Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres

Although basal cells function as human airway epithelial stem cells, analysis of these cells is limited by in vitro culture techniques that permit only minimal cell growth and differentiation. Here, we report a protocol that dramatically increases the long-term expansion of primary human airway basal cells while maintaining their genomic stability using 3T3-J2 fibroblast coculture and ROCK inhibition. We also describe techniques for the differentiation and imaging of these expanded airway stem cells as three-dimensional tracheospheres containing basal, ciliated, and mucosecretory cells. These procedures allow investigation of the airway epithelium under more physiologically relevant conditions than those found in undifferentiated monolayer cultures. Together these methods represent a novel platform for improved airway stem cell growth and differentiation that is compatible with high-throughput, high-content translational lung research as well as human airway tissue engineering and clinical cellular therapy

Ciliated Epithelial Cell Differentiation at Air-Liquid Interface Using Commercially Available Culture Media

The human nasal epithelium contains basal stem/progenitor cells that produce differentiated multiciliated and mucosecretory progeny. Basal epithelial cells can be expanded in cell culture and instructed to differentiate at an air-liquid interface using transwell membranes and differentiation media. For basal cell expansion, we have used 3T3-J2 co-culture in epithelial culture medium containing EGF, insulin, and a RHO-associated protein kinase (ROCK) inhibitor, Y-27632 (3T3 + Y). Here we describe our protocols for ciliated differentiation of these cultures at air-liquid interface and compare four commercially available differentiation media, across nine donor cell cultures (six healthy, two patients with chronic obstructive pulmonary disease (COPD), and one with primary ciliary dyskinesia (PCD)). Bright-field and immunofluorescence imaging suggested broad similarity between differentiation protocols. Subtle differences were seen in transepithelial electrical resistance (TEER), ciliary beat frequency, mucus production, and the extent to which basal cells are retained in differentiated cultures. Overall, the specific differentiation medium used in our air-liquid interface culture protocol was not a major determinant of ciliation, and our data suggest that the differentiation potential of basal cells at the outset is a more critical factor in air-liquid interface culture outcome. Detailed information on the constituents of the differentiation media was only available from one of the four manufacturers, a factor that may have profound implications in the interpretation of some research studies

Evaluation of SOVAT: An OLAP-GIS decision support system for community health assessment data analysis

Background. Data analysis in community health assessment (CHA) involves the collection, integration, and analysis of large numerical and spatial data sets in order to identify health priorities. Geographic Information Systems (GIS) enable for management and analysis using spatial data, but have limitations in performing analysis of numerical data because of its traditional database architecture. On-Line Analytical Processing (OLAP) is a multidimensional datawarehouse designed to facilitate querying of large numerical data. Coupling the spatial capabilities of GIS with the numerical analysis of OLAP, might enhance CHA data analysis. OLAP-GIS systems have been developed by university researchers and corporations, yet their potential for CHA data analysis is not well understood. To evaluate the potential of an OLAP-GIS decision support system for CHA problem solving, we compared OLAP-GIS to the standard information technology (IT) currently used by many public health professionals. Methods. SOVAT, an OLAP-GIS decision support system developed at the University of Pittsburgh, was compared against current IT for data analysis for CHA. For this study, current IT was considered the combined use of SPSS and GIS ("SPSS-GIS"). Graduate students, researchers, and faculty in the health sciences at the University of Pittsburgh were recruited. Each round consisted of: an instructional video of the system being evaluated, two practice tasks, five assessment tasks, and one post-study questionnaire. Objective and subjective measurement included: task completion time, success in answering the tasks, and system satisfaction. Results. Thirteen individuals participated. Inferential statistics were analyzed using linear mixed model analysis. SOVAT was statistically significant (α = .01) from SPSS-GIS for satisfaction and time (p < .002). Descriptive results indicated that participants had greater success in answering the tasks when using SOVAT as compared to SPSS-GIS. Conclusion. Using SOVAT, tasks were completed more efficiently, with a higher rate of success, and with greater satisfaction, than the combined use of SPSS and GIS. The results from this study indicate a potential for OLAP-GIS decision support systems as a valuable tool for CHA data analysis. © 2008 Scotch et al; licensee BioMed Central Ltd

The Road Less Traveled: Regulation of Leukocyte Migration Across Vascular and Lymphatic Endothelium by Galectins

