Ectomesenchymal Stem Cells Research Articles

Nasal mucosa derived-mesenchymal stem cells from mice reduce inflammation via modulating immune responses.

Mesenchymal stem cells (MSCs) have arisen the attention to be a new attractive therapeutic tool treating autoimmune diseases such as allergic rhinitis (AR). AR is a chronic reversible allergic inflammation caused by the excessive activation of T-helper 2 (Th2) cells. Recently, MSCs have been proposed as a new therapy of AR as it can suppress some cytokines to control allogeneic Th2 response and functions. However, how MSCs function to reduce inflammation remains unclear. In this study, we aimed to investigate the role of ectomesenchymal stem cells (ECTO-MSCs) derived from nasal mucosa in eosinophilic inflammation and how it affects some immunoglobulins and cytokines. We used ovalbumin (OVA) as a sensitizer to induce nasal inflammation in mice by both injection and inhalation. In order to obtain deeper insights into the influences of ECTO-MSCs on nasal inflammation, the migration of ECTO-MSCs was assessed, the numbers of eosinophils and sneezing were counted, and several immunoglobulins and cytokines were measured. Here we show the ECTO-MSCs are able to migrate to inflammation site via tail vein injection. Eosinophils and sneezing were suppressed by ECTO-MSCs. Interestingly, IgE, interleukin (IL)-4, IL-5 and IL-10 secreted by Th-2 cells were down-regulated by ECTO-MSCs whereas IgG2 and IFN-γ were up-regulated. In conclusion, we have observed that ECTO-MSCs are associated with enhanced Th-1 immune response to nasal inflammation and reduced Th-2 immune response. Given the contributions of Th-2 cells to AR, the injection of ECTO-MSCs can be a promising therapy of AR through balancing immune response.

Nasal ectomesenchymal stem cells: Multi-lineage differentiation and transformation effects on fibrin gels

Ectomesenchymal stem cells (EMSCs) are novel adult stem cells derived from the cranial neural crest. However, their stemness and multi-lineage differentiation potential on three-dimensional fibrin gels has not yet been explored. The objective of this study was to investigate induced differentiation of EMSCs on fibrin gels and their remodeling effects on the scaffolds during the induced differentiation process. The results indicated that CD133+/nestin+/CD44+ EMSCs were extensively distributed in the lamina propria of the nasal mucosa. The passaged cells could be induced to differentiate to a greater degree into neurons, Schwann cells and osteoblasts on three-dimensional fibrin gels than on two-dimensional glass slides. More importantly, the induced Schwann cells and osteoblasts exerted channelized and calcified remodeling effects, respectively, on the fibrin gels. Thus, these reshaped scaffolds have desirable biological properties, such as good cell adhesion, biocompatibility and guidance over the cell behavior, providing a tissue-committed niche for specific tissue generation.

The capacity and biological characteristic of the p75 neurotrophin receptor-positive ectomesenchymal stem cell in vitro

To investigate the biological characteristic of ectomesenchymal stem cells (EMSC) and the differentiation of p75(+)EMSC and influencing factors. The immunohistochemical staining method was used to observe the migration of EMSC from 12.5 d SD rat embryonic facial process. Then EMSC was labeled by p75 neurotrophin receptor, and the cell cycle and stem cell surface antigens of the p75(+) EMSC was examined. Immunohistochemical staining showed that stem cells from the cranial neural crest migrated to embryonic rat facial process at 12.5 days. The sorting rate of the p75(+)EMSC was 6.1%. The proportion of the p75(+)EMSCs' S/G2/M phase was stable during subculture. The special substances CD29, CD146 and Stro-1 were marked for p75(+)EMSC, and the expressions of the markers were all higher (> 90%) in p75(+) EMSC. The embryonic tooth did not start to grow on the conception 12.5 days of SD rats. The p75(+) EMSC after sorting had stable proliferation ability and had stem cell characteristics during subculture and didn't start differentiation.

A fibrin matrix promotes the differentiation of EMSCs isolated from nasal respiratory mucosa to myelinating phenotypical Schwann-like cells.

