The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, drug screening and modeling of lung disease, and studies of human lung development. We established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. Type II alveolar epithelial cells generated were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing agonists to signaling pathways critical for early lung development in the mouse—retinoic acid, Wnt and BMP—recapitulated defects in corresponding genetic mouse knockouts. The capability of this protocol to generate most cell types of the respiratory system suggests its utility for deriving patient-specific therapeutic cells.

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modeling, drug discovery and regenerative medicine1. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants2, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs3. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis4,5, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.

4E-BP is an important regulator of overall translation levels in cells. By binding eIF4E, 4E-BP impairs recruitment of the 40S ribosomal subunit to the cap structure present at the 5-end of all eukaryotic cellular mRNAs. 4E-BP activity is controlled by TOR (Target of Rapamycin). 4E-BP has been studied extensively in cell culture; however, the biological role of 4E-BP in developing organisms is unclear to date. Since TOR has been shown to control tissue growth during animal development, 4E-BP has also been assumed to serve as a growth regulator. Here, we study the relevance of 4E-BP function for organismal development, and present evidence for an alternate view. We show that 4E-BP strongly affects fat metabolism in Drosophila. We suggest that 4E-BP works as a metabolic brake that is activated under conditions of environmental stress to control fat metabolism. 4E-BP mutants lack this regulation, reducing their ability to survive under unfavorable conditions. The cellular activity and mechanism of action of 4E-BP as a translation inhibitor have been extensively studied. 4E-BP can bind eIF4E, and block its normal function of recruiting the initiation complex (containing the 40S ribosomal subunit) to the m 7 GpppX cap structure present at the 5Ј-end of all eukaryotic cellular mRNAs (for review, see Gingras et al. 1999b). In the absence of this cap-dependent recruitment, most cellular mRNAs are poorly translated Muthukrishnan et al. 1975). Therefore, when 4E-BP is active, overall translation levels are strongly dampened. 4E-BP activity is regulated through phosphorylation by the protein kinase TOR (Hay and Sonenberg 2004). 4E-BP is phosphorylated in a complex manner on a large number of sites . Briefly, when TOR activity is low, 4E-BP is hypophosphorylated allowing it to bind efficiently to eIF4E and block translation. When TOR activity increases, it phosphorylates 4E-BP, causing its affinity for eIF4E to drop and allowing cap-dependent translation to occur. TOR activity is regulated by growth factor signaling (especially insulin/PI3K), by amino acid availability, and by the energy state of the cell (for review, see Hay and Sonenberg 2004). Recently, several reports have indicated that 4E-BP activity is also regulated at the transcriptional level: 4E-BP transcription has been reported to be inhibited by the stress-dependent kinases ERK and p38 (Rolli-Derkinderen et al. 2003) and positively regulated by the forkhead transcription factor FOXO (Junger et al. 2003;Puig et al. 2003).The in vivo relevance of 4E-BP's activity remains a topic of conjecture, since almost all studies on 4E-BP have been done in cell culture. These studies have explained how 4E-BP functions molecularly, but not under what circumstances a living organism uses 4E-BP activity, nor what the consequences are for the whole animal when 4E-BP activity is lacking. A knockout mouse for one of the three mammalian 4E-BP proteins, 4E-BP1, has been described. These mice are viable and have elevated metabolism and reduced adipose tissue (Tsukiyama-Kohara et al...

The activation of matriptase requires proteolytic cleavage at a canonical activation motif that converts the enzyme from a one-chain zymogen to an active, twochain protease. In this study, matriptase bearing a mutation in its catalytic triad was unable to undergo this activational cleavage, suggesting that the activating cleavage occurs via a transactivation mechanism where interaction between matriptase zymogen molecules leads to activation of the protease. Using additional point and deletion mutants, we showed that activation of matriptase requires proteolytic processing at Gly-149 in the SEA domain of the protease, glycosylation of the first CUB domain and the serine protease domain, and intact low density lipoprotein receptor class A domains. Its cognate inhibitor, hepatocyte growth factor activator inhibitor-1, may also participate in the activation of matriptase, based on the observation that matriptase activation did not occur when the protease was co-expressed with hepatocyte growth factor activator inhibitor-1 mutated in its low density lipoprotein receptor class A domain. These results suggest that besides matriptase catalytic activity, matriptase activation requires post-translational modification of the protease, intact noncatalytic domains, and its cognate inhibitor.

Summary We report an unexpected role for protease signaling in neural tube closure and formation of the central nervous system. Mouse embryos lacking protease-activated receptor 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, Par2 selectively along the line of closure. Ablation of Gi/z and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often co-expressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.

Color vision in Drosophila relies on the comparison between two color-sensitive photoreceptors, R7 and R8. Two types of ommatidia in which R7 and R8 contain different rhodopsins are distributed stochastically in the retina and appear to discriminate short (p-subset) or long wavelengths (y-subset). The choice between p and y fates is made in R7, which then instructs R8 to follow the corresponding fate, thus leading to a tight coupling between rhodopsins expressed in R7 and R8. Here, we show that warts, encoding large tumor suppressor (Lats) and melted encoding a PH-domain protein, play opposite roles in defining the yR 8 or pR8 fates. By interacting antagonistically at the transcriptional level, they form a bistable loop that insures a robust commitment of R8 to a single fate, without allowing ambiguity. This represents an unexpected postmitotic role for genes controlling cell proliferation (warts and its partner hippo and salvador) and cell growth (melted).

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of human pluripotent stem cells into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development and takes approximately 50 days. First, definitive endoderm is induced in the presence of high concentrations of Activin A. Subsequently, lung-biased anterior foregut endoderm is specified by sequential inhibition of BMP, TGF-β and Wnt signaling. Anterior foregut endoderm is then ventralized by applying Wnt, BMP, FGF and RA signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, c-AMP and glucocorticoid agonism. This protocol is conducted in defined conditions, does not involve genetic manipulation of the cells, and results in cultures where the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.

SUMMARY The pathogenesis of idiopathic pulmonary fibrosis (IPF), an intractable interstitial lung disease, is unclear. Recessive mutations in some genes implicated in Hermansky-Pudlak syndrome (HPS) cause HPS-associated interstitial pneumonia (HPSIP), a clinical entity that is similar to IPF. We previously reported that HPS1−/−embryonic stem cell-derived 3D lung organoids showed fibrotic changes. Here, we show that the introduction of all HPS mutations associated with HPSIP promotes fibrotic changes in lung organoids, while the deletion of HPS8, which is not associated with HPSIP, does not. Genome-wide expression analysis revealed the upregulation of interleukin-11 (IL-11) in epithelial cells from HPS mutant fibrotic organoids. IL-11 was detected predominantly in type 2 alveolar epithelial cells in end-stage IPF, but was expressed more broadly in HPSIP. Finally, IL-11 induced fibrosis in WT organoids, while its deletion prevented fibrosis in HPS4−/− organoids, suggesting IL-11 as a therapeutic target. hPSC-derived 3D lung organoids are, therefore, a valuable resource to model fibrotic lung disease.