Alveolar type (AT)I and ATII cells are central to maintaining normal alveolar fluid homeostasis. exhibit indefinite cell proliferation that resembled a “conditionally reprogrammed cell” phenotype. Using this coculture system we found that primary human ATII cells (ATII cell proliferation barrier while retaining cell-specific functional properties. This work will allow for a significant increase in studies designed to elucidate ATII cell function with the goal of accelerating the development of novel therapies for alveolar diseases. alveolar type II cell proliferation barrier while retaining Polyphyllin VII cell-specific functional properties. This technique increases the supply of human primary alveolar type II cells and allows for additional studies to be performed focused on important biological and functional processes relevant to the physiology and pathophysiology of alveolar lung diseases. It is critical that this alveolar epithelium maintains a thin liquid layer lining to promote proper surface tension gas exchange and protection from inhaled toxins and pathogens. Alveolar type (AT)I and ATII epithelial cells are damaged during inflammation associated with acute lung injury (ALI) and acute respiratory distress syndrome (1-4). Resolution of ALI through removal of alveolar edema fluid has been the focus of many and studies (5-7). However one consistent limitation of these studies has been that ATII cells quickly transdifferentiate (i.e. drop their ATII cell-specific markers and gain “ATI-like”-specific markers) and do not proliferate under traditional culture conditions (8-10). The failure of human primary ATII cells to proliferate greatly limits the number of studies designed to elucidate the pathogenesis of human alveolar diseases. Previous attempts have been made to promote ATII cell Polyphyllin VII proliferation for extended periods suggesting that KGF (added or presumably secreted by fibroblasts) was in part responsible for retaining ATII cell differentiation (12). These studies also reported that KGF could stimulate ATII cell proliferation (14 16 17 which could be antagonized by the transforming growth factor (TGF)-β (18). However a precise genetic characterization of the proliferating cells was not reported nor were KGF-treated ATII cells amendable to serial passage and expansion. Recent published work has demonstrated the ability of human cells (e.g. keratinocytes and airway epithelial cells) to proliferate indefinitely without the transduction of exogenous viral or cellular genes by addition of a pharmacological inhibitor Polyphyllin VII of the Rho kinase signaling pathway (Y-27632 “Y”) (19-22) in the presence of mouse feeder cells (23). These “conditionally reprogrammed cells” Polyphyllin VII were shown to exhibit a stem cell-like phenotype with an up-regulation of adult stem cell genes (e.g. α6/β1 integrin ΔNp63α) (24). Passaged CRCs could revert to their initial epithelial cell phenotype on removal of the feeder cells and “Y.” This recently developed cell culture technology has the potential to accelerate alveolar epithelial research by expanding the availability of human alveolar cells and thus increasing cell-specific studies designed to target therapeutics against ALI and acute respiratory distress syndrome. We tested the hypothesis that primary human ATII cells cocultured with feeder cells and “Y” would exhibit a break in the ATII cell proliferation barrier and undergo serial passage and expansion. Due to the ATI transdifferentiation that affects most ATII cells < 0.05. Mouse monoclonal to CD80 Polyphyllin VII Results Feeder Cells and Rho Kinase Inhibitor Induce ATII Cell Proliferation and Growth coculture cell model in which isolated primary human ATII cells from rejected donated lungs were cultured with irradiated feeder cells (1:3) and “Y” and expanded on rat tail collagen-1-coated plastic dishes. To determine the contribution of each component to the culture process ATII cells were plated with base media alone (Physique 1Bi) with “Y” (Physique 1Bii) with feeder cells (Physique 1Biii) or with the combination of feeder cells and “Y” (Physique 1Biv) and imaged 4 days after seeding. Human ATII cells plated with base media alone did not proliferate and formed large round flat “ATI-like” cells as previously reported (25). In contrast ATII cells plated with the combination of feeder cells and “Y” generated islands of epithelial-like colonies surrounded by feeder cells. ATII cells plated with feeder cells or “Y” alone did not establish the.