Evaluation of Alginate Hydrogel Microstrands for Stromal Cell Encapsulation and Maintenance.

Mesenchymal stromal cells (MSCs) have displayed potential in regenerating organ function due to their anti-fibrotic, anti-inflammatory, and regenerative properties. However, there is a need for delivery systems to enhance MSC retention while maintaining their anti-fibrotic characteristics. This study investigates the feasibility of using alginate hydrogel microstrands as a cell delivery vehicle to maintain MSC viability and phenotype. To accommodate cell implantation needs, we invented a Syringe-in-Syringe approach to reproducibly fabricate microstrands in small numbers with a diameter of around 200 µm and a porous structure, which would allow for transporting nutrients to cells by diffusion. Using murine NIH 3T3 fibroblasts and primary embryonic 16 (E16) salivary mesenchyme cells as primary stromal cell models, we assessed cell viability, growth, and expression of mesenchymal and fibrotic markers in microstrands. Cell viability remained higher than 90% for both cell types. To determine cell number within the microstrands prior to in vivo implantation, we have further optimized the alamarBlue assay to measure viable cell growth in microstrands. We have shown the effect of initial cell seeding density and culture period on cell viability and growth to accommodate future stromal cell delivery and implantation. Additionally, we confirmed homeostatic phenotype maintenance for E16 mesenchyme cells in microstrands.

Identifying fibrogenic cells following salivary gland obstructive injury.

Fibrosis results from excess extracellular matrix accumulation, which alters normal tissue architecture and impedes function. In the salivary gland, fibrosis can be induced by irradiation treatment for cancer therapy, Sjögren's Disease, and other causes; however, it is unclear which stromal cells and signals participate in injury responses and disease progression. As hedgehog signaling has been implicated in fibrosis of the salivary gland and other organs, we examined contributions of the hedgehog effector, Gli1, to fibrotic responses in salivary glands. To experimentally induce a fibrotic response in female murine submandibular salivary glands, we performed ductal ligation surgery. We detected a progressive fibrotic response where both extracellular matrix accumulation and actively remodeled collagen significantly increased at 14 days post-ligation. Macrophages, which participate in extracellular matrix remodeling, and Gli1+ and PDGFRα+ stromal cells, which may deposit extracellular matrix, both increased with injury. Using single-cell RNA-sequencing, Gli1 + cells were not found in discrete clusters at embryonic day 16 but were found in clusters expressing the stromal genes Pdgfra and/or Pdgfrb. In adult mice, Gli1+ cells were similarly heterogeneous but more cells co-expressed PDGFRα and PDGFRß. Using Gli1-CreERT2; ROSA26tdTomato lineage-tracing mice, we found that Gli1-derived cells expand with ductal ligation injury. Although some of the Gli1 lineage-traced tdTomato+ cells expressed vimentin and PDGFRß following injury, there was no increase in the classic myofibroblast marker, smooth muscle alpha-actin. Additionally, there was little change in extracellular matrix area, remodeled collagen area, PDGFRα, PDGFRß, endothelial cells, neurons, or macrophages in Gli1 null salivary glands following injury when compared with controls, suggesting that Gli1 signaling and Gli1+ cells have only a minor contribution to mechanical injury-induced fibrotic changes in the salivary gland. We used scRNA-seq to examine cell populations that expand with ligation and/or showed increased expression of matrisome genes. Some Pdgfra + /Pdgfrb + stromal cell subpopulations expanded in response to ligation, with two stromal cell subpopulations showing increased expression of Col1a1 and a greater diversity of matrisome genes, consistent with these cells being fibrogenic. However, only a few cells in these subpopulations expressed Gli1, consistent with a minor contribution of these cells to extracellular matrix production. Defining the signaling pathways driving fibrotic responses in stromal cell sub-types could reveal future therapeutic targets.

Identifying Fibrogenic Cells Following Salivary Gland Obstructive Injury.

