Slow hydrogel matrix degradation enhances salivary gland mimetic phenotype.

We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland cells encapsulated in matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) hydrogels within arrays of ∼320 µm diameter spherical cavities molded in PDMS. The SGm provides a functional and physiologically relevant platform well-suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would benefit from improved retention of acinar cell phenotype and function. We hypothesized that tuning biochemical cues presented within the PEG hydrogel matrix would improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed >2-fold increase in sphere number formed at 48 h, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of adhesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm over 14 days of culture. Furthermore, combining this matrix with optimized media conditions synergistically prolonged the retention of the acinar cell phenotype in SGm. STATEMENT OF SIGNIFICANCE: Salivary gland (SG) dysfunction occurs due to off-target radiation due to head and neck cancer treatments. Progress in understanding gland dysfunction and developing therapeutic strategies for the SG are hampered by the lack of in vitro models, as salivary gland cells rapidly lose critical secretory function within 24 hours in vitro. Herein, we identify properties of poly(ethylene glycol) hydrogel matrices that enhance the secretory phenotype of SG tissue mimetics within the previously-described SG-microbubble tissue chip environment. Combining slow-degrading hydrogels with media conditions optimized for secretory marker expression further enhanced functional secretory response and secretory marker expression.

Encapsulation of Primary Salivary Gland Acinar Cell Clusters and Intercalated Ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-Degradable Hydrogels to Maintain Tissue Structure and Function.

Progress in the development of salivary gland regenerative strategies is limited by poor maintenance of the secretory function of salivary gland cells (SGCs) in vitro. To reduce the precipitous loss of secretory function, a modified approach to isolate intact acinar cell clusters and intercalated ducts (AIDUCs), rather than commonly used single cell suspension, is investigated. This isolation approach yields AIDUCs that maintain many of the cell-cell and cell-matrix interactions of intact glands. Encapsulation of AIDUCs in matrix metalloproteinase (MMP)-degradable PEG hydrogels promotes self-assembly into salivary gland mimetics (SGm) with acinar-like structure. Expression of Mist1, a transcription factor associated with secretory function, is detectable throughout the in vitro culture period up to 14 days. Immunohistochemistry also confirms expression of acinar cell markers (NKCC1, PIP and AQP5), duct cell markers (K7 and K5), and myoepithelial cell markers (SMA). Robust carbachol and ATP-stimulated calcium flux is observed within the SGm for up to 14 days after encapsulation, indicating that secretory function is maintained. Though some acinar-to-ductal metaplasia is observed within SGm, it is reduced compared to previous reports. In conclusion, cell-cell interactions maintained within AIDUCs together with the hydrogel microenvironment may be a promising platform for salivary gland regenerative strategies.

The critical role of collagen VI in lung development and chronic lung disease.

Type VI collagen (collagen VI) is an obligate extracellular matrix component found mainly in the basement membrane region of many mammalian tissues and organs, including skeletal muscle and throughout the respiratory system. Collagen VI is probably most recognized in medicine as the genetic cause of a spectrum of muscular dystrophies, including Ullrich Congenital Myopathy and Bethlem Myopathy. Collagen VI is thought to contribute to myopathy, at least in part, by mediating muscle fiber integrity by anchoring myoblasts to the muscle basement membrane. Interestingly, collagen VI myopathies present with restrictive respiratory insufficiency, thought to be due primarily to thoracic muscular weakening. Although it was recently recognized as one of the (if not the) most abundant collagens in the mammalian lung, there is a substantive knowledge gap concerning its role in respiratory system development and function. A few studies have suggested that collagen VI insufficiency is associated with airway epithelial cell survival and altered lung function. Our recent work suggested collagen VI may be a genomic risk factor for chronic lung disease in premature infants. Using this as motivation, we thoroughly assessed the role of collagen VI in lung development and in lung epithelial cell biology. Here, we describe the state-of-the-art for collagen VI cell and developmental biology within the respiratory system, and reveal its essential roles in normal developmental processes and airway epithelial cell phenotype and intracellular signaling.

Development of a functional salivary gland tissue chip with potential for high-content drug screening.

