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Background:
The olfactory mucosa has emerged as a promising source of mesenchymal stem cells with neurogenic potential. These cells exhibit neural, glial, and mesenchymal properties, making them attractive candidates for regenerative medicine, particularly in treating neurodegenerative and immunemediated disorders.
Methods:
This systematic review analyzed existing literature on the isolation, characterization, and therapeutic applications of olfactory mucosa mesenchymal stem cells. The review assessed variations in isolation techniques, culture conditions, and differentiation potential, as well as preclinical and clinical applications.
Results:
Olfactory mucosa mesenchymal stem cells express key neural and mesenchymal markers, including Nestin, SRY-box 2, Glial Fibrillary Acidic protein, CD44, and CD105, confirming their multilineage differentiation capacity. Their ability to secrete neurotrophic factors such as Brain-Derived Neurotrophic Factor, Nerve Growth Factor, and Glial Cell Derived Neurotrophic Factor underscores their role in neural repair. While most studies successfully isolated olfactory mucosa mesenchymal stem cells via biopsy, differences in sampling depth and culture media influenced cell yield and growth patterns. Preclinical studies suggest that olfactory mucosa mesenchymal stem cells (OM-MSCs) may represent a promising experimental model for neurological disorders—including Parkinson’s disease, spinal cord injury, schizophrenia, and retinal diseases—although current evidence remains preliminary and translational efficacy has not yet been established. However, challenges remain in standardizing protocols, addressing donor variability, and ensuring clinical safety.
Conclusion:
Olfactory mucosa mesenchymal stem cells represent a promising avenue for neurological and regenerative therapies. Despite their potential, further research is needed to optimize isolation techniques, enhance reproducibility, and navigate regulatory hurdles. Collaborative efforts between researchers, clinicians, and regulatory bodies will be essential to translating OM-MSC research into viable clinical applications.
IntroductionOver the past decades, stem cell research has advanced substantially, with its potential in healthcare and in expanding our understanding of human biology increasingly recognized by the scientific community. Stem cells derived from diverse tissues are being harvested, isolated, and studied, not only as reservoirs for clinical applications but also as models to investigate the cellular processes and signaling pathways that govern tissue function and regeneration, furthermore new approaches such as induced pluripotent stem cells (iPSCs) generation and differentiation, organoid production and CRISP-Cas9 editing changed the research in stem cells not only as therapeutic tools but also as potential models to study human development and disease progression (Bosio et al., 2009; Lee and Son, 2021; Bellon, 2024; Whiteley et al., 2022).
The olfactory neuroepithelium, located within the olfactory cleft and lining the upper nasal septum, dorsal nasal vault, and superior turbinate, performs the initial step of olfaction (Ross and Pawlina, 2006; Pinto, 2011). In mammals, chemosensory input from the olfactory system is essential for survival, feeding, reproduction, and predator avoidance, highlighting its evolutionary importance (Pinto, 2011; Agafonova et al., 2025; Díaz et al., 2013; Costanzo et al., 1992). Although in humans, olfaction plays a less dominant role, it still contributes to nutrition, safety, sensory pleasure, and overall well-being (Pinto, 2011).
In humans, the olfactory mucosa (OM) measures 60–80 μm in thickness, covers ~10 cm2, and is composed of a pseudostratified epithelium with basal, supporting, and olfactory receptor cells but lacking goblet cells (Ross and Pawlina, 2006; Patel and Pinto, 2014; Choi and Goldstein, 2018). Cells present in this region include olfactory ensheathing cells (OECs), a specialized glial population within the olfactory system, are among the cells found in this area. They share traits with both Schwann cells and astrocytes, which are linked with the peripheral and central nervous systems, respectively. OECs maintain close contact with the small, unmyelinated axons of olfactory receptor neurons, guiding them from the basal lamina of the epithelium to the olfactory bulb. OECs are also located in regions where other stem cell populations can be found; however, they are not the primary focus of this review, as their relevance is secondary to the stem cell types under discussion (Agafonova et al., 2025).
