Single-cell analysis of somatic mutations in human bronchial epithelial cells in relation to aging and smoking.

Although lung cancer risk among smokers is dependent on smoking dose, it remains unknown if this increased risk reflects an increased rate of somatic mutation accumulation in normal lung cells. Here, we applied single-cell whole-genome sequencing of proximal bronchial basal cells from 33 participants aged between 11 and 86 years with smoking histories varying from never-smoking to 116 pack-years. We found an increase in the frequency of single-nucleotide variants and small insertions and deletions with chronological age in never-smokers, with mutation frequencies significantly elevated among smokers. When plotted against smoking pack-years, mutations followed the linear increase in cancer risk until about 23 pack-years, after which no further increase in mutation frequency was observed, pointing toward individual selection for mutation avoidance. Known lung cancer-defined mutation signatures tracked with both age and smoking. No significant enrichment for somatic mutations in lung cancer driver genes was observed.

Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease.

Crohn's disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10-10) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10-8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson's disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases.

Very late antigen-5 facilitates stromal progenitor cell differentiation into myofibroblast.

Fibrotic disease is associated with abrogated stromal cell proliferation and activity. The precise identity of the cells that drive fibrosis remains obscure, in part because of a lack of information on their lineage development. To investigate the role of an early stromal progenitor cell (SPC) on the fibrotic process, we selected for, and monitored the stages of, fibroblast development from a previously reported free-floating anchorage-independent cell (AIC) progenitor population. Our findings demonstrate that organotypic pulmonary, cardiac, and renal fibroblast commitment follows a two-step process of attachment and remodeling in culture. Cell differentiation was confirmed by the inability of SPCs to revert to the free-floating state and functional mesenchymal stem/stromal cell (MSC) differentiation into osteoblast, adipocyte, chondrocyte, and fibroblastic lineages. The myofibroblastic phenotype was reflected by actin stress-fiber formation, α-smooth muscle production, and a greater than threefold increase in proliferative activity compared with that of the progenitors. SPC-derived pulmonary myofibroblasts demonstrated a more than 300-fold increase in fibronectin-1 (Fn1), collagen, type 1, α1, integrin α-5 (Itga5), and integrin ß-1 (Itgb1) transcript levels. Very late antigen-5 (ITGA5/ITGB1) protein cluster formations were also prevalent on the differentiated cells. Normalized SPC-derived myofibroblast expression patterns reflected those of primary cultured lung myofibroblasts. Intratracheal implantation of pulmonary AICs into recipient mouse lungs resulted in donor cell FN1 production and evidence of epithelial derivation. SPC derivation into stromal tissue in vitro and in vivo and the observation that MSC and fibroblast lineages share a common ancestor could potentially lead to personalized antifibrotic therapies.

Discrimination between lung homeostatic and injury-induced epithelial progenitor subsets by cell-density properties.

Stem/progenitor cells and their lineage derivatives are often identified by patterns and intensity of cell clusters of differentiation presentation. However, the cell biochemical façade can prove to be elusive, transient, and subject to interlaboratory disparities. To enhance current methods of lung stem cell isolation and identification and to investigate biophysical changes, which occur during homeostasis and in response to acute lung injury, we separated cells on a discontinuous density gradient, of 1.025-1.074 g/cm(3), and characterized the eluted lineages. At homeostasis, surfactant protein-C (SFTPC)-expressing cells of the alveolar type (AT)-2 lineage possessed average densities ≥1.039 g/cm(3) and aquaporin-5 producing AT1 cells equilibrated at densities <1.039 g/cm(3). While 0.74%±0.32% of lung cells were determined proliferating or postmitotic by BrdU nucleotide uptake, 73% of CD49f-, 72% of c-KIT-, and 61% of SCA-1-positive cells (putative alveolar progenitor lineage markers) showed densities ≤1.039 g/cm(3). CD49f/EpCAM(hi) progenitors, as well as c-KIT(pos)/CD45(neg) cells, could be enriched at the 1.039 g/cm(3) interface. Following acute bleomycin-induced injury, the frequency of BrdU-incorporating cells rose to 0.92%±0.36% and density could largely explain cell-lineage distribution. Specifically, a decline in the density of mitotic/postmitotic SFTPC-positive cells to ≤1.029 g/cm(3), in conjunction with an increase in CD45-positive, and proliferating CD45 and c-KIT cells in the heaviest fraction (≥1.074 g/cm(3)) were observed. These data attest to the generation of AT2 cells from low-density precursors and emphasize a relationship between cell density and molecular expression following injury, expanding on our current understanding of lung and progenitor cell dynamics.