Because Schwann cells perform the triple tasks of myelination, axon guidance and neurotrophin synthesis, they are candidates for cell transplantation that might cure some types of nervous-system degenerative diseases or injuries. However, Schwann cells are difficult to obtain. As another option, ectomesenchymal stem cells (EMSCs) can be easily harvested from the nasal respiratory mucosa. Whether fibrin, an important transplantation vehicle, can improve the differentiation of EMSCs into Schwann-like cells (SLCs) deserves further research. EMSCs were isolated from rat nasal respiratory mucosa and were purified using anti-CD133 magnetic cell sorting. The purified cells strongly expressed HNK-1, nestin, p75NTR, S-100, and vimentin. Using nuclear staining, the MTT assay and Western blotting analysis of the expression of cell-cycle markers, the proliferation rate of EMSCs on a fibrin matrix was found to be significantly higher than that of cells grown on a plastic surface but insignificantly lower than that of cells grown on fibronectin. Additionally, the EMSCs grown on the fibrin matrix expressed myelination-related molecules, including myelin basic protein (MBP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and galactocerebrosides (GalCer), more strongly than did those grown on fibronectin or a plastic surface. Furthermore, the EMSCs grown on the fibrin matrix synthesized more neurotrophins compared with those grown on fibronectin or a plastic surface. The expression level of integrin in EMSCs grown on fibrin was similar to that of cells grown on fibronectin but was higher than that of cells grown on a plastic surface. These results demonstrated that fibrin not only promoted EMSC proliferation but also the differentiation of EMSCs into the SLCs. Our findings suggested that fibrin has great promise as a cell transplantation vehicle for the treatment of some types of nervous system diseases or injuries.

Rat Nasal Respiratory Mucosa-Derived Ectomesenchymal Stem Cells Differentiate into Schwann-Like Cells Promoting the Differentiation of PC12 Cells and Forming Myelin In Vitro.

Schwann cell (SC) transplantation as a cell-based therapy can enhance peripheral and central nerve repair experimentally, but it is limited by the donor site morbidity for clinical application. We investigated weather respiratory mucosa stem cells (REMSCs), a kind of ectomesenchymal stem cells (EMSCs), isolated from rat nasal septum can differentiate into functional Schwann-like cells (SC-like cells). REMSCs proliferated quickly in vitro and expressed the neural crest markers (nestin, vimentin, SOX10, and CD44). Treated with a mixture of glial growth factors for 7 days, REMSCs differentiated into SC-like cells. The differentiated REMSCs (dREMSCs) exhibited a spindle-like morphology similar to SC cells. Immunocytochemical staining and Western blotting indicated that SC-like cells expressed the glial markers (GFAP, S100β, Galc, and P75) and CNPase. When cocultured with dREMSCs for 5 days, PC12 cells differentiated into mature neuron-like cells with long neurites. More importantly, dREMSCs could form myelin structures with the neurites of PC12 cells at 21 days in vitro. Our data indicated that REMSCs, a kind of EMSCs, could differentiate into SC-like cells and have the ability to promote the differentiation of PC12 cells and form myelin in vitro.

Translational Research: Palatal-derived Ecto-mesenchymal Stem Cells from Human Palate: A New Hope for Alveolar Bone and Cranio-Facial Bone Reconstruction.

The management of facial defects has rapidly changed in the last decade. Functional and esthetic requirements have steadily increased along with the refinements of surgery. In the case of advanced atrophy or jaw defects, extensive horizontal and vertical bone augmentation is often unavoidable to enable patients to be fitted with implants. Loss of vertical alveolar bone height is the most common cause for a non primary stability of dental implants in adults. At present, there is no ideal therapeutic approach to cure loss of vertical alveolar bone height and achieve optimal pre-implantological bone regeneration before dental implant placement. Recently, it has been found that specific populations of stem cells and/or progenitor cells could be isolated from different dental resources, namely the dental follicle, the dental pulp and the periodontal ligament. Our research group has cultured palatal-derived stem cells (paldSCs) as dentospheres and further differentiated into various cells of the neuronal and osteogenic lineage, thereby demonstrating their stem cell state. In this publication will be shown whether paldSCs could be differentiated into the osteogenic lineage and, if so, whether these cells are able to regenerate alveolar bone tissue in vivo in an athymic rat model. Furthermore, using these data we have started a proof of principle clinical- and histological controlled study using stem cell-rich palatal tissues for improving the vertical alveolar bone augmentation in critical size defects. The initial results of the study demonstrate the feasibility of using stem cell-mediated tissue engineering to treat alveolar bone defects in humans.