A hallmark of the olfactory system is its lifelong neurogenesis. Because olfactory sensory neurons (OSNs) are exposed to environmental insults (including pathogens, toxins, and trauma) and survive for only a few months before apoptosis, they must be continuously replaced by newly generated neurons derived from resident Neural Stem Cells (NSCs) (Mackay-Sim, 2010; Choi and Goldstein, 2018).
The persistence of olfaction throughout life, despite this vulnerability, underscores the regenerative capacity of the olfactory epithelium. This capacity is mediated by stem cells capable of self-renewal and progression through intermediate progenitors to form multiple differentiated cell types, including neurons (Berger et al., 2020; Chen et al., 2019; Durante et al., 2020). Two main populations are found in the basal layer: globose basal cells (GBCs), which actively replenish neurons, and horizontal basal cells (HBCs), quiescent reserve cells that activate after severe epithelial injury (Choi and Goldstein, 2018; Mackay-Sim, 2010; Calof et al., 1998; Carter et al., 2004; Schwob et al., 2017). Another population, olfactory epithelium mesenchymal stem cells (OE-MSCs), resides in the lamina propria. These cells combine mesenchymal and NSCs features, with the ability to differentiate into mesodermal lineages or cross the basement membrane to generate OSNs (Calof et al., 1998; Schwob et al., 2017).
Multiple terms have been described in literature to define OM-derived stem/progenitor populations. In this review, we use the following operational definitions: (1) Neural stem/progenitor cells (NSCs): basal epithelial stem/progenitor cells within the olfactory epithelium (including GBCs and HBCs), primarily generating neuronal and supporting lineages; (2) Olfactory epithelium / olfactory mucosa mesenchymal stem cells (OE-MSCs / OM-MSCs; also referred to as ecto-MSCs): mesenchymal-like stromal cells predominantly located in the lamina propria, typically adherent and expressing MSC markers (e.g., CD73/CD90/CD105) with variable neural/glial markers (Nestin/Sox2/GFAP); (3) Olfactory stem cells (OSCs)/olfactory neurosphere-derived cells: an umbrella term often used for mixed sphere-forming cultures containing epithelial progenitors and stromal components, whose phenotype is strongly protocol-dependent. Where possible, we report the original authors’ terminology but interpret results according to anatomical origin and marker profile to avoid conflating distinct cell populations.
The presence of these diverse stem cell types has been confirmed in both humans and other mammals, suggesting that similar mechanisms of stem cell activity extend across species (Calof et al., 1998; Schwob et al., 2017; Mackay-Sim, 2010). A unique advantage of the olfactory system is its anatomical accessibility in humans and other mammals, permitting direct sampling of NSCs and progenitors. Because these cells share developmental origins with central nervous system neurons and glia, they provide a valuable model for studying neural development and regeneration without invasive brain procedures.
Despite this promise, methods for isolating OM-derived stem cells remain heterogeneous. Most studies have used biopsy, a technique that provides adequate tissue but is invasive and difficult to standardize (Schwob et al., 2017; Lanza et al., 1993; Fletcher et al., 2017). To reduce invasiveness, nasal brushing was introduced more than 30 years ago, yielding high cell numbers with minimal trauma by using nylon-bristled brushes (Jafek et al., 2002; Stokes et al., 2014; Orrú et al., 2014; Féron et al., 1998). However, relatively few studies have adopted this method, limiting comparability across protocols. The objective of this systematic review is to critically evaluate harvesting and culture methods for olfactory mucosa stem cells, highlight methodological differences, and assess how these influence outcomes. Specifically, we examine approaches to sampling, pre-culture processing, and culture conditions, as well as the impact of added growth factors or supplements. We also analyze the reported success of isolations in terms of neuronal lineage differentiation. To our knowledge, no prior systematic review has synthesized these aspects, and we aim to provide a comprehensive framework for future research.
