Analysis of a human fungiform papillae cDNA library and identification of taste-related genes

Various genes related to early events in human gustation have recently been discovered, yet a thorough understanding of taste transduction is hampered by gaps in our knowledge of the signaling chain. As a first step toward gaining additional insight, the expression specificity of genes in human taste tissue needs to be determined. To this end, a fungiform papillae cDNA library has been generated and analyzed. For validation of the library, taste-related gene probes were used to detect known molecules. Subsequently, DNA sequence analysis was performed to identify further candidates. Of 987 clones sequenced, clustering results in 288 contigs. Comparison of these contigs with genomic databases reveals that 207 contigs (71.9%) match known genes, 16 (5.6%) match hypothetical genes, eight (2.8%) match repetitive sequences and 57 (19.8%) have no or low similarity to annotated genes. The results indicate that despite a high level of redundancy, this human fungiform cDNA library contains specific taste markers and is valuable for investigation of both known and novel taste-related genes.     

Disruption of sonic hedgehog signaling alters growth and patterning of lingual taste papillae

Taste buds on the anterior part of the tongue develop in conjunction with epithelial-mesenchymal specializations in the form of gustatory (taste) papillae. Sonic hedgehog (Shh) and Bone Morphogenetic Protein 4 (BMP4) are expressed in developing taste papillae, but the roles of these signaling molecules in specification of taste bud progenitors and in papillary morphogenesis are unclear. We show here that BMP4 is not expressed in the early tongue, but is precisely coexpressed with Shh in papillary placodes, which serve as a signaling center for both gustatory and papillary development. To elucidate the role of Shh, we used an in vitro model of mouse fungiform papillary development to determine the effects of two functional inhibitors of Shh signaling: anti-Shh (5E1) antibody and cyclopamine. Cultured E11.5 tongue explants express Shh and BMP4(LacZ) in a pattern similar to that of intact embryos, localizing to developing papillary placodes after 2 days in culture. Tongues cultured with 5E1 antibody continue to express these genes in papillary patterns but develop more papillae that are larger and closer together than in controls. Tongues cultured with cyclopamine have a dose-dependent expansion of Shh and BMP4(LacZ) expression domains. Both antibody-treated and cyclopamine-treated tongue explants also are smaller than controls. Taken together, these results suggest that, although Shh is not involved in the initial specification of papillary placodes, Shh does play two key roles during pmcry development: (1) as a morphogen that directs cells toward a nonpapillary fate, and (2) as a mitogen, causing expansion of the interplacodal epithelium and underlying mesenchyme.     

Isolation and culture of rat embryonic neural cells: a quick protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.     

Bitter taste thresholds, numbers and diameters of circumvallate papillae and their relation with age in a Turkish population

Objectives: This study analyses the relationship between the sense of bitter taste and age. The relationships between these and the numbers and diameters of Circumvallate Papillae (CP) are also analysed. Subjects: Twenty-four elderly subjects (from 65 to 85 years) and 30 young subjects (from 17 to 25 years) were studied. Method: Bitter taste thresholds were determined by the three drop method with an ascending series of concentrations. The numbers and diameters of CP were observed by direct naked eye observation using a wooden tongue depressor, a gauge and a light source. Results: The bitter taste acuity was significantly poorer in the older sample. There was no relationship between the bitter taste acuity and number of papillae but an inverse relationship was observed between the bitter taste acuity and diameters of papillae, thus lower acuity was associated with larger papillae. It was also observed that the diameters of papillae were inversely related to the numbers of CP. The distribution of numbers and diameters of CP were not significantly different between these samples with age.     

Isolation and culture of neural crest cells from embryonic murine neural tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from protocols optimized for the culture of rat NC. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC.     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Isolation and characterization of dendritic cells and macrophages from the mouse intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.     

