SUMMARY Functional modeling of many adult epithelia is limited by the difficulty of maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We found that TGFβ/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD inhibition in a feeder-free culture system we were able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium that is physiologically responsive to clinically relevant drugs such as CFTR modulators. This approach is effective for clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers, and may therefore be broadly applicable for modeling of epithelia.

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.

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We have isolated a full-length cDNA, PPI1 (pepper-PMMV interaction 1), encoding a novel basic region-leucine zipper (bZIP) DNA-binding protein, from expressed sequence tags differentially expressed in Capsicum chinense PI257284 infected with Pepper mild mottle virus (PMMV). PPI1 encodes a predicted protein of 170 amino acids and contains a putative DNA-binding domain that shares significant amino acid identity with ACGT-binding domains of members of the bZIP DNA-binding protein family. PPI1 was localized in the nucleus and had transcriptional activation activity in yeast. Transcripts of the PPI1 gene were preferentially induced during an incompatible interaction by inoculation with PMMV, Pseudomonas syringae pv. syringae 61, and Xanthomonas campestris pv. vesicatoria race 3. However, the PPI1 gene was not induced by abiotic stressors that activate the plant defense-signaling pathway. Our data provide the first evidence that a bZIP transcription factor is preferentially induced by pathogen attack, suggesting that PPI1 may play a specific functional role in the regulation of expression of plant defense-related genes.

The CMV (cucumber mosaic virus) is the most frequently occurring virus in chili pepper farms. A variety of peppers that are resistant to CMVP0 were developed in the middle of 1990s through a breeding program, and commercial cultivars have since been able to control the spread of CMVP0. However, a new pathotype (CMVP1) that breaks the resistance of CMVP0-resistant peppers has recently appeared and caused a heavy loss in productivity. Since no genetic source of this new pathotype was available, a traditional breeding method cannot be used to generate a CMVP1-resistant pepper variety. Therefore, we set up a transformation system of pepper using Agrobacterium that had been transfected with the coat protein gene, CMVP0-CP, with the aim of developing a new CMVP1-resistant pepper line. A large number of transgenic peppers (T(1), T(2) and T(3)) were screened for CMVP1 tolerance using CMVP1 inoculation. Transgenic peppers tolerant to CMVP1 were selected in a plastic house as well as in the field. Three independent T(3) pepper lines highly tolerant to the CMVP1 pathogen were found to also be tolerant to the CMVP0 pathogen. These selected T(3) pepper lines were phenotypically identical or close to the non-transformed lines. However, after CMVP1 infection, the height and fruit size of the non-transformed lines became shorter and smaller, respectively, while the T(3) pepper lines maintained a normal phenotype.