Materials and methodsSearch strategyA systematic review was conducted between May 1, 2022, and November 10, 2024, according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guidelines (Liberati et al., 2009). A systematic electronic search for studies was conducted that reported research done on mammals in the last 12 years focusing entirely or partly on the isolation of stem cells from the nasal olfactory epithelium in mammals. Only articles in the English language were selected.
We searched PubMed, Scopus, and Embase databases with wide search strategies for Isolation of Stem cells from nasal olfactory epithelium from humans and other mammals, with samples obtained by either brushing or biopsy. The details of our full literature search strategy and the number of unique terms retrieved from each database are available in Table 1.
Study and yearType of mammal, ageSample populationSample harvesting techniqueDetail of preparation of sample for cultureBenítez-King et al. (2011)Human mean age 27.5 years8Nasal brushingKrolewski et al. (2011)B6.129F1 MiceN/ANasal biopsy(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Mechanical dissociation.
(4) Complex medium.
(5) Spheres.
Toft et al. (2012)Fischer ratsN/ANasal biopsy(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Enzymatic and mechanical dissociation.
(4) Complex medium.
(5) Spheres.
Lindsay et al. (2013)Human(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Enzymatic and mechanical dissociation.
(4) Complex medium.
(5) Spheres.
Stamegna et al. (2014)Sprague Dawley Rat(1) Simple dissection and mincing, direct plating without dissociation or with enzymatic and mechanical dissociation.
(2) Complex medium.
(3) Spheres (dissociated by trypsin).
Thakur et al. (2014)Albino Wistar Rat6Nasal biopsyOhnishi et al. (2015)Human(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Direct plating of the undissociated tissue in basic medium.
(4) Recover of the cells.
(5) Plating in complex medium.
(6) Spheres.
Ercolin et al. (2016)Rabbit(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Direct plating of the undissociated tissue in basic medium (15% FBS).
(4) Plating in basic medium. After 72 h of incubation remove non-adherent cells.
(5) Cultivate adherent cells until 70% confluence in basic medium (15% FBS).
Batioglu-Karaaltin et al. (2016)HumanComplex dissection and mincing.
Plating in basic medium, refreshed every other day, and cell adhesion is observed after 2 weeks.
Ge et al. (2016)Human(1) Complex dissection and mincing.
(2) Direct plating of the tissue, simple medium was changed every 2 or 4 days.
(3) Seven to 8 days after, stem cells will begin to invade the culture dish and after 2–3 weeks.
(4) When confluency is reached, passage and transfer the cells to culture flasks.
(5) After the first harvesting plate in basic medium added with FGF2 and EGF, medium was added every 2 to 4 days.
Tanos et al. (2017)HumanComplex dissection and mincing.
Enzymatic dissociation.
Plating in basic medium.
When adherent cells reached confluency, they were passaged using trypsin.
Sphere cells were allowed to grow for 7 days.
Franco et al. (2017)Humans and Swiss-Webster Mice (5 weeks)N/ANasal brushing in humans:Exfoliation.
Tissue washing with antibiotics.
Enzymatic dissociation.
Plating in complex medium.
Spheres were weekly passaged.
Lu et al. (2017)Human20Nasal Biopsy(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics:
(3) Direct tissue plating in basic medium.
(4) Layer.
Muniswami et al. (2017)Rat10Nasal biopsy(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Enzymatic dissociation.
(4) Plating in complex medium.
(5) Change the medium every 2 days.
Veron et al. (2018)6 genera: Mouse, Rat, Rabbit, Sheep, Dog, Horse, Gray mouse lemur, MacaqueTotal 34(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Enzymatic dissociation.
(4) Plating in complex medium.
(5) Change the medium every 2 days.
Li et al. (2018)Wistar Rats (Embryonic day 17)N/ANasal biopsy(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Enzymatic dissociation.
(4) Plating in complex medium.
(5) Change the medium every 2 days.
(6) Neurospheres.
Jiménez-Vaca et al. (2018)Human(1) Exfoliation.
