Mary T. Lucero

Properties of four human embryonic stem cell lines maintained in a feeder‐free culture system

Several laboratories have begun evaluating human ES (hES) cell lines; however, direct comparisons between different hES cell lines have not been performed. We have characterized the properties of four human cell lines maintained in feeder‐free culture conditions. Quantitative assessment of surface markers, microarray analysis of gene expression patterns, expression of SOX‐2, UTF‐1, Rex‐1, OCT3/4, CRIPTO, and telomerase activity demonstrated similar patterns in all hES cell lines examined. Undifferentiated hES cells do not respond to neurotransmitters such as acetylcholine, glutamate, and gamma‐aminobutyric acid. In addition, the undifferentiated hES cells possess gap junctions. Although similarities in marker expression were observed, allotyping showed that all four lines have a distinct HLA profile, predicting differences in transplantation responses. These data provide the first detailed comparison of different hES cell lines and demonstrate remarkable similarities among lines maintained in identical culture conditions. Developmental Dynamics 229:243–258, 2004.

Identification and characterization of neuronal precursors and their progeny from human fetal tissue

We have examined primary human neuronal precursors (HNPs) from 18–22‐week‐old fetuses. We showed that E‐NCAM/MAP2/β‐III tubulin‐immunoreactive neuronal precursors divide in vitro and could be induced to differentiate into mature neurons in 2 weeks. HNPs did not express nestin and differentiated slowly compared to rodent neuronal restricted precursors (NRPs, 5 days). Immunocytochemical and physiological analyses showed that HNPs could generate a heterogeneous population of neurons that expressed neurofilament‐associated protein and various neurotransmitters, neurotransmitter synthesizing enzymes, voltage‐gated ion channels, and ligand‐gated neurotransmitter receptors and could fire action potentials. Undifferentiated and differentiated HNPs did not coexpress glial markers. Only a subset of cells that expressed GFP under the control of the Tα1 tubulin promoter was E‐NCAM/β‐III tubulin‐immunoreactive, indicating nonexclusive overlap between these two HNP cell populations. Overall, HNPs resemble NRPs isolated from rodent tissue and appear to be a neuronal precursor population. J. Neurosci. Res. 66:356–368, 2001.

Calcium store-mediated signaling in sustentacular cells of the mouse olfactory epithelium.

Sustentacular cells have structural features that allude to functions of secretion, absorption, phagocytosis, maintenance of extracellular ionic gradients, metabolism of noxious chemicals, and regulation of cell turnover. We present data detailing their dynamic activity. We show, using a mouse olfactory epithelium slice model, that sustentacular cells are capable of generating two types of calcium signals: intercellular calcium waves where elevations in intracellular calcium propagate between neighboring cells, and intracellular calcium oscillations consisting of repetitive elevations in intracellular calcium confined to single cells. Sustentacular cells exhibited rapid, robust increases in intracellular calcium in response to G‐protein coupled muscarinic and purinergic receptor stimulation. In a subpopulation of sustentacular cells, oscillatory calcium transients were evoked. We pharmacologically characterized the properties of purinergic‐evoked increases in intracellular calcium. Calcium transients were elicited by release from intracellular stores and were not dependent on extracellular calcium. BAPTA‐AM, a cytosolic calcium chelator, and cyclopiazonic acid, an endoplasmic reticulum Ca2+‐ATPase inhibitor irreversibly blocked the purinergic‐induced calcium transient. Phospholipase C antagonist U73122 inhibited the purinergic‐evoked calcium transient. 2‐Aminoethoxydiphenyl borate, an inositol‐1,4,5‐trisphosphate (IP3) receptor antagonist, and the ryanodine receptor (RyR) antagonists tetracaine and ryanodine, inhibited the UTP‐induced calcium transients. Collectively, these data suggest that activation of the phospholipase C pathway, IP3‐mediated calcium release, and subsequent calcium‐induced‐calcium release is involved in ATP‐elicited increases in intracellular calcium. Our findings indicate that sustentacular cells are not static support cells, and, like glia in the central nervous system, have complex calcium signaling.