Leukocyte entry from the blood into inflamed tissues, exit into the lymphatics, and migration to regional lymph nodes are all crucial processes for mounting an effective adaptive immune response. Leukocytes must cross two endothelial cell layers, the vascular and the lymphatic endothelial cell layers, during the journey from the blood to the lymph node. The proteins and cellular interactions which regulate leukocyte migration across the vascular endothelium are well studied; however, little is known about the factors that regulate leukocyte migration across the lymphatic endothelium. Here, we will summarize evidence for a role for galectins, a family of carbohydrate-binding proteins, in regulating leukocyte migration across the vascular endothelium and propose that galectins are also involved in leukocyte migration across the lymphatic endothelium

Multipotent Capacity of Immortalized Human Bronchial Epithelial Cells

While the adult murine lung utilizes multiple compartmentally restricted progenitor cells during homeostasis and repair, much less is known about the progenitor cells from the human lung. Translating the murine stem cell model to humans is hindered by anatomical differences between species. Here we show that human bronchial epithelial cells (HBECs) display characteristics of multipotent stem cells of the lung. These HBECs express markers indicative of several epithelial types of the adult lung when experimentally tested in cell culture. When cultured in three different three-dimensional (3D) systems, subtle changes in the microenvironment result in unique responses including the ability of HBECs to differentiate into multiple central and peripheral lung cell types. These new findings indicate that the adult human lung contains a multipotent progenitor cell whose differentiation potential is primarily dictated by the microenvironment. The HBEC system is not only important in understanding mechanisms for specific cell lineage differentiation, but also for examining changes that correlate with human lung diseases including lung cancer

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics

© 2017 The Author(s). Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range

Children’s Gender Identity in Lesbian and Heterosexual Two-Parent Families

This study compared gender identity, anticipated future heterosexual romantic involvement, and psychosocial adjustment of children in lesbian and heterosexual families; it was furthermore assessed whether associations between these aspects differed between family types. Data were obtained in the Netherlands from children in 63 lesbian families and 68 heterosexual families. All children were between 8 and 12 years old. Children in lesbian families felt less parental pressure to conform to gender stereotypes, were less likely to experience their own gender as superior and were more likely to be uncertain about future heterosexual romantic involvement. No differences were found on psychosocial adjustment. Gender typicality, gender contentedness and anticipated future heterosexual romantic involvement were significant predictors of psychosocial adjustment in both family types

AAV Exploits Subcellular Stress Associated with Inflammation, Endoplasmic Reticulum Expansion, and Misfolded Proteins in Models of Cystic Fibrosis

Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, β-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency

HIV-1 Residual Viremia Correlates with Persistent T-Cell Activation in Poor Immunological Responders to Combination Antiretroviral Therapy

BACKGROUND:The clinical significance and cellular sources of residual human immunodeficiency virus type 1 (HIV-1) production despite suppressive combination antiretroviral therapy (cART) remain unclear and the effect of low-level viremia on T-cell homeostasis is still debated. METHODOLOGY/PRINCIPAL FINDINGS:We characterized the recently produced residual viruses in the plasma and short-lived blood monocytes of 23 patients with various immunological responses to sustained suppressive cART. We quantified the residual HIV-1 in the plasma below 50 copies/ml, and in the CD14(high) CD16(-) and CD16+ monocyte subsets sorted by flow cytometry, and predicted coreceptor usage by genotyping V3 env sequences. We detected residual viremia in the plasma of 8 of 10 patients with poor CD4+ T-cell reconstitution in response to cART and in only 5 of 13 patients with good CD4+ T-cell reconstitution. CXCR4-using viruses were frequent among the recently produced viruses in the plasma and in the main CD14(high) CD16(-) monocyte subset. Finally, the residual viremia was correlated with persistent CD4+ and CD8+ T-cell activation in patients with poor immune reconstitution. CONCLUSIONS:Low-level viremia could result from the release of archived viruses from cellular reservoirs and/or from ongoing virus replication in some patients. The compartmentalization of the viruses between the plasma and the blood monocytes suggests at least two origins of residual virus production during effective cART. CXCR4-using viruses might be produced preferentially in patients on cART. Our results also suggest that low-level HIV-1 production in some patients may contribute to persistent immune dysfunction despite cART