Ecto-mesenchymal stem cells from facial process: potential for muscle regeneration.

Ecto-mesenchymal stem cells (EMSCs) originate from the cranial neural crest and participate in the formation of tooth, salivary, and muscle in early development stage. The transplantation of EMSCs, a potential source of myoblast stem cell, might improve muscle regeneration. The purpose of this study was to explore whether EMSCs have the potential to differentiate and display a myogenic phenotype in vitro the in vitro. Here, we characterized the EMSCs isolated from the facial process, and p75 + EMSCs were collected by a FACS calibur flow cytometer. In vitro, p75 + EMSCs induced by DMSO can accumulate and fuse into multinucleated myotubes and further differentiate into the skeletal muscle cells in form of cell sheet. Functional myoblast phenotypes of p75 + EMSCs were found in vivo model of muscle injury. The remarkable ability of stem cells to regenerate skeletal muscle indicated their potential role in the cell therapy and tissue engineering of the skeletal muscle.

A Site-Specific Phosphorylation of the Focal Adhesion Kinase Controls the Formation of Spheroid Cell Clusters

Human dental follicle cells (DFCs) are ectomesenchymal multipotent stem cells that form spheroid cell clusters (SCCs) under serum free medium cell culture conditions (SFM). Until today, molecular mechanisms for the formation of SCCs are unknown. In this study a quantitative phosphoproteomics approach revealed regulated phosphorylated proteins in SCCs, which were derived from DFCs after 24 and 48h in SFM. These regulated proteins were categorized using the Kyoto encyclopedia of genes and genomes program. Here, cellular processes and signaling pathway were identified such as the focal adhesion kinase (FAK) signaling pathway. In addition to the phosphoproteomics approach we showed that a specific phosphorylation of FAK (Y397) was required for the formation of SCCs. In conclusion, this study disclosed the phosphoproteome of SCCs for the first time and showed that the FAK signaling pathway is required for the formation of SCCs.

Prolactin (PRL) and prolactin receptor (PRLR) genes and their role in poultry production traits.

Prolactin (PRL), secreted from the anterior pituitary, plays extensive roles in osmoregulation, corpus luteum formation, mammogenesis, lactogenesis, lactopoiesis, and production of crop milk. In birds, prolactin (PRL) is generally accepted as crucial to the onset and maintenance of broodiness. All the actions of prolactin (PRL) hormone are mediated by its receptor (PRLR), which plays an important role in the PRL signal transduction cascade. It has been well established that the PRL gene is closely associated to the onset and maintenance of broody behavior, and could be a genetic marker in breeding against broodiness in chickens. Meanwhile, the prolactin receptor (PRLR) gene is regarded as a candidate genetic marker for reproductive traits. PRLR is also an important regulator gene for cell growth and differentiation. The identified polymorphism of this gene is mainly viewed in terms of egg production traits. Due to different biological activities attributed to PRL and PRLR, they can be used as major candidate genes in molecular animal breeding programs. Characterization of PRL and PRLR genes helps to elucidate their roles in birds and provides insights into the regulatory mechanisms of PRL and PRLR expression conserved in birds and mammals.

