Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic H. pylori infection1–3. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis4, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells (hPSCs). We identified that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signaling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signaling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signaling and induction of epithelial proliferation. Together, these studies describe a novel and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.

Despite the global prevalence of gastric disease, there are few adequate models to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (PSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/β-catenin signaling in mouse embryos led to conversion of fundic to antral epithelium, while β-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signaling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. While hFGOs are a powerful new model for studying the development of the human fundus and its lineages, they also represent a critical new model system to study the molecular basis of human gastric physiology, pathophysiology, and drug discovery.

Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT, BMP, retinoic acid (RA) and hedgehog (Hh), but how these pathways interact in a gene regulatory network is less clear. Using Xenopus embryology, mouse genetics, and human ES cell cultures we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then it promotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally, RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis.

In this study, the culturability of indoor and outdoor airborne fungi was determined through longterm sampling (24-h) using a Button Personal Inhalable Aerosol Sampler. The air samples were collected during three seasons in six Cincinnati area homes that were free from moisture damage or visible mold. Cultivation and total microscopic enumeration methods were employed for the sample analysis. The geometric means of indoor and outdoor culturable fungal concentrations were 88 and 102 colony-forming units (CFU) m -3 , respectively, with a geometric mean of the I/O ratio equal to 0.66. Overall, 26 genera of culturable fungi were recovered from the indoor and outdoor samples. For total fungal spores, the indoor and outdoor geometric means were 211 and 605 spores m -3 , respectively, with a geometric mean of I/O ratio equal to 0.32. The identification revealed 37 fungal genera from indoor and outdoor samples based on the total spore analysis. Indoor and outdoor concentrations of culturable and total fungal spores showed significant correlations (r = 0.655, p<0.0001 and r = 0.633, p<0.0001, respectively). The indoor and outdoor median viabilities of fungi were 55% and 25%, respectively, which indicates that indoor environment provides more favorable survival conditions for the aerosolized fungi. Among the seasons, the highest indoor and outdoor culturability of fungi was observed in the fall. Cladosporium had a highest median value of culturability (38% and 33% for indoor and outdoor, respectively) followed by Aspergillus/ Penicillium (9% and 2%) among predominant genera of fungi. Increased culturability of fungi inside the homes may have important implications because of the potential increase in the release of allergens from viable spores and pathogenicity of viable fungi on immunocompromised individuals.

This field study investigated the relationship between indoor and outdoor concentrations of airborne actinomycetes, fungal spores, and pollen. Air samples were collected for 24 h with a button inhalable aerosol sampler inside and outside of six single-family homes located in the Cincinnati area (overall, 15 pairs of samples were taken in each home). The measurements were conducted during three seasons - spring and fall 2004, and winter 2005. The concentration of culturable actinomycetes was mostly below the detection limit. The median indoor/outdoor ratio (I/O) for actinomycetes was the highest: 2.857. The indoor of fungal and pollen concentrations followed the outdoor concentrations while indoor levels were mostly lower than the outdoor ones. The I/O ratio of total fungal spores (median=0.345) in six homes was greater than that of pollen grains (median=0.025). The low I/O ratios obtained for pollen during the peak ambient pollination season (spring) suggest that only a small fraction penetrated from outdoor to indoor environment. This is attributed to the larger size of pollen grains. Higher indoor concentration levels and variability in the I/O ratio observed for airborne fungi may be associated with indoor sources and/or higher outdoor-to-indoor penetration of fungal spores compared to pollen grains. Practical Implication This study addresses the relationship between indoor and outdoor concentrations of three different types of bio-aerosols, namely actinomycetes, fungal spores, and pollen grains. The results show that actinomycetes are rare in indoor and outdoor air in Midwest, USA. Exposure to pollen occurs mainly in the outdoor air even during peak pollen season. Unexpectedly high fungal spore concentrations were measured outdoors during winter. The presented pilot database on the inhalable levels of indoor and outdoor bio-aerosols can help apportion and better characterize the inhalation exposure to these bio-aerosols. Furthermore, the data can be incorporated into existing models to quantify the penetration of biological particles into indoor environments from outdoors.

Smaller-sized fungal fragments (<1 μm) may contribute to mold-related health effects. Previous laboratory-based studies have shown that the number concentration of fungal fragments can be up to 500 times higher than that of fungal spores, but this has not yet been confirmed in a field study due to lack of suitable methodology. We have recently developed a field-compatible method for the sampling and analysis of airborne fungal fragments. The new methodology was utilized for characterizing fungal fragment exposures in mold-contaminated homes selected in New Orleans, Louisiana and Southern Ohio. Airborne fungal particles were separated into three distinct size fractions: (i) >2.25 μm (spores); (ii) 1.05-2.25 μm (mixture); and (iii) < 1.0 μm (submicrometersized fragments). Samples were collected in five homes in summer and winter and analyzed for (1→3)-β-D-glucan. The total (1→3)-β-D-glucan varied from 0.2 to 16.0 ng m −3 . The ratio of (1→3)-β-D-glucan mass in fragment size fraction to that in spore size fraction (F/S) varied from 0.011 to 2.163. The mass ratio was higher in winter (average = 1.017) than in summer (0.227) coinciding with a lower relative humidity in the winter. Assuming a mass-based F/S-ratio=1 and the spore size = 3 μm, the corresponding number-based F/S-ratio (fragment number/spore number) would be 10 3 and 10 6 , for the fragment sizes of 0.3 and 0.03 μm, respectively. These results indicate that the actual (field) contribution of fungal fragments to the overall exposure may be very high, even much greater than that estimated in our earlier laboratory-based studies.

Characterizing the variation in bioaerosol concentrations is important for the estimation of health effects associated with bioaerosols and planning exposure assessment strategies. This investigation was conducted in order to develop a better understanding of exposure to fungal spores, pollen, and (1?3)-b-D-glucan, by determining the variations of their concentrations between and within homes. In the study, 24-h air sampling was performed on five consecutive days in four Cincinnati area homes. The samples (a total of 160) were taken simultaneously in four different rooms inside each home and at four different outside locations near the home using Button Personal Inhalable Aerosol Samplers. The relative sizes of the between-and within-home variability to the total variability were calculated for each outcome. The relative sizes of the betweenand within-home variability in indoor air ranged from 0.10 to 0.52 and 0.09 to 0.10, respectively. For outdoor air, the between-and within-home variability ranged from 0.27 to 0.50 and 0.09 to 0.10, respectively. Thus, the ranges of within-home variability, both indoors and outdoors, were much less than the variability between different homes. The results suggest that, if long-term sampling methods are employed to characterize the bioaerosol exposure for a population, the sampling should be repeated in a larger number of homes as an alternative to replicate sampling in a fewer number of homes. When characterizing exposure within one home, the sampling should be repeated in different rooms, rather than repeating it on different days.

No abstract