Conditionally immortalized clonal cell lines from the mouse olfactory placode differentiate into olfactory receptor neurons

To test extracellular signals that direct the development of the olfactory system, we have generated clonal temperature-sensitive cell lines that represent distinct cellular lineages derived from the E10 mouse olfactory placode. Two of these lines, OP6 and OP27, express (at the permissive temperature), a transcriptional profile representing intermediate-late developmental stages in the olfactory receptor neuron (ORN) lineage. At the nonpermissive temperature, both OP6 and OP27 cells can be induced by all-trans retinoic acid to differentiate into a population of mature bipolar ORN-like cells. In response to retinoic acid, differentiated OP6 and OP27 down-regulate neuron-specific transcription factors required for early stages of neuronal differentiation, and shift active components of the neurotrophin signaling cascade (Trk receptors) into a kinase inactive state. When morphologically mature, OP6 and OP27 express the mature ORN chemosensory signaling components, olfactory G-protein (G(olf)), Type III adenylate cyclase (ACIII), OCNC1, and the olfactory marker protein (OMP). OP27 expresses one odorant receptor, OR 27-3. OP6 expresses two very closely related receptors, OR 6-13 and OR 6-8. Voltage-gated sodium and potassium channels resembling those recorded from primary cultures of ORNs can also be recorded from a subset of differentiated OP6 cells.

Purinergic Receptor Antagonists Inhibit Odorant-Induced Heat Shock Protein 25 Induction in Mouse Olfactory Epithelium

Heat shock proteins (HSPs) accumulate in cells exposed to a variety of physiological and environmental factors, such as heat shock, oxidative stress, toxicants, and odorants. Ischemic, stressed, and injured cells release ATP in large amounts. Our hypothesis is that noxious stimulation (in this case, strong odorant) evokes the release of ATP in the olfactory epithelium (OE). Extracellular ATP, a signal of cellular stress, induces the expression of HSPs via purinergic receptors. In the present study, in vivo odorant exposure (heptanal or R‐carvone) led to a selective induction of HSP25 in glia‐like sustentacular cells in the Swiss Webster mouse OE, as previously shown in rats (Carr et al., 2001). Furthermore, in vitro and in vivo administration of purinergic receptor antagonists suramin and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS) blocked the expression of HSP25 immunoreactivity in sustentacular cells. ATP released by acutely injured cells could act as an early signal of cell and tissue damage, causing HSP expression and initiating a stress signaling cascade to protect against further damage. Sustentacular cells have a high capacity to detoxify xenobiotics and thereby protect the olfactory epithelium from airborne pollutants. Thus, the robust, rapid induction of HSPs in sustentacular cells may help maintain the integrity of the OE during exposure to toxicants.

Centrin 2 Is Required for Mouse Olfactory Ciliary Trafficking and Development of Ependymal Cilia Planar Polarity

Centrins are ancient calmodulin-related Ca2+-binding proteins associated with basal bodies. In lower eukaryotes, Centrin2 (CETN2) is required for basal body replication and positioning, although its function in mammals is undefined. We generated a germline CETN2 knock-out (KO) mouse presenting with syndromic ciliopathy including dysosmia and hydrocephalus. Absence of CETN2 leads to olfactory cilia loss, impaired ciliary trafficking of olfactory signaling proteins, adenylate cyclase III (ACIII), and cyclic nucleotide-gated (CNG) channel, as well as disrupted basal body apical migration in postnatal olfactory sensory neurons (OSNs). In mutant OSNs, cilia base-anchoring of intraflagellar transport components IFT88, the kinesin-II subunit KIF3A, and cytoplasmic dynein 2 appeared compromised. Although the densities of mutant ependymal and respiratory cilia were largely normal, the planar polarity of mutant ependymal cilia was disrupted, resulting in uncoordinated flow of CSF. Transgenic expression of GFP-CETN2 rescued the Cetn2-deficiency phenotype. These results indicate that mammalian basal body replication and ciliogenesis occur independently of CETN2; however, mouse CETN2 regulates protein trafficking of olfactory cilia and participates in specifying planar polarity of ependymal cilia.

Catecholamine concentrations in rat nasal mucus are modulated by trigeminal stimulation of the nasal cavity

Olfactory mucus provides the perireceptor environment in which the initial steps of olfactory signal transduction occur [5]. Extrinsic autonomic and trigeminal innervation controls mucus secretion and may release neurotransmitters into nasal mucus [13]. We quantitated catecholamines in rat nasal mucus and found that catecholamine levels first increased and then declined with trigeminal stimulation. These data indicate that catecholamine levels are regulated in nasal mucus and could modulate the odor sensitivity of olfactory sensory neurons.

Ionic conductances in sustentacular cells of the mouse olfactory epithelium

The electrical properties of sustentacular cells (SCs) in the olfactory epithelium (OE) were investigated in tissue slices taken from neonatal mice (P0–P4). Conventional whole‐cell recordings were obtained from SCs and also from olfactory receptor neurones (ORNs) insitu. SCs had a larger apparent cell capacitance (Ccell) (18.6 ± 0.5 pF) than ORNs (4.4 ± 0.4 pF) and a lower apparent membrane resistance (Rm) (160 ± 11 MΩversus 664 ± 195 MΩ, respectively). When corrected for a seal resistance of 1 GΩ, these mean Rm values were increased to 190 MΩ and 2 GΩ in SCs and ORNs, respectively. SCs generated a TTX (1 μm)‐resistant voltage‐activated Na+ current (INa) that had a peak density at −38 mV of −44 pA pF−1 and supported action potential firing. Peak current density of INa in neurones was 510 ± 96 pA pF−1. The outward K+ current in SCs was composed (> 70%) of a TEA (2 mm)‐sensitive component that was mediated by the opening of large‐conductance (237 ± 10 pS; BK) channels. The resting leak conductance (gL) of SCs was permeable to monovalent cations and anions and was largely inhibited by substitution of external Na+ with NMDG and by internal F− with gluconate. gL deactivated up to 50% at potentials negative of −70 mV and was inhibited by 18β‐glycyrrhetinic acid (20 μm). SCs were identified using fluorescent dyes (Lucifer Yellow and Alexa Fluor 488) in the whole‐cell patch pipette‐filling solution. Our findings indicate that SCs in the OE of neonates are electrically excitable and are distinguishable from neurones by a having a resting gL.

Calcium Signalling in Squid Olfactory Receptor Neurons

Isolated squid olfactory receptor neurons respond to dopamine and betaine with hyperpolarizing conductances. We used Ca²⁺ imaging techniques to determine if changes in intracellular Ca²⁺ were involved in transducing the hyperpolarizing odor responses. We found that dopamine activated release of Ca²⁺ from intracellular stores while betaine did not change internal Ca²⁺ concentrations. Application of 10 mM caffeine also released Ca²⁺ from intracellular stores, suggesting the presence of ryanodine-like receptors. Depletion of intracellular stores with 100 μM thapsigargin revealed the presence of a Ca²⁺ store depletion-activated Ca²⁺ influx. The influx of Ca²⁺ through the store-operated channel was reversibly blocked by 10 mM Cd²⁺. Taken together, these data suggest a novel odor transduction system in squid olfactory receptor neurons involving Ca²⁺ release from intracellular stores.

Peripheral modulation of smell: Fact or fiction?

Despite studies dating back 30 or more years showing modulation of odorant responses at the level of the olfactory epithelium, most descriptions of the olfactory system infer that odorant signals make their way from detection by cilia on olfactory sensory neurons to the olfactory bulb unaltered. Recent identification of multiple subtypes of microvillar cells and identification of neuropeptide and neurotransmitter expression in the olfactory mucosa add to the growing body of literature for peripheral modulation in the sense of smell. Complex mechanisms including perireceptor events, modulation of sniff rates, and changes in the properties of sensory neurons match the sensitivity of olfactory sensory neurons to the external odorant environment, internal nutritional status, reproductive status, and levels of arousal or stress. By furthering our understanding of the players mediating peripheral olfaction, we may open the door to novel approaches for modulating the sense of smell in both health and disease.