Immune Regulatory Properties Are a Common Feature Of Stem Cells

Allogeneic stem cell-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some stem cell (SC) types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow-MSCs, olfactory ecto-mesenchymal stem cells, leptomeningeal stem cells, and three different c-Kit-positive SC types, i.e. amniotic fluid SCs, cardiac SCs, and lung SCs. We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers, modulatory activity towards immune effector cells, immunogenicity and molecular inhibitory pathways, with some SC type-related peculiarities. In addition, we found that the inhibitory behaviour is not a constitutive property of SCs, but is acquired as a consequence of immune effector cell activation, as previously described for MSCs. Thus, immune regulation is a general property of stem cells and the characterization of this phenomenon may be useful for a proper therapeutical use of SCs. Disclosures:No relevant conflicts of interest to declare.

Fibrin Scaffolds Containing Ectomesenchymal Stem Cells Enhance Behavioral and Histological Improvement in a Rat Model of Spinal Cord Injury

Fibrin has been widely used in wound healing. However, its benefit for spinal cord injury (SCI) is limited. In this study, we investigated the impact of fibrin scaffolds containing ectomesenchymal stem cells (EMSCs) on histological and behavioral recovery after SCI and compared it with fibrin alone. To achieve this, EMSCs derived from adult rat nasal respiratory mucosa were cultured, characterized and transfected with green fluorescent protein adenovirus before transplantation. Then, Sprague-Dawley host rats were randomly assigned into four groups: the control group (laminectomy); the SCI group (laminectomy and transection of spinal cords); the fibrin group (fibrin was transplanted immediately after SCI), and the fibrin cell (FC) group (fibrin scaffolds containing EMSCs were transplanted after SCI). Three days after the operation, a TUNEL assay indicated less apoptotic cells in the FC group than in the fibrin group. Two weeks after SCI, fluorescence staining demonstrated not only the survival and migration of EMSCs into the lesion sites, but also a higher number of nerve fibers in the FC group than in the fibrin group. Histological examination including immunohistochemistry and transmission electron microscopy 12 weeks after the operation showed more nerve fibers and a thicker myelin sheath in the FC group compared to the fibrin group. Western blotting confirmed these morphological results. Consistent with the histological results, Basso, Beattie and Bresnahan locomotor scores of the FC group were higher than those of the fibrin group. These results suggest that fibrin scaffolds containing EMSCs can improve the behavioral and histological recovery after SCI better than fibrin alone.

Role of Matrix Metalloproteinases in Migration and Neurotrophic Properties of Nasal Olfactory Stem and Ensheathing Cells

Adult olfactory ectomesenchymal stem cells (OE-MSCs) and olfactory ensheathing cells (OECs), both from the nasal olfactory lamina propria, display robust regenerative properties when transplanted into the nervous system, but the mechanisms supporting such therapeutic effects remain unknown. Matrix metalloproteinases (MMPs) are an important family of proteinases contributing to cell motility and axonal outgrowth across the extracellular matrix (ECM) in physiological and pathological conditions. In this study, we have characterized for the first time in nasal human OE-MSCs the expression profile of some MMPs currently associated with cell migration and invasiveness. We demonstrate different patterns of expression for MMP-1, MMP-2, MMP-9, and MT1-MMP upon cell migration when compared with nonmigrating cells. Our results establish a correspondence between the localization of these proteinases in the migration front with the ability of cells to migrate. Using various modulators of MMP activity, we also show that at least MMP-2, MMP-9, and MT1-MMP contribute to OE-MSC migration in an in vitro 3D test. Furthermore, we demonstrate under the same conditions of culture used for in vivo transplantation that OE-MSCs and OECs secrete neurotrophic factors that promote neurite outgrowth of cortical and dorsal root ganglia (DRG) neurons, as well as axo-dendritic differentiation of cortical neurons. These effects were abolished by the depletion of MMP-2 and MMP-9 from the culture conditioned media. Altogether, our results provide the first evidence that MMPs may contribute to the therapeutic features of OE-MSCs and OECs through the control of their motility and/or their neurotrophic properties. Our data provide new insight into the mechanisms of neuroregeneration and will contribute to optimization of cell therapy strategies.