CD45/CD11b positive subsets of adult lung anchorage-independent cells harness epithelial stem cells in culture.

Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between stem/progenitor cells that proliferate or differentiate and somatic cells of the lung, we used a novel organotypic ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free-floating anchorage-independent cells (AICs). AICs did not express integrin proteins α5, ß3 and ß7, and constituted 37% of the total culture at day 14, yielding a mixed yet conservative population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60-fold increase in cell numbers by day 21. More than 50% of the AIC population was c-KIT(+) or double-positive for CD45(+) and CD11b(+) antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein-C and SCGB1A1 expressing putative stem cells and with aquaporin-5 producing cells, characteristic of terminally differentiated alveolar epithelial type-1 pneumocytes. At the air/gel interface, AICs undergo remodeling to form a cellular lining, whereas TGF(ß)1 treatment modifies protein expression properties to further imply a robust effect of the microenvironment on AIC phenotypic changes. These data confirm the active participation of clonogenic hematopoietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which license sustained cell viability, proliferation and differentiation, for use in studies of compensatory pulmonary growth.

Epidermal growth factor receptor and claudin-2 participate in A549 permeability and remodeling: implications for non-small cell lung cancer tumor colonization.

Tumor colonization involves changes in cell permeability and remodeling. Paracellular permeability is regulated by claudins, integrated tight junction (TJ) proteins, located on the apicolateral portion of epithelial cells. Epidermal growth factor (EGF) was reported to modify cellular claudin levels and induce remodeling. To investigate a role for EGF receptor (EGFR) activation in tumor colonization we studied the effect of EGF and claudin-2 overexpression on permeability and cell reorganization in the human A549 non-small cell lung cancer (NSCLC) cell line. Our data demonstrated that A549 cells possess functional TJs and that EGF treatment increased levels of claudin-2 expression by 46%. Furthermore, EGFR signaling reduced monolayer permeability to choline and triggered cellular remodeling. The mitogen-activated protein kinase inhibitor PD98059 blocked the effect on A549 permeability and remodeling. EGF stimulation also exacerbated a fourfold increase in cell colonization elicited by claudin-2 upregulation. Our findings are consistent with the hypothesis that EGFR signaling plays an important role in A549 cell physiology and acts synergistically with claudin-2 to accelerate tumor colonization. Understanding the influence of EGF on A549 cell permeability and reorganization will help shed light on NSCLC tumor colonization and contribute to the development of novel anti-cancer treatments.

Tracheotomy: a method for transplantation of stem cells to the lung.

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Human Gene-Centric Databases at the Weizmann Institute of Science: GeneCards, UDB, CroW 21 and HORDE.

Recent enhancements and current research in the GeneCards (GC) (http://bioinfo.weizmann.ac.il/cards/) project are described, including the addition of gene expression profiles and integrated gene locations. Also highlighted are the contributions of specialized associated human gene-centric databases developed at the Weizmann Institute. These include the Unified Database (UDB) (http://bioinfo.weizmann.ac.il/udb) for human genome mapping, the human Chromosome 21 database at the Weizmann Insti-tute (CroW 21) (http://bioinfo.weizmann.ac.il/crow21), and the Human Olfactory Receptor Data Explora-torium (HORDE) (http://bioinfo.weizmann.ac.il/HORDE). The synergistic relationships amongst these efforts have positively impacted the quality, quantity and usefulness of the GeneCards gene compendium.