Craig R. Rackley

Gas exchange in the lung occurs within alveoli, air-filled sacs composed of type 2 and type 1 epithelial cells (AEC2s and AEC1s), capillaries, and various resident mesenchymal cells. Here, we use a combination of in vivo clonal lineage analysis, different injury/repair systems, and in vitro culture of purified cell populations to obtain new information about the contribution of AEC2s to alveolar maintenance and repair. Genetic lineage-tracing experiments showed that surfactant protein C-positive (SFTPC-positive) AEC2s self renew and differentiate over about a year, consistent with the population containing long-term alveolar stem cells. Moreover, if many AEC2s were specifically ablated, high-resolution imaging of intact lungs showed that individual survivors undergo rapid clonal expansion and daughter cell dispersal. Individual lineage-labeled AEC2s placed into 3D culture gave rise to self-renewing "alveolospheres," which contained both AEC2s and cells expressing multiple AEC1 markers, including HOPX, a new marker for AEC1s. Growth and differentiation of the alveolospheres occurred most readily when cocultured with primary PDGFRα⁺ lung stromal cells. This population included lipofibroblasts that normally reside close to AEC2s and may therefore contribute to a stem cell niche in the murine lung. Results suggest that a similar dynamic exists between AEC2s and mesenchymal cells in the human lung.

Airspaces of the lung are lined by an epithelium whose cellular composition changes along the proximal-to-distal axis to meet local functional needs for mucociliary clearance, hydration, host defense, and gas exchange. Advances in cell isolation, in vitro culture techniques, and genetic manipulation of animal models have increased our understanding of the development and maintenance of the pulmonary epithelium. This review discusses basic cellular mechanisms that regulate establishment of the conducting airway and gas exchange systems as well as the functional maintenance of the epithelium during postnatal life.

Mechanisms that regulate regional epithelial cell diversity and pathologic remodeling in airways are poorly understood. We hypothesized that regional differences in cell composition and injury-related tissue remodeling result from the type and composition of local progenitors. We used surface markers and the spatial expression pattern of an SFTPC-GFP transgene to subset epithelial progenitors by airway region. Green fluorescent protein (GFP) expression ranged from undetectable to high in a proximal-to-distal gradient. GFP(hi) cells were subdivided by CD24 staining into alveolar (CD24(neg)) and conducting airway (CD24(low)) populations. This allowed for the segregation of three types of progenitors displaying distinct clonal behavior in vitro. GFP(neg) and GFP(low) progenitors both yielded lumen containing colonies but displayed transcriptomes reflective of pseudostratified and distal conducting airways, respectively. CD24(low)GFP(hi) progenitors were present in an overlapping distribution with GFP(low) progenitors in distal airways, yet expressed lower levels of Sox2 and expanded in culture to yield undifferentiated self-renewing progeny. Colony-forming ability was reduced for each progenitor cell type after in vivo bleomycin exposure, but only CD24(low) GFP(hi) progenitors showed robust expansion during tissue remodeling. These data reveal intrinsic differences in the properties of regional progenitors and suggest that their unique responses to tissue damage drive local tissue remodeling.

Background Assessing functional impairment, therapeutic response and disease progression in patients with idiopathic pulmonary fibrosis (IPF) continues to be challenging. Hyperpolarized 129 Xe MRI can address this gap through its unique capability to image gas transfer three-dimensionally from airspaces to interstitial barrier tissues to red blood cells (RBCs). This must be validated by testing the degree to which it correlates with pulmonary function tests (PFTs) and CT scores, and its spatial distribution reflects known physiology and patterns of disease. Methods 13 healthy individuals (33.6±15.7 years) and 12 patients with IPF (66.0±6.4 years) underwent 129 Xe MRI to generate three-dimensional quantitative maps depicting the 129 Xe ventilation distribution, its uptake in interstitial barrier tissues and its transfer to RBCs. For each map, mean values were correlated with PFTs and CT fibrosis scores, and their patterns were tested for the ability to depict functional gravitational gradients in healthy lung and to detect the known basal and peripheral predominance of disease in IPF. Results 129 Xe MRI depicted functional impairment in patients with IPF, whose mean barrier uptake increased by 188% compared with the healthy reference population. 129 Xe MRI metrics correlated poorly and insignificantly with CT fibrosis scores but strongly with PFTs. Barrier uptake and RBC transfer both correlated significantly with diffusing capacity of the lungs for carbon monoxide (r=−0.75, p<0.01 and r=0.72, p<0.01), while their ratio (RBC/barrier) correlated most strongly (r=0.94, p<0.01). RBC transfer exhibited significant anterior-posterior gravitational gradients in healthy volunteers, but not in IPF, where it was significantly impaired in the basal (p=0.02) and subpleural (p<0.01) lung. Conclusions Hyperpolarized 129 Xe MRI is a rapid and well-tolerated exam that provides region-specific quantification of interstitial barrier thickness and RBC transfer efficiency. With further development, it could become a robust tool for measuring disease progression and therapeutic response in patients with IPF, sensitively and non-invasively.

Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with the bld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walked 720 kb and identified a 13.2-kb fragment that contains epsilon-tubulin and rescues the Bld2 defects. The bld2-1 allele has a premature stop codon and intragenic revertants replace the stop codon with glutamine, glutamate, or lysine. Polyclonal antibodies to epsilon-tubulin show peripheral labeling of full-length basal bodies and centrioles. Thus, epsilon-tubulin is encoded by the BLD2 allele and epsilon-tubulin plays a role in basal body/centriole morphogenesis.