In vitro Maintenance of Olfactory Mucosa: with Enriched Olfactory Ensheathing Cells

Human Olfactory Mucosa (OM) regulates olfaction through axonal regeneration and myelination mediated by stem cells and Olfactory Ensheathing Cells (OECs) resident in the niche. Purified OECs/olfactory biopsies have been utilized for functional recovery in different Spinal Cord Injury (SCI) models. However, recent reports find this debatable where we propose primary culture of OM, basal cells of olfactory epithelium and olfactory ecto-mesenchymal stem cells. Our defined culture conditions improve the life span of OM with enrichment of OECs providing a strategy for employment for SCI/cochlear damage repair. Briefly, OM post-collection, was non-enzymatically sliced, cultured for 6 weeks and cells characterized morphologically, immuno-cytochemically and western blotting. By day 21, ~70% GFAP and p75NTR stained, spindle shaped astrocyte-like and flattened sheet-like OECs displayed axonal remyelination. By day 30, caspase 3, 8, 9 (gene-product and activity), phospho-p53 negative; GFAP and p75NTR positive dense, overlapping mass of cells was found. This was accompanied with degenerative changes by 6 weeks through GFAP staining. Conversely, trypsination on day 21 resulted in >95% OECs with flattened morphology, GFAP and p75NTR positivity. The human derived OECs were compared with the 2-day SD rat Olfactory Bulb Cells cultured for 2 weeks in F12 media (GFAP and p75NTR positive). Hence, cultured olfactory mucosa displaying axonal regeneration with OECs in culture provides a vehicle for SCI/cochlear damage repair studies.

Development of A Three-Dimensional Tissue Construct from Dental Human Ectomesenchymal Stem Cells: In Vitro and In Vivo Study

Application of regenerative medicine technology provides treatment for patients with several clinical problems, like loss of tissue and its function. The investigation of biological tooth replacement, dental tissue engineering and cell culture, scaffolds and growth factors are considered essential. Currently, studies reported on the making of threedimensional tissue constructs focused on the use of animal cells in the early stages of embryogenesis applied to young biomodels. The purpose of this study was the development and characterization of a three-dimensional tissue construct from human dental cells. The construct was detached, cultured and characterized in mesenchymal and epithelial cells of a human tooth germ of a 12 year old patient. The cells were characterized by specific membrane markers (STRO1, CD44), making a biocomplex using Pura Matrix as a scaffold, and it was incubated for four days and transplanted into 30 adult immunosuppressed male Wistar rats. They were evaluated at 6 days, 10 days and 2 months, obtaining histological sections stained with hematoxylin and eosin. Cell cultures were positive for specific membrane markers, showing evident deviations in morphology under phase contrast microscope. Differentiation and organization were noted at 10 days, while the constructs at 2 months showed a clear difference in morphology, organization and cell type. It was possible to obtain a three-dimensional tissue construct from human dental ectomesenchymal cells achieving a degree of tissue organization that corresponds to the presence of cellular stratification and extracellular matrix.

Characterization of p75+ ectomesenchymal stem cells from rat embryonic facial process tissue

Several populations of stem cells, including those from the dental pulp and periodontal ligament, have been isolated from different parts of the tooth and periodontium. The characteristics of such stem cells have been reported as well. However, as a common progenitor of these cells, ectomesenchymal stem cells (EMSCs), derived from the cranial neural crest have yet to be fully characterized. The aim of this study was to better understand the characteristics of EMSCs isolated from rat embryonic facial processes. Immunohistochemical staining showed that EMSCs had migrated to rat facial processes at E11.5, while the absence of epithelial invagination or tooth-like epithelium suggested that any epithelial–mesenchymal interactions were limited at this stage. The p75 neurotrophin receptor (p75NTR), a typical neural crest marker, was used to select p75NTR-positive EMSCs (p75+ EMSCs), which were found to show a homogeneous fibroblast-like morphology and little change in the growth curve, proliferation capacity, and cell phenotype during cell passage. They also displayed the capacity to differentiate into diverse cell types under chemically defined conditions in vitro. p75+ EMSCs proved to be homogeneous, stable in vitro and potentially capable of multiple lineages, suggesting their potential for application in dental or orofacial tissue engineering.