Fibrosis results from excess extracellular matrix accumulation, which alters normal tissue architecture and impedes function. In the salivary gland, fibrosis can be induced by irradiation treatment for cancer therapy, Sjögren's Disease, and other causes; however, it is unclear which stromal cells and signals participate in injury responses and disease progression. As hedgehog signaling has been implicated in fibrosis of the salivary gland and other organs, we examined contributions of the hedgehog effector, Gli1, to fibrotic responses in salivary glands. To experimentally induce a fibrotic response in female murine submandibular salivary glands, we performed ductal ligation surgery. We detected a progressive fibrotic response where both extracellular matrix accumulation and actively remodeled collagen trended upwards at 7 days and significantly increased at 14 days post- ligation. Macrophages, which participate in extracellular matrix remodeling, Gli1 + and PDGFRα + stromal cells, which may deposit extracellular matrix, both increased with injury. Using single-cell RNA-sequencing, we found that a majority of Gli1 + cells at embryonic day 16 also express Pdgfra and/or Pdgfrb. However, in adult mice, only a small subset of Gli1 + cells express PDGFRα and/or PDGFRß at the protein level. Using lineage-tracing mice, we found that Gli1-derived cells expand with ductal ligation injury. Although some of the Gli1 lineage-traced tdTomato + cells expressed vimentin and PDGFRß following injury, there was no increase in the classic myofibroblast marker, smooth muscle alpha-actin. Additionally, there was little change in extracellular matrix area, remodeled collagen area, PDGFRα, PDGFRß, endothelial cells, neurons, or macrophages in Gli1 null salivary glands following injury when compared with controls, suggesting that Gli1 signaling and Gli1 + cells have only a minor contribution to mechanical injury-induced fibrotic changes in the salivary gland. We used scRNA-seq to examine cell populations that expand with ligation and/or showed increased expression of matrisome genes. Pdgfra + /Pdgfrb + stromal cell subpopulations both expanded in response to ligation, showed increased expression and a greater diversity of matrisome genes expressed, consistent with these cells being fibrogenic. Defining the signaling pathways driving fibrotic responses in stromal cell sub-types could reveal future therapeutic targets.

Canine as a Comparative and Translational Model for Human Mammary Tumor.

Despite the advances in research and treatment of human breast cancer, its incidence rate continues to increase by 0.5% per year, and the discovery of novel therapeutic strategies for specific subtypes of human breast cancer remains challenging. Traditional laboratory mouse models have contributed tremendously to human breast cancer research. However, mice do not develop tumors spontaneously; consequently, genetically engineered mouse models or patient-derived xenograft models are often relied upon for more sophisticated human breast cancer studies. Since human breast cancer develops spontaneously, there is a need for alternative, yet complementary, models that can better recapitulate the features of human breast cancer to better understand the molecular and clinical complexities of the disease in developing new therapeutic strategies. Canine mammary tumors are one such alternative model that share features with human breast cancer, including prevalence rate, subtype classification, treatment, and mutational profiles, all of which are described in this review.

Raman microspectroscopy fingerprinting of organoid differentiation state.

BACKGROUND: Organoids, which are organs grown in a dish from stem or progenitor cells, model the structure and function of organs and can be used to define molecular events during organ formation, model human disease, assess drug responses, and perform grafting in vivo for regenerative medicine approaches. For therapeutic applications, there is a need for nondestructive methods to identify the differentiation state of unlabeled organoids in response to treatment with growth factors or pharmacologicals. METHODS: Using complex 3D submandibular salivary gland organoids developed from embryonic progenitor cells, which respond to EGF by proliferating and FGF2 by undergoing branching morphogenesis and proacinar differentiation, we developed Raman confocal microspectroscopy methods to define Raman signatures for each of these organoid states using both fixed and live organoids. RESULTS: Three separate quantitative comparisons, Raman spectral features, multivariate analysis, and machine learning, classified distinct organoid differentiation signatures and revealed that the Raman spectral signatures were predictive of organoid phenotype. CONCLUSIONS: As the organoids were unlabeled, intact, and hydrated at the time of imaging, Raman spectral fingerprints can be used to noninvasively distinguish between different organoid phenotypes for future applications in disease modeling, drug screening, and regenerative medicine.

Regulation of myoepithelial differentiation.

The salivary gland can be permanently impaired by radiation treatment for head and neck cancers. Efforts at tissue regeneration have focused on saliva-producing acinar cells. However, myoepithelial cells are also critical to gland function, but mechanisms that regulate their differentiation are poorly defined. To study myoepithelial differentiation, we employed mSG-PAC1 murine salivary gland epithelial cells. We demonstrate that mSG-PAC1 spheroids exhibit phenotypic plasticity between pro-acinar and myoepithelial cell fates. Increased expression of pro-acinar/acinar or myoepithelial RNAs was identified from spheroids cultured under different media conditions by microarray followed by gene-set enrichment analysis. Spheroids cultured with different medium components expressed proteins typical of either acinar or myoepithelial cells, as detected by immunocytochemistry. We demonstrate that the pattern of TAZ expression in the epithelial compartment of the differentiating murine salivary gland correlates with the expression of the myoepithelial marker alpha-SMA, as is the case for TAZ expression in mSG-PAC1 spheroids. Our analysis also indicates that YAP/TAZ target genes are upregulated together with myoepithelial markers. Importantly, siRNA targeting of TAZ expression in mSG-PAC1 spheroids diminished the expression of myoepithelial markers. Our results in this in vitro cell model implicate TAZ signaling in myoepithelial differentiation.

