**2. Olfactory Neuroepithelium and the Non-Invasive Isolation of ONPs**

The olfactory neuroepithelium is a key structure for odor sensing. It consists of a pseudostratified columnar epithelium located on the outer domain of the olfactory mucosa settled on the basement membrane (BM) and the lamina propria (LP) [33]. The cellular composition of these layers has been widely documented based on morphological analysis and the use of characteristic markers for each cell type [34–37]. Figure 1 schematizes the location, cellular components, and molecular markers of the human olfactory mucosa.

**Figure 1.** Cytoarchitecture and cellular components of the human olfactory mucosa. Lamina propria components. Olfactory Ensheathing Cells, Bowman's gland and Olfactory Ectomesenchymal Stem Cells (OE-MSCs). The image indicates the OE-MSCs markers: CD29, CD90, CD44, Nestin, and Vimentin. Olfactory epithelium components. Basal Cells, Olfactory sensory neurons (OSNs) or Olfactory receptor neurons (ORNs), Sustentacular cells, and Microvillar cells. The figure shows basal cell markers: K5 (Keratin 5), K17 (Keratin 17), p63, Sox-2 (SRY-Box Transcription Factor 2), Nestin, BrdU (Bromodeoxyuridine), and Ki-67; ORNs markers: GAP-43 (Growth Associated Protein 43), β-tubulin, OMP (Olfactory Marker Protein), GNG8 (Guanine Nucleotide-binding protein subunit Gamma), and GNG13 (Guanine Nucleotide-binding protein G(I)/G(S)/G(O) subunit Gamma-13)); sustentacular cell markers (SUS-1, Cbr2 (Carbonyl Reductase 2) and Cyp2g1 (Cytochrome P450, family 2, subfamily G, polypeptide 1)) and, microvillar cell marker: (spot-35 proteins). Created with BioRender.com.

The olfactory neuroepithelium is also a source of stem cells, which are capable of self-renewal and can generate neuronal precursors throughout the entire human lifetime. These precursors include neural stem cells known as basal cells. As expected for neural stem cells, basal cells are multipotent and allow the continuous replacement of neuronal and non-neuronal cells such as olfactory receptor neurons (ORNs) and sustentacular cells (of astrocytic lineage), respectively [38–40]. In addition, the LP contains another less accessible population of stem cells, whose features meet most of the minimum criteria of the mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy [41]. As such, they are named as olfactory ectomesenchymal stem cells (OE-MSCs) [42–44].

Isolation of cells of the olfactory neuroepithelium from patients provides a source of cultured neural stem cells, which has been used to model different brain disorders such as schizophrenia, Parkinson's disease, autism, ataxia-telangiectasia, hereditary spastic paraplegia (HSP), and AD [7,45–49]. These neural stem cells can be frozen and stored for subsequent use and tolerate several passages without significantly losing their main

properties. Furthermore, purified cultures obtained by cloning selection through limiting dilution significantly increases cell viability at least until passage 60 [50]. In this work, we will refer to neural stem cells isolated from the olfactory neuroepithelium as olfactory neuronal precursors (ONPs), similar to [8,9,50,51].

Different strategies have been used to isolate and culture patient-derived ONPs, ranging from biopsies to non-invasive exfoliation of the nasal turbinate. Human ONPs were first isolated by Wolozin et al. from the olfactory neuroepithelium of cadavers or from adult biopsied samples [10,52]. Another similar isolation approach demonstrated that a significant subpopulation of these cells express markers of mature olfactory neurons such as OMP, Golf, NCAM, and NST and look small and bright to the microscope, in contrast to the remaining "dark phase" cells that do not express OMP, but glial markers [53]. However, a systematic characterization of these cultures has shown that after a few days in vitro, both dark and bright phase cells show an intracellular calcium increase in response to odorants, highlighting the neuronal features of these cells [54]. In addition, cells with features of ONPs have also been obtained from dissociated neurospheres, which have been denominated "olfactory neurosphere-derived" (ONS) cells [43]. Alternatively, ONPs can be non-invasively isolated by an exfoliation of the nasal cavity [51]. These exfoliated cells can be cultured in a modified media to propitiate neural lineage maintenance and proliferation. Notably, these neuronal precursors conserve their capability to differentiate into ORNs in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate (Db-cAMP) and, strikingly, maintain their electrical response to odorants [51]. Thus, non-invasively isolated ONPs retain neuronal features similar to those obtained by biopsy. A simplified extraction protocol and the molecular characterization of non-invasively isolated ONPs is shown in Figure 2.

**Figure 2.** Non-invasive isolation of olfactory neuronal precursors (ONPs). (**A**) Schematic cartoon of the isolation protocol based on the extraction of nasal exfoliate with the subsequent adherent culture and enrichment of ONPs. (**B**) Left, the nasal exfoliate is directly seeded on adherent plates, showing a mixture of cell morphologies. Right, after 1–2 weeks ONPs dividing colonies are easily observed with their characteristic morphologies. (**C**) Upper panel, immunofluorescence of cultured ONPs, depicting the stem cell marker Nestin and Ki67 (yellow arrows) to show active cell proliferation. Lower panel, cultured ONPs express neuronal markers such as β3 tubulin. Cell nuclei are shown by DAPI staining. All scale bars = 100 μm. All images were generated in our lab. Created with BioRender.com.
