Method Article

Generation and Differentiation of Human Olfactory Epithelial Organoids from Adult Stem Cells

DOI:

10.3791/70917

⸱

April 3rd, 2026

 ,  ,  ,  ,  ,  ,  , 
1The Department of Otolaryngology, People's Hospital of Tibet Autonomous Region, 2The Department of Otolaryngology-Head & Neck Surgery, West China Hospital, Sichuan University, 3The Department of Medicine and Engineering Interdisciplinary Research Laboratory of Nursing & Materials, West China Hospital, Sichuan University, 4The Department of Otolaryngology, People's Hospital of Shannan City in Tibet Autonomous Region, 5The Upper Airways Research Laboratory, Department of Otolaryngology, Ghent University

Corresponding Authors: Luo Ba <bhanor@163.com>, Jintao Du <dujt@scu.edu.cn>

* These authors contributed equally

In This Article

Summary

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This protocol outlines an established approach for isolating olfactory progenitor cells from adult human superior turbinate tissue to generate olfactory epithelial organoids. It also details a chemical induction strategy to drive the differentiation of these organoids into mature, olfactory sensory neurons, thereby enabling the modeling of human olfactory neurogenesis.

Abstract

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The human olfactory epithelium possesses remarkable neurogenic capacity, sustained by a resident population of basal stem cells; however, establishing a dynamic in vitro model that faithfully recapitulates this regenerative process has been proven challenging. Here, we present an established protocol for generating olfactory organoids from progenitor cells isolated from adult human superior turbinate tissue. These organoids maintain stemness, as evidenced by SOX2 expression, and display a spatially organized differentiation pattern, characterized by uniform expression of the immature neuronal marker GAP43 and peripheral localization of the mature olfactory sensory neuron marker OMP. Subsequent targeted differentiation with the Notch inhibitor LY411575, the Wnt activator CHIR-99021, and retinoic acid generated marker-positive olfactory sensory neuron–like cells that co-expressed OMP and the neuronal marker Tuj1 (class III β-tubulin). This human-derived model circumvents the limitations of murine systems and provides a robust in vitro platform for studying olfactory neurogenesis, COVID-19–associated anosmia, and other olfactory-related neurodegenerative disorders.

Introduction

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The human olfactory system supports continuous neurogenesis,1 a process primarily driven by basal stem cells in the olfactory epithelium, specifically, horizontal basal cells (HBCs) and globose basal cells (GBCs).2,3 GBCs constitutively differentiate into olfactory sensory neurons (OSNs), whereas HBCs remain quiescent until activated by injury.4,5 Olfactory perception begins when odorant molecules bind to receptors on the cilia of OSNs, triggering a signal transduction cascade that relays information to the olfactory bulb and co....

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Protocol

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This study was conducted with approval from the Institutional Review Board (IRB) of West China Hospital of Sichuan University (Approval No. 2024 (31)). Written informed consent was obtained from all participating patients prior to tissue collection and data publication. Superior turbinate tissue specimens were obtained from patients undergoing endoscopic skull base surgery or ethmoid sinus surgery for cerebrospinal fluid rhinorrhea.

1. Tissue sample processing and transport

  1. Immediately after collection, place the tissue samples in a prechilled, sterile transport medium, composed of DMEM/F12/HEPES basal medium su....

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Results

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Isolation of olfactory epithelial tissue and culture of primary olfactory epithelial organoids
To establish a culture system for olfactory epithelial organoids, we isolated olfactory epithelium from human superior turbinate tissue and embedded it in a three-dimensional Matrigel matrix (Figure 1). Building on the defined culture conditions reported by Ren et al., we validated and fine-tuned the concentrations of key growth factors, EGF, Noggin, Wnt3a, and R-pondin 1, toge.......

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Discussion

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Organoids represent a unique in vitro model because their self-organizing, three-dimensional architecture closely recapitulates that of native human tissues and, in some cases, can be histologically indistinguishable from actual organs.14 Establishing human organoid models is therefore critical for studying human diseases. Here, we present a protocol for generating an organoid culture system from primary human olfactory epithelial cells that reliably differentiate into mature olfactory se.......

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Disclosures

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The authors have no conflicts of interest to declare.

Acknowledgements

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The authors sincerely thank all patients who generously donated superior turbinate tissue specimens, this study would not have been possible without their contribution. Financial support for the research, authorship, and/or publication of this article was received. This work was supported by the National Natural Science Foundation of China (Ba L: 82160209 and 82460217), the Sichuan University–Zigong Municipal Government Science and Technology Cooperation Special Fund (Yang H: 2023CDZG-18), and the Natural Science Foundation of the Tibet Autonomous Region (Cong TC: XZ2023ZR-ZY10(Z)).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
ALEXA FLUOR 488 DONKEYThermoA110551:500
ALEXA FLUOR 555 DONKEYThermoA315721:500
Anti-GAP43 antibodyAbcamab75810 1:160
Anti-OMP antibodySANTA CRUZsc-3658181:50
Anti-Sox-2 antibodySANTA CRUZsc-3658231:50
B-27 SupplementGibco175040442%
CHIR99021NovoproteinHY-101823 μM
DMEM/F12/HEPESProcellPM150310/
Glutamax (L-glutamine supplement)Gibco350500611%
LY411575NovoproteinHY-507525 μM
Matrigel (basement membrane matrix)Corning3562314% (Vol/Vol)
N-2 SupplementGibco175020481%
Organoid Dissociation ReagentPrecedoPRS-ODR/
Organoid Recovery SolutionThewellbioMS04-100/
Primocin (primary antimicrobial agent)Invivogenant-pm-05100 μg/mL
Recombinant Human EGFNovoproteinC02950 ng/mL
Recombinant Human NogginNovoproteinCB89100 ng/mL
Recombinant Human RSPO1NovoproteinCX83200 ng/mL
Recombinant Human Wnt3aNovoproteinC18K50 ng/mL
Retinoic acidMCEHY-146491 μM
Y27632MCEHY-1007110 μM

References

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  1. Hummel, T., et al. Olfactory dysfunction: etiology, diagnosis, and treatment. Dtsch Arztebl Int. 120 (9), 146-154 (2023).
  2. Firestein, S. How the olfactory system makes sense of scents. Nature. 413 (68....

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Tags

Olfactory OrganoidsHuman Stem CellsOlfactory EpitheliumOrganoid DifferentiationBasal Stem CellsProgenitor Cell IsolationSOX2 ExpressionOlfactory Sensory NeuronsNotch Inhibitor DifferentiationNeurogenesis Model

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