Isolation and culture of airway epithelial cells from chronically infected human lungs

We describe procedures for isolating and culturing airway epithelial cells from chronically infected human lungs. Experience in our laboratory demonstrated the need to balance pathogen eradication against antibiotic toxicity to epithelial cells. To provide a logical basis for antibiotic selection and dose, we systematically analyzed the cytotoxicity of antibiotics useful against typical pathogens. Alone, colistin, ciprofloxacin, doxycycline, and tobramycin were moderately toxic at concentrations close to those used in cell culture, whereas amphotericin, ceftazidime, chloramphenicol, imipenem, meropenem, piperacillin, sulfamethoxazole/trimethoprim, and vancomycin were nontoxic even at concentrations many times the antimicrobial level. Epithelial cytotoxicity of combined antibiotics was additive, with no evidence of competition or synergism. Antibiotics had little effect on initial cell attachment and did not acutely lyse cells, but inhibited subsequent growth. Interestingly, cytotoxicity decreased markedly with increasing epithelial cell density. Cystic fibrosis (CF) and non-CF epithelial cells showed no differences in sensitivity to the antibiotics tested and initial exposure to antibiotics did not affect the electrophysiologic properties of resistance or short circuit current in well-differentiated cells. Tailored combinations of antibiotics at appropriate doses killed even multidrug-resistant bacteria. Thus, epithelial cells can usually be cultured from chronically infected CF airways.     

Isolation and monolayer culture of human fallopian tube epithelial cells

Summary In the present study we have established a pure monolayer culture system of human fallopian tube epithelial cells. The cells were isolated using collagenase digestion, and were cultured in Medium 199 supplemented with 15% fetal bovine serum. The epithelial cells derived from primary and secondary culture were characterized using immunocytochemical staining and electron microscopy. The cells continued to grow for 2 to 3 wk once the monolayer culture of the cells was established. It is currently possible to maintain the cultures until the third generation. Proliferation of these cells was enhanced by epidermal growth factor but not by basic-fibroblast growth factor, insulin, transferrin, estradiol-17β, or progesterone. This culture system offers a good model for determining characteristics of the tubal epithelium and would permit effective study of co-culture with embryos.     

Isolation and long-term culture of rat,rabbit, and human nasal turbinate epithelial cells

Summary Nasal turbinate epithelial cells were isolated from rats, rabbits, and humans using either a surgical or an in situ enzyme incubation technique. The culture conditions that permit optimal cell attachment and selective growth of the nasal epithelial cells were determined. These conditions will permit the long-term culture of these cells where typically 20 to 30 population doublings were observed. Differences between rat and human nasal epithelial cells were seen in substrate requirements, colony-forming efficiency, and response to fetal bovine serum and bovine serum albumin. These methodology and results will permit mechanistic studies of normal and abnormal cellular function and comparative response studies between nasal epithelial cells from rats and humans. This work was supported under U.S. Environmental Protection Agency contract 68-02-4032.     

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.     

Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture

In an organ culture system under a three-dimensional microenvironment that provides the conditions needed for odontoblast differentiation, a row of odontoblasts can be induced (Kikuchi et al. 1996, 2001). Therefore, in a newly designed three-dimensional cell culture system that fulfils the conditions necessary for odontoblast differentiation (Kikuchi et al. 2002), we examined whether dental papilla cells in rat mandibular incisors could differentiate into tubular dentine-forming cells. In our previously established organ culture system, CM-Dil-labeled cells that were microinjected into isolated dental papillae were replaced by a row of odontoblasts. In a three-dimensional cell culture system, which consists of two kinds of type I collagen in the upper layer over multi-layered cells seeded onto collagen containing Matrigel in the lower layer and which acts as a structural meshwork, dental papilla cells were incubated as multi-layered cells in an artificial extracellular matrix (ECM). The cells aggregated to form a cell mass and invaginated as a cell mass into the ECM. The cells also extended fine fibrillar processes into the ECM. With regard to invagination, the proteolytic activities of matrix metalloproteinase-2 (MMP-2)/membrane type 1-matrix metalloproteinase (MT 1-MMP) were observed on the outer multi-layers of cells within a cell mass adjacent to the ECM. The cell mass progressively shrank to about one-half to one-third of its original diameter and was organized as a tissue surrounded by a newly secreted ECM, like dental pulp-dentine. The cells adjacent to the secreted ECM were constructed as a row of polarized columnar cells. They extended slender processes into the new ECM, which is characteristic of tubular matrix. Dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP 1) genes, which are specific for odontoblast differentiation, were expressed in an aggregated cell mass where tubular matrix-forming cells were induced. Furthermore, the tubular matrix became mineralized under prolonged culture. These results imply that the putative progenitor cells/stem cells residing in dental papillae can differentiate into odontoblasts under appropriate conditions in vitro.     