Radiation therapy for head and neck cancers causes salivary gland dysfunction leading to permanent xerostomia. Limited progress in the discovery of new therapeutic strategies is attributed to the lack of in vitro models that mimic salivary gland function and allow high-throughput drug screening. We address this limitation by combining engineered extracellular matrices with microbubble (MB) array technology to develop functional tissue mimetics for mouse and human salivary glands. We demonstrate that mouse and human salivary tissues encapsulated within matrix metalloproteinase-degradable poly(ethylene glycol) hydrogels formed in MB arrays are viable, express key salivary gland markers, and exhibit polarized localization of functional proteins. The salivary gland mimetics (SGm) respond to calcium signaling agonists and secrete salivary proteins. SGm were then used to evaluate radiosensitivity and mitigation of radiation damage using a radioprotective compound. Altogether, SGm exhibit phenotypic and functional parameters of salivary glands, and provide an enabling technology for high-content/throughput drug testing.

3D iPSC modeling of the retinal pigment epithelium-choriocapillaris complex identifies factors involved in the pathology of macular degeneration.

The retinal pigment epithelium (RPE)-choriocapillaris (CC) complex in the eye is compromised in age-related macular degeneration (AMD) and related macular dystrophies (MDs), yet in vitro models of RPE-CC complex that enable investigation of AMD/MD pathophysiology are lacking. By incorporating iPSC-derived cells into a hydrogel-based extracellular matrix, we developed a 3D RPE-CC model that recapitulates key features of both healthy and AMD/MD eyes and provides modular control over RPE and CC layers. Using this 3D RPE-CC model, we demonstrated that both RPE- and mesenchyme-secreted factors are necessary for the formation of fenestrated CC-like vasculature. Our data show that choroidal neovascularization (CNV) and CC atrophy occur in the absence of endothelial cell dysfunction and are not necessarily secondary to drusen deposits underneath RPE cells, and CC atrophy and/or CNV can be initiated systemically by patient serum or locally by mutant RPE-secreted factors. Finally, we identify FGF2 and matrix metalloproteinases as potential therapeutic targets for AMD/MDs.

Rare Pulmonary Connective Tissue Type Mast Cells Regulate Lung Endothelial Cell Angiogenesis.

Within the human lung, mast cells typically reside adjacent to the conducting airway and assume a mucosal phenotype (MCT). In rare pathologic conditions, connective tissue phenotype mast cells (MCTCs) can be found in the lung parenchyma. MCTCs accumulate in the lungs of infants with severe bronchopulmonary dysplasia, a chronic lung disease associated with preterm birth, which is characterized by pulmonary vascular dysmorphia. The human mast cell line (LUVA) was used to model MCTCs or MCTs. The ability of MCTCs to affect vascular organization during fetal lung development was tested in mouse lung explant cultures. The effect of MCTCs on in vitro tube formation and barrier function was studied using primary fetal human pulmonary microvascular endothelial cells. The mechanistic role of MCTC proteases was tested using inhibitors. MCTCLUVA but not MCTLUVA was associated with vascular dysmorphia in lung explants. In vitro, the addition of MCTCLUVA potentiated fetal human pulmonary microvascular endothelial cell interactions, inhibited tube stability, and disrupted endothelial cell junctions. Protease inhibitors ameliorated the ability of MCTCLUVA to alter endothelial cell angiogenic activities in vitro and ex vivo. These data indicate that MCTCs may directly contribute to disrupted angiogenesis in bronchopulmonary dysplasia. A better understanding of factors that regulate mast cell subtype and their different effector functions is essential.

Lymphocyte-Specific Biomarkers Associated With Preterm Birth and Bronchopulmonary Dysplasia.

Many premature babies who are born with neonatal respiratory distress syndrome (RDS) go on to develop Bronchopulmonary Dysplasia (BPD) and later Post-Prematurity Respiratory Disease (PRD) at one year corrected age, characterized by persistent or recurrent lower respiratory tract symptoms frequently related to inflammation and viral infection. Transcriptomic profiles were generated from sorted peripheral blood CD8+ T cells of preterm and full-term infants enrolled with consent in the NHLBI Prematurity and Respiratory Outcomes Program (PROP) at the University of Rochester and the University at Buffalo. We identified outcome-related gene expression patterns following standard methods to identify markers for oxygen utilization and BPD as outcomes in extremely premature infants. We further identified predictor gene sets for BPD based on transcriptomic data adjusted for gestational age at birth (GAB). RNA-Seq analysis was completed for CD8+ T cells from 145 subjects. Among the subjects with highest risk for BPD (born at <29 weeks gestational age (GA); n=72), 501 genes were associated with oxygen utilization. In the same set of subjects, 571 genes were differentially expressed in subjects with a diagnosis of BPD and 105 genes were different in BPD subjects as defined by physiologic challenge. A set of 92 genes could predict BPD with a moderately high degree of accuracy. We consistently observed dysregulation of TGFB, NRF2, HIPPO, and CD40-associated pathways in BPD. Using gene expression data from both premature and full-term subjects (n=116), we identified a 28 gene set that predicted the PRD status with a moderately high level of accuracy, which also were involved in TGFB signaling. Transcriptomic data from sort-purified peripheral blood CD8+ T cells from 145 preterm and full-term infants identified sets of molecular markers of inflammation associated with independent development of BPD in extremely premature infants at high risk for the disease and of PRD among the preterm and full-term subjects.