Engineering cryoelectrospun elastin-alginate scaffolds to serve as stromal extracellular matrices.

Scaffold-based regenerative strategies that emulate physical, biochemical, and mechanical properties of the native extracellular matrix (ECM) of the region of interest can influence cell growth and function. Existing ECM-mimicking scaffolds, including nanofiber (NF) mats, sponges, hydrogels, and NF-hydrogel composites are unable to simultaneously mimic typical composition, topography, pore size, porosity, and viscoelastic properties of healthy soft-tissue ECM. In this work, we used cryoelectrospinning to fabricate 3D porous scaffolds with minimal fibrous backbone, pore size and mechanical properties similar to soft-tissue connective tissue ECM. We used salivary glands as our soft tissue model and found the decellularized adult salivary gland (DSG) matrix to have a fibrous backbone, 10-30µm pores, 120 Pa indentation modulus, and ∼200 s relaxation half time. We used elastin and alginate as natural, compliant biomaterials and water as the solvent for cryoelectrospinning scaffolds to mimic the structure and viscoelasticity of the connective tissue ECM of the DSG. Process parameters were optimized to produce scaffolds with desirable topography and compliance similar to DSG, with a high yield of >100 scaffolds/run. Using water as solvent, rather than organic solvents, was critical to generate biocompatible scaffolds with desirable topography; further, it permitted a green chemistry fabrication process. Here, we demonstrate that cryoelectrospun scaffolds (CESs) support penetration of NIH 3T3 fibroblasts 250-450µm into the scaffold, cell survival, and maintenance of a stromal cell phenotype. Thus, we demonstrate that elastin-alginate CESs mimic many structural and functional properties of ECM and have potential for future use in regenerative medicine applications.

Single-cell RNA sequencing reveals PDGFRα+ stromal cell subpopulations that promote proacinar cell differentiation in embryonic salivary gland organoids.

Stromal cells can direct the differentiation of epithelial progenitor cells during organ development. Fibroblast growth factor (FGF) signaling is essential for submandibular salivary gland development. Through stromal fibroblast cells, FGF2 can indirectly regulate proacinar cell differentiation in organoids, but the mechanisms are not understood. We performed single-cell RNA-sequencing and identified multiple stromal cell subsets, including Pdgfra+ stromal subsets expressing both Fgf2 and Fgf10. When combined with epithelial progenitor cells in organoids, magnetic-activated cell-sorted PDGFRα+ cells promoted proacinar cell differentiation similarly to total stroma. Gene expression analysis revealed that FGF2 increased the expression of multiple stromal genes, including Bmp2 and Bmp7. Both BMP2 and BMP7 synergized with FGF2, stimulating proacinar cell differentiation but not branching. However, stromal cells grown without FGF2 did not support proacinar organoid differentiation and instead differentiated into myofibroblasts. In organoids, TGFß1 treatment stimulated myofibroblast differentiation and inhibited the proacinar cell differentiation of epithelial progenitor cells. Conversely, FGF2 reversed the effects of TGFß1. We also demonstrated that adult salivary stromal cells were FGF2 responsive and could promote proacinar cell differentiation. These FGF2 signaling pathways may have applications in future regenerative therapies.

Alginate Hydrogel Microtubes for Salivary Gland Cell Organization and Cavitation.

Understanding the different regulatory functions of epithelial and mesenchymal cell types in salivary gland development and cellular organization is essential for proper organoid formation and salivary gland tissue regeneration. Here, we demonstrate a biocompatible platform using pre-formed alginate hydrogel microtubes to facilitate direct epithelial-mesenchymal cell interaction for 3D salivary gland cell organization, which allows for monitoring cellular organization while providing a protective barrier from cell-cluster loss during medium changes. Using mouse salivary gland ductal epithelial SIMS cells as the epithelial model cell type and NIH 3T3 fibroblasts or primary E16 salivary mesenchyme cells as the stromal model cell types, self-organization from epithelial-mesenchymal interaction was examined. We observed that epithelial and mesenchymal cells undergo aggregation on day 1, cavitation by day 4, and generation of an EpCAM-expressing epithelial cell layer as early as day 7 of the co-culture in hydrogel microtubes, demonstrating the utility of hydrogel microtubes to facilitate heterotypic cell-cell interactions to form cavitated organoids. Thus, pre-formed alginate microtubes are a promising co-culture method for further understanding epithelial and mesenchymal interaction during tissue morphogenesis and for future practical applications in regenerative medicine.

