Planar organization of airway epithelial cell morphology using hydrogel grooves during ciliogenesis fails to induce ciliary alignment.

Topographical cues are known to influence cell organization both in native tissues and in vitro. In the trachea, the matrix beneath the epithelial lining is composed of collagen fibres that run along the long axis of the airway. Previous studies have shown that grooved topography can induce morphological and cytoskeletal alignment in epithelial cell lines. In the present work we assessed the impact of substrate topography on the organization of primary human tracheal epithelial cells (HTECs) and human induced pluripotent stem cell (hiPSC)-derived airway progenitors and the resulting alignment of cilia after maturation of the airway cells under Air-Liquid-Interface (ALI) culture. Grooves with optimized dimensions were imprinted into collagen vitrigel membranes (CVM) to produce gel inserts for ALI culture. Grooved CVM substrates induced cell alignment in HTECs and hiPSC airway progenitors in submerged culture. Further, both cell types were able to terminally differentiate into a multi-ciliated epithelium on both flat and groove CVM substrates. When exposed to ALI conditions, HTECs lost alignment after 14 days. Meanwhile, hiPSC-derived airway progenitors maintained their alignment throughout 31 days of ALI culture. Interestingly, neither initial alignment on the grooves, nor maintained alignment on the grooves induced alignment of cilia basal bodies, an indication of the direction of ciliary beating direction in the airway cells. Planar organization of airway cells during or prior to ciliogenesis therefore does not appear to be a feasible strategy to control cilia organization and subsequent airway epithelial function and additional cues are likely necessary to produce cilia alignment.

Design of biomimetic substrates for long-term maintenance of alveolar epithelial cells.

There is a need to establish in vitro lung alveolar epithelial culture models to better understand the fundamental biological mechanisms that drive lung diseases. While primary alveolar epithelial cells (AEC) are a useful option to study mature lung biology, they have limited utility in vitro. Cells that survive demonstrate limited proliferative capacity and loss of phenotype over the first 3-5 days in traditional culture conditions. To address this limitation, we generated a novel physiologically relevant cell culture system for enhanced viability and maintenance of phenotype. Here we describe a method utilizing e-beam lithography, reactive ion etching, and replica molding to generate poly-dimethylsiloxane (PDMS) substrates containing hemispherical cavities that mimic the architecture and size of mouse and human alveoli. Primary AECs grown on these cavity-containing substrates form a monolayer that conforms to the substrate enabling precise control over cell sheet architecture. AECs grown in cavity culture conditions remain viable and maintain their phenotype over one week. Specifically, cells grown on substrates consisting of 50 µm diameter cavities remained 96 ± 4% viable and maintained expression of surfactant protein C (SPC), a marker of type 2 AEC over 7 days. While this report focuses on primary lung alveolar epithelial cells, our culture platform is potentially relevant and useful for growing primary cells from other tissues with similar cavity-like architecture and could be further adapted to other biomimetic shapes or contours.

Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam.

Germline stem cells in the Drosophila ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. We find that the large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2-3 germline stem cells. In particular, we focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. We conclude that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. Further, we propose that Tj controls the morphogenetic behavior of cap cells as they adopted the shape and spatial organization of terminal filament cells but otherwise appeared to retain their fate when Tj expression was only partially reduced. Our data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. We propose that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors.

Challenges and opportunities for tissue-engineering polarized epithelium.

The epithelium is one of the most important tissue types in the body and the specific organization of the epithelial cells in these tissues is important for achieving appropriate function. Since many tissues contain an epithelial component, engineering functional epithelium and understanding the factors that control epithelial maturation and organization are important for generating whole artificial organ replacements. Furthermore, disruption of the cellular organization leads to tissue malfunction and disease; therefore, engineered epithelium could provide a valuable in vitro model to study disease phenotypes. Despite the importance of epithelial tissues, a surprisingly limited amount of effort has been focused on organizing epithelial cells into artificial polarized epithelium with an appropriate structure that resembles that seen in vivo. In this review, we provide an overview of epithelial tissue organization and highlight the importance of cell polarization to achieve appropriate epithelium function. We next describe the in vitro models that exist to create polarized epithelium and summarize attempts to engineer artificial epithelium for clinical use. Finally, we highlight the opportunities that exist to translate strategies from tissue engineering other tissues to generate polarized epithelium with a functional structure.

Hydrolysis of S-aryl-cysteinylglycine conjugates catalyzed by porcine kidney cortex membrane dipeptidase.

Following conjugation with glutathione, xenobiotics are converted into cysteinylglycine conjugates, cysteine conjugates, and finally, mercapturic acids. The structural factors determining the activities of dipeptidases for the metabolism of toxicologically-relevant cysteinylglycine conjugates are not well understood. We purified porcine kidney cortex membrane dipeptidase (MDP) to homogeneity, via phosphatidylinositol-specific phospholipase C-mediated cleavage of the protein's membrane anchor and cilastatin affinity chromatography. The homodimeric structure of the MDP protein was confirmed by mass spectrometry. The cysteinylglycine conjugates of 1-(chloromethyl)naphthalene, 4-nitrobenzyl chloride, and 1-chloro-2,4-dinitrobenzene were synthesized and HPLC separation methods for their quantitation were developed. MDP catalyzed the hydrolysis of all three conjugates, but the rate of this activity was strongly dependent on the nature of the substituent on the cysteine sulfur atom.