A novel in vitro model of primary human pediatric lung epithelial cells.

BACKGROUND: Current in vitro human lung epithelial cell models derived from adult tissues may not accurately represent all attributes that define homeostatic and disease mechanisms relevant to the pediatric lung. METHODS: We report methods for growing and differentiating primary Pediatric Human Lung Epithelial (PHLE) cells from organ donor infant lung tissues. We use immunohistochemistry, flow cytometry, quantitative RT-PCR, and single cell RNA sequencing (scRNAseq) analysis to characterize the cellular and transcriptional heterogeneity of PHLE cells. RESULTS: PHLE cells can be expanded in culture up to passage 6, with a doubling time of ~4 days, and retain attributes of highly enriched epithelial cells. PHLE cells can form resistant monolayers, and undergo differentiation when placed at air-liquid interface. When grown at Air-Liquid Interface (ALI), PHLE cells expressed markers of airway epithelial cell lineages. scRNAseq suggests the cultures contained 4 main sub-phenotypes defined by expression of FOXJ1, KRT5, MUC5B, and SFTPB. These cells are available to the research community through the Developing Lung Molecular Atlas Program Human Tissue Core. CONCLUSION: Our data demonstrate that PHLE cells provide a novel in vitro human cell model that represents the pediatric airway epithelium, which can be used to study perinatal developmental and pediatric disease mechanisms.

Collagen VI Deficiency Results in Structural Abnormalities in the Mouse Lung.

Collagen VI (COL6) is known for its role in a spectrum of congenital muscular dystrophies, which are often accompanied by respiratory dysfunction. However, little is known regarding the function of COL6 in the lung. We confirmed the presence of COL6 throughout the basement membrane region of mouse lung tissue. Lung structure and organization were studied in a previously described Col6a1-/- mouse, which does not produce detectable COL6 in the lung. The Col6a1-/- mouse displayed histopathologic alveolar and airway abnormalities. The airspaces of Col6a1-/- lungs appeared simplified, with larger (29%; P < 0.01) and fewer (31%; P < 0.001) alveoli. These airspace abnormalities included reduced isolectin B4+ alveolar capillaries and surfactant protein C-positive alveolar epithelial type-II cells. Alterations in lung function consistent with these histopathologic changes were evident. Col6a1-/- mice also displayed multiple airway changes, including increased branching (59%; P < 0.001), increased mucosal thickness (34%; P < 0.001), and increased epithelial cell density (13%; P < 0.001). Comprehensive transcriptome analysis revealed that the loss of COL6 is associated with reductions in integrin-paxillin-phosphatidylinositol 3-kinase signaling in vivo. In vitro, COL6 promoted steady-state phosphorylated paxillin levels and reduced cell density (16% to 28%; P < 0.05) at confluence. Inhibition of phosphatidylinositol 3-kinase, or its downstream effectors, resulted in increased cell density to a level similar to that seen on matrices lacking COL6.

CX3CR1 as a respiratory syncytial virus receptor in pediatric human lung.