Genome-wide analysis of familial dysautonomia and kinetin target genes with patient olfactory ecto-mesenchymal stem cells

Familial dysautonomia (FD) is a rare inherited neurodegenerative disorder. The most common mutation is a c.2204+6T>C transition in the 5' splice site (5'ss) of IKBKAP intron 20, which causes a tissue-specific skipping of exon 20, resulting in lower synthesis of IKAP/hELP1 protein. To better understand the specificity of neuron loss in FD, we modeled the molecular mechanisms of IKBKAP mRNA splicing by studying human olfactory ecto-mesenchymal stem cells (hOE-MSCs) derived from FD patient nasal biopsies. We explored how the modulation of IKBKAP mRNA alternative splicing impacts the transcriptome at the genome-wide level. We found that the FD transcriptional signature was highly associated with biological functions related to the development of the nervous system. In addition, we identified target genes of kinetin, a plant cytokinin that corrects IKBKAP mRNA splicing and increases the expression of IKAP/hELP1. We identified this compound as a putative regulator of splicing factors and added new evidence for a sequence-specific correction of splicing. In conclusion, hOE-MSCs isolated from FD patients represent a promising avenue for modeling the altered genetic expression of FD, demonstrating a methodology that can be applied to a host of other genetic disorders to test the therapeutic potential of candidate molecules.

Neural Crest Stem Cells from Dental Tissues: A New Hope for Dental and Neural Regeneration

Several stem cell sources persist in the adult human body, which opens the doors to both allogeneic and autologous cell therapies. Tooth tissues have proven to be a surprisingly rich and accessible source of neural crest-derived ectomesenchymal stem cells (EMSCs), which may be employed to repair disease-affected oral tissues in advanced regenerative dentistry. Additionally, one area of medicine that demands intensive research on new sources of stem cells is nervous system regeneration, since this constitutes a therapeutic hope for patients affected by highly invalidating conditions such as spinal cord injury, stroke, or neurodegenerative diseases. However, endogenous adult sources of neural stem cells present major drawbacks, such as their scarcity and complicated obtention. In this context, EMSCs from dental tissues emerge as good alternative candidates, since they are preserved in adult human individuals, and retain both high proliferation ability and a neural-like phenotype in vitro. In this paper, we discuss some important aspects of tissue regeneration by cell therapy and point out some advantages that EMSCs provide for dental and neural regeneration. We will finally review some of the latest research featuring experimental approaches and benefits of dental stem cell therapy.

The response of human ectomesenchymal dental pulp stem cells to cisplatin treatment

To determine the response of dental pulp stem cells (DPSCs) to DNA-damaging cytostatic cisplatin and compare it with the response of normal human dermal fibroblasts (HDFs). Dental pulp stem cells were exposed to 5, 10, 20 or 40 μmol L(-1) of cisplatin. The proliferation of affected cells was assessed by a Z2 Counter and viability was assessed by means of a Vi-Cell XR using Trypan blue exclusion staining. Cell cycle analysis and induction of apoptosis were performed by flow cytometry. Induction of apoptosis was determined by monitoring the activities of caspases. The expression of proteins was detected by electrophoresis and Western blotting. The descriptive statistics of the results was analyzed by Student's t-test. Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. All three main Mitogen-activated protein kinases (MAPK) families - extracellular signal-regulated kinases (ERK), c-Jun-N-terminal kinase (JNK) and p38 were activated after treatment of DPSCs with cisplatin. The activation of MAPK pathways was not observed in HDFs exposed to cisplatin. The exposure of DPSCs and HDFs to cisplatin provoked an increase in p53 and p21 expression and p53 phosphorylation of serine 15. Higher concentrations of cisplatin reduced the viability of DPSCs and HDFs and induced the activation of caspases 3/7 and 9. Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. Cisplatin in higher concentrations triggered activation of MAPK and apoptosis in DPSCs but not in HDFs.

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7. The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11. Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16. In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.