Functions of human bitter taste receptors depend on N-glycosylation

Human bitter taste receptors of the TAS2R gene family play a crucial role as warning sensors against the ingestion of toxic food compounds. Moreover, the genetically highly polymorphic hTAS2Rs recognize an enormous number of structurally diverse toxic and non-toxic bitter substances, and hence, may substantially influence our individual eating habits. Heterologous expression in mammalian cells is a useful tool to investigate interactions between these receptors and their agonists. However, many bitter taste receptors are poorly expressed at the cell surface of heterologous cells requiring the addition of plasma membrane export promoting epitopes to the native receptor proteins. Currently, nothing is known about amino acid motifs or other receptor-intrinsic features of TAS2Rs affecting plasma membrane association. In the present study, we analyzed the Asn-linked glycosylation of hTAS2Rs at a consensus sequence in the second extracellular loop, which is conserved among all 25 hTAS2Rs. Non-glycosylated receptors exhibit substantially lower cell surface localization and reduced association with the cellular chaperone calnexin. As the auxiliary factors receptor transporting proteins 3 and 4 are able to restore the function of non-glycosylated hTAS2R16 partially, we conclude that glycosylation is important for receptor maturation but not for its function per se .     

Efficient derivation of human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small molecule induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging(1-3). Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells(1-3). Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration(1,4-7). However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity(7-10). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic(11-13). To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules(14) (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders(1, 14). And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons(1, 14, 15). However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.     

Isolation and culture of rat microvascular endothelial cells

The purpose of this study is to identify the separation techniques that result in pure cultures of rat microvascular endothelial cells (MECs). A multistep process is used to optimize the separation of the cells from rat epididymal fat pads, obtaining as pure a culture as possible within a relatively short processing time. The process initially employs the digestion, filtration, and density gradient separation steps. We further describe the use of an attachment phase that allows the differential adherence of contaminating cell types. Immunomagnetic purification is the final step in the process and is performed using anti-PECAM-1 (CD31) monoclonal antibody-labeled DynaBeads.     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.     

Primary culture of gustatory receptor neurons from the blowfly, Phormia regina

Flies provide a powerful model system for exploring signaling systems in gustatory receptor neurons (GRNs). To elucidate the cellular and molecular bases of these signaling systems, we sought to develop techniques to dissociate GRNs. We developed a primary culture of GRNs isolated from the labella of the blowfly, Phormia regina, 4-5 days after pupation. Dissected labella were treated with papain in a low Ca2+ saline solution and shaken in Leibovitz's L-15 medium supplemented with 20-hydroxyecdysone, L-ascorbic acid, and trehalose with a test tube mixer. Released cells were plated and kept at 29 degrees C in a medium containing fetal bovine serum. After a minimum of 2 days in culture, we observed survival or growth of bipolar cells with the characteristic morphology of GRNs. We also examined taste responsiveness by monitoring intracellular Ca2+ with a Ca2+-sensitive fluorescent dye, fluo-3. For some bipolar cells, application of sucrose, NaCl, or LiCl for 5-20 s transiently increased the intracellular Ca2+ levels in cell bodies for 20-30 s. The primary cell culture described here is useful for functional analysis of GRNs.     

A new culture medium for human skin epithelial cells

Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.