BACKGROUND: Data on the host factors that contribute to infection of young children by respiratory syncytial virus (RSV) are limited. The human chemokine receptor, CX3CR1, has recently been implicated as an RSV receptor. Here we evaluate a role for CX3CR1 in pediatric lung RSV infections. METHODS: CX3CR1 transcript levels in the upper and lower pediatric airways were assessed. Tissue localization and cell-specific expression was confirmed using in situ hybridization and immunohistochemistry. The role of CX3CR1 in RSV infection was also investigated using a novel physiological model of pediatric epithelial cells. RESULTS: Low levels of CX3CR1 transcript were often, but not always, expressed in both upper (62%) and lower airways (36%) of pediatric subjects. CX3CR1 transcript and protein expression was detected in epithelial cells of normal human pediatric lung tissues. CX3CR1 expression was readily detected on primary cultures of differentiated pediatric/infant human lung epithelial cells. RSV demonstrated preferential infection of CX3CR1-positive cells, and blocking CX3CR1/RSV interaction significantly decreased viral load. CONCLUSION: CX3CR1 is present in the airways of pediatric subjects where it may serve as a receptor for RSV infection. Furthermore, CX3CR1 appears to play a mechanistic role in mediating viral infection of pediatric airway epithelial cells in vitro.

Type VI collagen promotes lung epithelial cell spreading and wound-closure.

Basement membrane (BM) is an essential part of the extracellular matrix (ECM) that plays a crucial role in mechanical support and signaling to epithelial cells during lung development, homeostasis and repair. Abnormal composition and remodeling of the lung ECM have been associated with developmental abnormalities observed in multiple pediatric and adult respiratory diseases. Collagen VI (COL6) is a well-studied muscle BM component, but its role in the lung and its effect on pulmonary epithelium is largely undetermined. We report the presence of COLVI immediately subjacent to human airway and alveolar epithelium in the pediatric lung, in a location where it is likely to interact with epithelial cells. In vitro, both primary human lung epithelial cells and human lung epithelial cell lines displayed an increased rate of "wound healing" in response to a scratch injury when plated on COL6 as compared to other matrices. For the 16HBE cell line, wounds remained >5-fold larger for cells on COL1 (p<0.001) and >6-fold larger on matrigel (p<0.001), a prototypical basement membrane, when compared to COL6 (>96% closure at 10 hr). The effect of COL6 upon lung epithelial cell phenotype was associated with an increase in cell spreading. Three hours after initial plating, 16HBE cells showed >7-fold less spreading on matrigel (p<0.01), and >4-fold less spreading on COL1 (p<0.01) when compared to COL6. Importantly, the addition of COL6 to other matrices also enhanced cell spreading. Similar responses were observed for primary cells. Inhibitor studies indicated both integrin ß1 activity and activation of multiple signaling pathways was required for enhanced spreading on all matrices, with the PI3K/AKT pathway (PI3K, CDC42, RAC1) showing both significant and specific effects for spreading on COL6. Genetic gain-of-function experiments demonstrated enhanced PI3K/AKT pathway activity was sufficient to confer equivalent cell spreading on other matrices as compared to COL6. We conclude that COL6 has significant and specific effects upon human lung epithelial cell-autonomous functions.

Interpreting in vitro micronucleus positive results: simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents.

The specificity of in vitro mammalian cell genotoxicity assays is low, as they yield a high incidence of positive results that are not observed in animal genotoxicity and carcinogenicity tests, that is, "misleading" or "irrelevant" positives. We set out to develop a rapid and effective follow-up testing strategy that would predict whether apparent in vitro micronucleus-inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of micronucleus frequencies, or that could be accomplished in parallel using a homogeneous-type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positive chemicals was studied. These experiments were conducted with human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to micronucleus frequency, the following endpoints were investigated, most often at time of harvest: cleaved Parp-positive chromatin, cleaved caspase 3-positive chromatin, ethidium monoazide bromide-positive chromatin, polyploid nuclei, phospho-histone H3-positive (metaphase) cells, tetramethylrhodamine ethyl ester-negative cells, cellular ATP levels, cell cycle perturbation, and shift in γ-H2AX fluorescence relative to solvent control. Logistic regression was used to identify endpoints that effectively predict chemicals' a priori classification. Cross validation using a leave-one-out approach indicated that a promising base model includes γ-H2AX shift and change in phospho-histone H3-positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26-30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and also by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput-compatible biomarkers can effectively delineate two important modes of genotoxic action as well as identify cytotoxicity that can lead to irrelevant positive results.

Interlaboratory Pig-a gene mutation assay trial: Studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes.

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (∼20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [2011]: Mutat Res 721:163-170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN-RET) counts. 1,3-PS was found to increase Pig-a mutation and MN-RET frequencies in both 3- and 28-day study designs. While the greatest induction of MN-RETs was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one-third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated.