Human olfactory epithelial cells generated in vitro express diverse neuronal characteristics

Abstract

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.

Figure 1. hOE culture cells express protein markers and mRNA that are characteristic of olfactory sensory neurons

(A) Protein extracts of dissociated hOE culture cells and of rat olfactory epithelium were immunoblotted with antibodies for ACIII, OcNc1, and TrkB. Anti-ACIII (SC-588) pointed to 110 and 145kDa bands. Anti-OcNc1 (Alomone APC-045) identified three bands between 70kDa and 100kDa in hOE cultures, the lower two of which were suppressed by peptide blockade. Anti-TrkB (SC-12) recognized 80, 110, 145kDa bands, all of which were abolished with peptide adsorption. (B) PCR products of the correct sizes for neuronal mRNAs: OcNc1 (CNGA2), ACIII (ADCY3) and TrkB (NTRK2) were generated from cDNA derived from hOE culture cells. A) CNGA2 (157bp) C) ADCY3 (167bp) E) NTRK2 (232bp) No RT controls: B, D, F. Abbreviations: ACIII; adenylate cyclase type 3, OcNc1; olfactory cyclic nucleotide gated channel 1.

Figure 2. Immunocytochemical detection of ACIII, OcNc1 and TrkB

HOE culture cells were immunostained as described with the antibodies used for western blotting: (A) anti-ACIII, (B) anti-OcNc1 and (C) anti-TrkB. Images representative of experiments with cultures from two individuals are shown. Specificity of OcNc1 (D) and TrkB (E) antibodies were determined by pre-incubation with specific peptides. Scale bars = 50μm.

Figure 3. hOE culture cells express mRNA for OMP, OcNc1 and odorant receptors

OMP probe was detected using Vector Red. OcNc1 probe was detected with TSA-FITC. OR3A1 probe was detected with TSA-FITC. The OR1A1 probe was detected with Vector Red when used with the OR3A1 probe. (A) Cell reactive with probes for OMP and OcNc1. (B) Cell hybridized with probes for both OR3A1 and OR1A1, labeling only with OR3A1. (C) Cell hybridized with probes for both OR3A1 and OR1A1, labeling only with OR1A1. Exposure times are noted on each image. Scale bar = 50μm.

Figure 4. Cultured human olfactory cells respond to helional, a ligand for the OR3A1

Cultured hOE cells were loaded with Fura-2/AM and imaged as described in Experimental Procedures. Stimuli were added for 10 sec, followed by a 2 minute rinse with Ringer's solution. Figure 4 depicts traces from a subset of 165 cells assayed from two experiments using cells derived from the same source as that used for in situ hybridization. Traces for one helional-responsive and three unresponsive cells are shown. Additional experiments using two independent cultures yielded similar results.

Figure 5. hOE cultures express the neurotransmitter receptors 5HT2C, dopamine D2R, and NMDAR1

Membrane extracts of hOE cultures were immunoblotted with antibodies for: 5HT2C, D2R and NMDAR1. HOE cultures showed robust expression of 55 kDa bands for 5HT2C receptor, a 48kDa band for D2R and a 110 kDa band for NMDAR1 with an additional double band at approximately 90kDa.

Figure 6. Ligand stimulation of 5-HT and dopamine receptors activate signaling pathways in hOE cultures

(A) 5HT receptor function. HOE cultures were incubated with DOI, a 5-HT2 agonist, at 37°C for 5 min, in the presence of 0.5 nM [³⁵S]GTPγS. To test the role of specific subtypes in 5-HT mediated activation, the cultured cells were pre-incubated with spiperone (5-HT2A receptor antagonist), SB 204741 (5-HT2B receptor antagonist) or RS 102221 (5-HT2C receptor antagonist) for 10 min. Membrane extracts were immunoprecipitated with antibodies for Gαs/olf, -Gαi, -Gαo or -Gαq/11 and the levels of [³⁵S]GTPγS within the immunoprecipitates were measured by scintillation spectrometry. Statistical differences were analyzed using Newman-Keul's test that followed ANOVA. *p < 0.01. (B) Dopamine receptor function. HOE culture cells were pre-incubated with sch23390 or sulpiride at 37°C for 10 min, which was followed by stimulation with 1 μM dopamine and 1μM ascorbic acid, at 37°C for 5 min in the presence of 0.5 nM [³⁵S]GTPγS. Isoform specific activation of G proteins was assessed for Gαs/olf, -Gαi, -Gαo or -Gαq/11 as described for 5HT receptors in A. Statistical differences were analyzed using Newman-Keul's test that followed ANOVA. *p < 0.01.

Figure 7. Functional integrity of NMDAR signaling in hOE cultures

(A) HOE culture cells were incubated with or without 10μM NMDA +1μM glycine. Protein extracts were immunoprecipitated with antibodies for NMDAR1 (NR1) or NMDAR2A/2B (NR2A/2B) and the immunoprecipitates were analyzed by Western blotting for signaling partners, PLCγ1 and pY220-Src, for NR1 and phosphotyrosine for NR2A/2B. (B) Representative traces from fura-2 loaded hOE culture cells stimulated with 10μM NMDA + 1μM glycine. Of 78 cells imaged in three independent experiments, ∼80% responded to depolarization with high potassium (KCl, 50 mM) with a transient increase in intracellular calcium, indicating the presence of voltage-sensitive calcium channels. Of these, 12% exhibited a transient increase in calcium following 60-90 sec exposure to NMDA (10μM + 1μM Glycine) and ∼35% exhibited gradual increases in calcium which were more accelerated than what is typically seen during a similar time period in the absence of NMDA stimulation (n= 74). Abbreviations; PLCγ1: phospholipase C-γ1, pY-NR2A/2B: phosphotyrosine-NMDAR1A/2B.