(2) Tissue washing with antibiotics.
(3) Mechanical dissociation.
(4) Plating in Complex medium.
(5) Spheres.
Bagher et al. (2018)Human(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Direct plating of the tissue.
(4) Basic medium plating.
(5) Layer.
Simorgh et al. (2019)Wistar Rats(1) Complex dissection and mincing.
(2) Tissue direct plating.
(3) Basic medium plating.
(4) Replating of cells in the following passages.
Kim et al. (2019)BALB/c Mice(1) Simple dissection and mincing (pull 5 mice).
(2) Tissue washing with antibiotics.
(3) Enzymatic and mechanical dissociation.
(4) Complex medium plating.
(5) Spheres mechanical dissociation.
Esaki et al. (2019)Mice(1) Simple dissection and mincing (pull 5 mice).
(2) Tissue washing with antibiotics.
(3) Dissociation by 70 μm cell strainer.
(4) Mechanical dissociation.
(5) Complex medium plating.
(6) Fresh medium was added to the cultures every third day.
(7) Spheres.
Salehi et al. (2019)HumanN/ANasal biopsy(1) Simple dissection and mincing (pull 5 mice).
(2) Tissue washing with antibiotics.
(3) Direct tissue plating.
(4) Basic medium plating.
(6) Change the medium every 3 days.
(7) Layer.
Alizadeh et al. (2019b)HumanN/ANasal biopsy(1) Complex dissection and mincing, enzymatic treatment.
(2) Direct tissue plating.
(3) Basic medium plating.
(4) Layer.
Bagher et al. (2019)Human2Nasal biopsy(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Direct tissue plating.
(4) Basic medium plating.
(5) Dissociation replating.
(6) Change the medium every 3 days.
(7) Layer.
Alizadeh et al. (2019a)Human(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Direct tissue plating.
(4) Basic medium plating.
(5) Dissociation replating.
(6) Change the medium every 3 days.
(7) Layer.
Solís-Chagoyán et al. (2019)Human54 years old healthy womanNasal brush(1) Exfoliation.
(2) Cells washing with antibiotics.
(3) Basic medium plating.
(4) Dissociation replating.
(5) Change the medium every 3 days.
(6) Layer.
Pérez-Luz et al. (2020)Human7 total(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Direct tissue and cells plating.
(4) Complex medium plating.
(5) After 3 days growing remove non adherent cells.
(6) Dissociation replating.
(7) Change the medium every 3 days.
(8) Layer.
Alvites et al. (2020)Sprague Dawley Rat(1) Simple dissection and mincing.
(2) Direct tissue plating.
(3) Basic medium plating.
(4) Passaged by trypsin.
(5) Layer.
Jafari et al. (2020)Human10Nasal biopsy(1) Simple dissection and mincing.
(2) Enzymatic dissociation.
(3) Basic medium plating.
(4) After 72 h, non-adherent cells removed with PBS.
(5) Medium changed every 3 days.
(6) Layer.
Tian et al. (2020)C57BL/6 Mice(1) Simple dissection and mincing.
(2) Direct tissue plating for 7 days.
(3) Basic medium plating.
(4) Passaged by trypsin.
(5) Layer.
Liu et al. (2020)Human(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Direct tissue plating.
(4) Basic medium plating.
(5) Passaged by trypsin.
(6) Layer.
Voronova et al. (2020)Human40Nasal biopsy(1) Complex dissection and mincing.
(2) Direct tissue plating.
(3) Basic medium plating.
(4) Passaged by trypsin.
(5) Basic medium plating added with FGF2 and EGF.
(6) Layer.
Farhadi et al. (2021)HumanN/ANasal biopsy(1) Simple dissection and mincing.
(2) Tissue washing with antibiotics.
(3) Direct tissue plating.
(4) Basic medium plating.
(5) Remove non adherent cells.
(6) Passaged by trypsin.
(7) Layer.
Hamidabadi et al. (2021)HumanN/A
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