Time course of structural and functional maturation of human olfactory epithelial cells in vitro

Abstract

The unique ability of olfactory neurons to regenerate in vitro has allowed their use for the study of olfactory function, regeneration, and neurodegenerative disorders; thus, characterization of their properties is important. This present study attempts to establish the timeline of structural (protein expression) and functional (odorant sensitivity) maturation of human olfactory epithelial cells (hOE) in vitro using biopsy-derived cultured tissue. Cells were grown for 7 days; on each day, cells were tested for odorant sensitivity using calcium imaging techniques and then protein expression of each cell was tested using immunocytochemistry for proteins typically used for characterizing olfactory cells. Previous studies have shown that mature olfactory neurons in vitro attain a unique "phase-bright" morphology and express the olfactory marker protein (OMP). By day 3 in vitro, a variety of cells were odorant-sensitive, including both "phase-bright" and "phase-dark" cells that have previously been considered glial-like cells. The functional maturation of these hOEs appears to take place within 4 days. Interestingly, the emergence of an odorant sensitivity profile of both phase-bright and phase-dark cells preceded the expression of marker protein expression for OMP (which is expressed only by mature neurons in vivo). This structural maturation took 5 days, suggesting that the development of odorant sensitivity is not coincident with the expression of marker molecules that are hallmarks of structural maturation. These results have important implications for the use of hOEs as in vitro models of olfactory and neuronal function.

Chronic odorant exposure upregulates acquisition of functional properties in cultured embryonic chick olfactory sensory neurons

“…In this example (Fig B), both OSNs responded to high K + depolarization, and only one cell responded to odorant stimulation. The magnitude and frequency of the [Ca 2+ ] i responses seen in our study were similar to those seen in our previously published work (Gomez and Celii, ; Yazinski and Gomez, ).…”

“…The relative number of neurons increased steadily through day 4; after this point, the growth of the non‐neuronal cells tended to outpace the rate of neuron differentiation, resulting in a plateau. This timeline of neuronal differentiation was similar to that seen in other olfactory culture systems (Yazinski and Gomez, ). The proportion of NST + cells in untreated vs. odorant‐treated cultures at each time point was compared using a Student t ‐test.…”

“…The total number of NST + cells was counted and expressed as a proportion of total number of cells (determined using DAPI‐stained nuclei). The timeline of maturation was similar to that seen in other in vitro studies on the development of olfactory function (Yazinski and Gomez, ). Note that odorant treatment did not result in an increase in the overall number of neurons.…”

“…In contrast, G olf was expressed in 50% of the untreated cells and in a significantly higher proportion of the odorant‐treated cells (AA‐treated: t 8 = 2.79; P = 0.02; PEA‐treated: t 8 = 8.22; P < 0.0001). The difference in the time course of OMP and G olf expression has been seen previously (Yazinski and Gomez, ) and suggests that the genes that regulate expression of primary odorant signaling are modulated differently from those that regulate signal modulation. At day 5, odorant treatment resulted in significant increases in both OMP (AA‐treated: t 8 = 9.85; P < 0.0001; PEA‐treated: t 8 = 6.86; P = 0.0001) and G olf (AA‐treated: t 8 = 13.80; P < 0.0001; PEA‐treated: t 8 = 15.00; P < 0.001) expression.…”

“…Under these conditions, cells remained viable for several minutes up to 1 hr. Data were analyzed as described previously (Jung et al, ; Yazinski and Gomez, ). Briefly, individual cells were visually identified and selected by drawing a region of interest (ROI) to restrict calcium measurements within the individual ROIs.…”