Vi Luan Ha

Dynamic changes in intracellular ROS levels regulate airway basal stem cell homeostasis through Nrf2-dependent Notch signaling

Airways are exposed to myriad environmental and damaging agents such as reactive oxygen species (ROS), which also have physiological roles as signaling molecules that regulate stem cell function. However, the functional significance of both steady and dynamically changing ROS levels in different stem cell populations, as well as downstream mechanisms that integrate ROS sensing into decisions regarding stem cell homeostasis, are unclear. Here, we show in mouse and human airway basal stem cells (ABSCs) that intracellular flux from low to moderate ROS levels is required for stem cell self-renewal and proliferation. Changing ROS levels activate Nrf2, which activates the Notch pathway to stimulate ABSC self-renewal and an antioxidant program that scavenges intracellular ROS, returning overall ROS levels to a low state to maintain homeostatic balance. This redox-mediated regulation of lung stem cell function has significant implications for stem cell biology, repair of lung injuries, and diseases such as cancer.

Novel stem/progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential.

The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic‐ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self‐renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell‐based therapies for patients with airway diseases. STEM CELLS 2011;29:1283–1293

Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.

Smoking is the most important known risk factor for the development of lung cancer. Tobacco exposure results in chronic inflammation, tissue injury, and repair. A recent hypothesis argues for a stem/progenitor cell involved in airway epithelial repair that may be a tumor-initiating cell in lung cancer and which may be associated with recurrence and metastasis. We used immunostaining, quantitative real-time PCR, Western blots, and lung cancer tissue microarrays to identify subpopulations of airway epithelial stem/progenitor cells under steady-state conditions, normal repair, aberrant repair with premalignant lesions and lung cancer, and their correlation with injury and prognosis. We identified a population of keratin 14 (K14)-expressing progenitor epithelial cells that was involved in repair after injury. Dysregulated repair resulted in the persistence of K14+ cells in the airway epithelium in potentially premalignant lesions. The presence of K14+ progenitor airway epithelial cells in NSCLC predicted a poor prognosis, and this predictive value was strongest in smokers, in which it also correlated with metastasis. This suggests that reparative K14+ progenitor cells may be tumor-initiating cells in this subgroup of smokers with NSCLC.

Isolation and In Vitro Characterization of Basal and Submucosal Gland Duct Stem/Progenitor Cells from Human Proximal Airways

Basal cells and submucosal gland (SMG) duct cells have been isolated and shown to be stem/progenitor cell populations for the murine airway epithelium. However, methods for the isolation of basal and SMG duct cells from human airways have not been defined. We used an optimized two‐step enzyme digestion protocol to strip the surface epithelium from tracheal specimens separate from SMG cells, and we then sorted the basal and duct stem/progenitors using fluorescence‐activated cell sorting. We used nerve growth factor receptor, as well as a combination of CD166 and CD44, to sort basal cells and also used CD166 to isolate SMG duct cells. Sorted stem/progenitor cells were cultured to characterize their self‐renewal and differentiation ability. Both basal and SMG duct cells grew into spheres. Immunostaining of the spheres showed mostly dense spheres with little to no central lumen. The spheres expressed cytokeratins 5 and 14, with some mucus‐ and serous‐secreting cells. The sphere‐forming efficiency and the rate of growth of the spheres varied widely between patient samples and correlated with the degree of hyperplasia of the epithelium. We found that only aldehyde dehydrogenase (ALDH)hi basal and duct cells were capable of sphere formation. Global inhibition of ALDH, as well as specific inhibition of the ALDH2 isoform, inhibited self‐renewal of both basal and duct cells, thereby producing fewer and smaller spheres. In conclusion, we have developed methods to isolate basal and SMG duct cells from the surface epithelium and SMGs of human tracheas and have developed an in vitro model to characterize their self‐renewal and differentiation.

Repair and regeneration of tracheal surface epithelium and submucosal glands in a mouse model of hypoxic-ischemic injury

Background and objective:  The heterotopic syngeneic tracheal transplant mouse model is an acute hypoxic‐ischemic injury model that undergoes complete repair and regeneration. We hypothesized that the repair and regeneration process of the surface epithelium and submucosal glands would occur in a reproducible pattern that could be followed by the expression of specific markers of epithelial cell types.

Aldehyde Dehydrogenase Activity Enriches for Proximal Airway Basal Stem Cells and Promotes Their Proliferation

Both basal and submucosal gland (SMG) duct stem cells of the airway epithelium are capable of sphere formation in the in vitro sphere assay, although the efficiency at which this occurs is very low. We sought to improve this efficiency of sphere formation by identifying subpopulations of airway basal stem cells (ABSC) and SMG duct cells based on their aldehyde dehydrogenase (ALDH) activity. ALDH(hi) ABSCs and SMG duct cells were highly enriched for the population of cells that could make spheres, while the co-culture of ALDH(hi) differentiated cells with the ALDH(hi) ABSCs increased their sphere-forming efficiency. Specific ALDH agonists and antagonists were used to show that airway specific ALDH isozymes are important for ABSC proliferation. Pathway analysis of gene expression profiling of ALDH(hi) and ALDH(lo) ABSCs revealed a significant upregulation of the arachidonic acid (AA) metabolism pathway in ALDH(hi) ABSCs. We confirmed the importance of this pathway in the metabolism of proliferating ALDH(hi) ABSCs using bioenergetics studies as well as agonists and antagonists of the AA pathway. These studies could lead to the development of novel strategies for altering ABSC proliferation in the airway epithelium.

Isolation of basal cells and submucosal gland duct cells from mouse trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.