2) response to cigarette smoke exposure, and 3) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated, and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. In addition, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.

SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways.

SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium, SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium.

A novel method for expansion and differentiation of mouse tracheal epithelial cells in culture.

Air-liquid interface (ALI) cultures of mouse tracheal epithelial cells (MTEC) are a well-established model to study airway epithelial cells, but current methods require large numbers of animals which is unwanted in view of the 3R principle and introduces variation. Moreover, stringent breeding schemes are frequently needed to generate sufficient numbers of genetically modified animals. Current protocols do not incorporate expansion of MTEC, and therefore we developed a protocol to expand MTEC while maintaining their differentiation capacity. MTEC were isolated and expanded using the ROCK inhibitor Y-27632 in presence or absence of the γ-secretase inhibitor DAPT, a Notch pathway inhibitor. Whereas MTEC proliferated without DAPT, growth rate and cell morphology improved in presence of DAPT. ALI-induced differentiation of expanded MTEC resulted in an altered capacity of basal cells to differentiate into ciliated cells, whereas IL-13-induced goblet cell differentiation remained unaffected. Ciliated cell differentiation improved by prolonging the ALI differentiation or by adding DAPT, suggesting that basal cells retain their ability to differentiate. This technique using expansion of MTEC and subsequent ALI differentiation drastically reduces animal numbers and costs for in vitro experiments, and will reduce biological variation. Additionally, we provide novel insights in the dynamics of basal cell populations in vitro.

Autophagy linked FYVE (Alfy/WDFY3) is required for establishing neuronal connectivity in the mammalian brain.

The regulation of protein degradation is essential for maintaining the appropriate environment to coordinate complex cell signaling events and to promote cellular remodeling. The Autophagy linked FYVE protein (Alfy), previously identified as a molecular scaffold between the ubiquitinated cargo and the autophagic machinery, is highly expressed in the developing central nervous system, indicating that this pathway may have yet unexplored roles in neurodevelopment. To examine this possibility, we used mouse genetics to eliminate Alfy expression. We report that this evolutionarily conserved protein is required for the formation of axonal tracts throughout the brain and spinal cord, including the formation of the major forebrain commissures. Consistent with a phenotype reflecting a failure in axon guidance, the loss of Alfy in mice disrupts localization of glial guidepost cells, and attenuates axon outgrowth in response to Netrin-1. These findings further support the growing indication that macroautophagy plays a key role in the developing CNS.

Regeneration of the lung: Lung stem cells and the development of lung mimicking devices.

Inspired by the increasing burden of lung associated diseases in society and an growing demand to accommodate patients, great efforts by the scientific community produce an increasing stream of data that are focused on delineating the basic principles of lung development and growth, as well as understanding the biomechanical properties to build artificial lung devices. In addition, the continuing efforts to better define the disease origin, progression and pathology by basic scientists and clinicians contributes to insights in the basic principles of lung biology. However, the use of different model systems, experimental approaches and readout systems may generate somewhat conflicting or contradictory results. In an effort to summarize the latest developments in the lung epithelial stem cell biology, we provide an overview of the current status of the field. We first describe the different stem cells, or progenitor cells, residing in the homeostatic lung. Next, we focus on the plasticity of the different cell types upon several injury-induced activation or repair models, and highlight the regenerative capacity of lung cells. Lastly, we summarize the generation of lung mimics, such as air-liquid interface cultures, organoids and lung on a chip, that are required to test emerging hypotheses. Moreover, the increasing collaboration between distinct specializations will contribute to the eventual development of an artificial lung device capable of assisting reduced lung function and capacity in human patients.