Generating Embryonic Salivary Gland Organoids.

Organoids are important research tools for studying organ morphogenesis and differentiation because they recapitulate ex vivo the native 3D organization of cells that is essential for proper cell and organ function. The composition of organoids can be manipulated to incorporate specific cell types to facilitate molecular interrogation of cell-cell interactions during organoid formation. A method for generating organoids derived from both embryonic salivary gland epithelial progenitor cells and mesenchymal support cells is described. Methods for isolating enriched populations of the epithelial cells as clusters and the mesenchyme cells as single cells from mouse embryonic submandibular salivary glands are also provided. Separating the epithelial and mesenchymal cell populations allows for independent molecular manipulation of each cell type. In addition, methods for lentiviral transduction of the mesenchyme cells and quantitative image analysis of organoids are provided. The methods described here are useful for exploring mechanisms driving organ formation. © 2018 by John Wiley & Sons, Inc.

A 'smart' tube holder enables real-time sample monitoring in a standard lab centrifuge.

The centrifuge is among the oldest and most widely used pieces of laboratory equipment, with significant applications that include clinical diagnostics and biomedical research. A major limitation of laboratory centrifuges is their "black box" nature, limiting sample observation to before and after centrifugation. Thus, optimized protocols require significant trial and error, while unoptimized protocols waste time by centrifuging longer than necessary or material due to incomplete sedimentation. Here, we developed an instrumented centrifuge tube receptacle compatible with several commercial benchtop centrifuges that can provide real-time sample analysis during centrifugation. We demonstrated the system by monitoring cell separations during centrifugation for different spin speeds, concentrations, buffers, cell types, and temperatures. We show that the collected data are valuable for analytical purposes (e.g. quality control), or as feedback to the user or the instrument. For the latter, we verified an adaptation where complete sedimentation turned off the centrifuge and notified the user by a text message. Our system adds new functionality to existing laboratory centrifuges, saving users time and providing useful feedback. This add-on potentially enables new analytical applications for an instrument that has remained largely unchanged for decades.

FGF2-dependent mesenchyme and laminin-111 are niche factors in salivary gland organoids.

Epithelial progenitor cells are dependent upon a complex 3D niche to promote their proliferation and differentiation during development, which can be recapitulated in organoids. The specific requirements of the niche remain unclear for many cell types, including the proacinar cells that give rise to secretory acinar epithelial cells that produce saliva. Here, using ex vivo cultures of E16 primary mouse submandibular salivary gland epithelial cell clusters, we investigated the requirement for mesenchymal cells and other factors in producing salivary organoids in culture. Native E16 salivary mesenchyme, but not NIH3T3 cells or mesenchymal cell conditioned medium, supported robust protein expression of the progenitor marker Kit and the acinar/proacinar marker AQP5, with a requirement for FGF2 expression by the mesenchyme. Enriched salivary epithelial clusters that were grown in laminin-enriched basement membrane extract or laminin-111 together with exogenous FGF2, but not with EGF, underwent morphogenesis to form organoids that displayed robust expression of AQP5 in terminal buds. Knockdown of FGF2 in the mesenchyme or depletion of mesenchyme cells from the organoids significantly reduced AQP5 levels even in the presence of FGF2, suggesting a requirement for autocrine FGF2 signaling in the mesenchyme cells for AQP5 expression. We conclude that basement membrane proteins and mesenchyme cells function as niche factors in salivary organoids.

Development of eight mitochondrial markers for Funariaceae (Musci) and their amplification success in other mosses.

PREMISE OF THE STUDY: In comparison to the wide use of chloroplast markers, few mitochondrial markers are available for phylogenetic studies in bryophytes. We investigated the phylogenetic suitability of several mtDNA markers within the Funariaceae and across mosses. METHODS AND RESULTS: By comparing mitochondrial genomes of two mosses, eight regions with higher substitution rates were identified and sequenced for three species in the Funariaceae and one outgroup taxon. Variations in the substitution rate of these new loci were compared to previously used markers. Thirty-four samples representing all major moss lineages were targeted to assess the universality of the newly designed primers. CONCLUSIONS: The new markers provided similar or more sequence variations in Funariaceae compared to previously developed mtDNA markers. Five out of eight loci were amplified in 70% of other taxa, indicating that these markers may be suitable for phylogenetic studies in other moss lineages.