Prospects for the use of olfactory mucosa cells in bioprinting for the treatment of spinal cord injuries

The review focuses on the most important areas of cell therapy for spinal cord injuries.Olfactory mucosa cells are promising for transplantation.Obtaining these cells is safe for patients.The use of olfactory mucosa cells is effective in restoring motor function due to the remyelination and regeneration of axons after spinal cord injuries.These cells express neurotrophic factors that play an important role in the functional recovery of nerve tissue after spinal cord injuries.In addition,it is possible to increase the content of neurotrophic factors,at the site of injury,exogenously by the direct injection of neurotrophic factors or their delivery using gene therapy.The advantages of olfactory mucosa cells,in combination with neurotrophic factors,open up wide possibilities for their application in threedimensional and four-dimensional bioprinting technology treating spinal cord injuries.

Differentiation of human olfactory mucosa mesenchymal stem cells into photoreceptor cells in vitro

AIM：To investigate whether the human olfactory mucosa mesenchymal stem cells（OM-MSCs）can differentiate into photoreceptor cells in vitro.METHODS：Through the olfactory mucosa adherent method,olfactory mucosa was isolated,cultured and identified in vitro among mesenchymal stem cells.The cell surface markers were analyzed by flow cytometry,induced to differentiate into retinal photoreceptor cells in vitro,and the expression of rhodopsin was observed and identified by Immunofluorescence and Western blot methods.RESULTS：OM-MSCs from human were spindle cellbased,and showing radial colony arrangement.OM-MSCs were negative for CD34,CD45 and CD105,but positive for CD73 and CD90.Following induction,a strong positive reaction was produced by photoreceptor specific marker rhodopsin in the cells.CONSLUSION：This novel finding demonstrates that OM-MSCs can be cultured and expanded in vitro.They possess biological characteristics of mesenchymal stem cells,and have the ability to be induced into retinal cells.

Gene and protein expression profiles of olfactory ensheathing cells from olfactory bulb versus olfactory mucosa

Olfactory ensheathing cells(OECs) from the olfactory bulb(OB) and the olfactory mucosa(OM) have the capacity to repair nerve injury. However, the difference in the therapeutic effect between OB-derived OECs and OM-derived OECs remains unclear. In this study, we extracted OECs from OB and OM and compared the gene and protein expression profiles of the cells using transcriptomics and non-quantitative proteomics techniques. The results revealed that both OB-derived OECs and OM-derived OECs highly expressed genes and proteins that regulate cell growth, proliferation, apoptosis and vascular endothelial cell regeneration. The differentially expressed genes and proteins of OB-derived OECs play a key role in regulation of nerve regeneration and axon regeneration and extension, transmission of nerve impulses and response to axon injury. The differentially expressed genes and proteins of OM-derived OECs mainly participate in the positive regulation of inflammatory response, defense response, cytokine binding, cell migration and wound healing. These findings suggest that differentially expressed genes and proteins may explain why OB-derived OECs and OM-derived OECs exhibit different therapeutic roles. This study was approved by the Animal Ethics Committee of the General Hospital of Ningxia Medical University(approval No. 2017-073) on February 13, 2017.

Nogo-A expression in injured spinal cord following human olfactory mucosa-derived olfactory ensheathing cells transplantation

Transplantation of olfactory bulb-derived olfactory ensheathing cells （OECs） promotes motor functional recovery in rats with acute spinal cord injury, possibly by Nogo-A expression changes at the injury site. The present study transplanted OECs derived from the olfactory mucosa （OM） of rats OM-derived OEC （OM-OEC） transplantation significantly reduced the increase of Nogo-A protein and mRNA expression caused by spinal cord injury, supporting the hypothesis that OM-OECs improve spinal cord regeneration by reducing Nogo-A expression.

Isolation,culture,and purification of olfactory mucosa-derived olfactory ensheathing cells using modified differential attachment with low concentration serum

BACKGROUND：Studies have demonstrated that olfactory mucosa can promote the regeneration and formation of axonal medullary sheath of injured neurons. To date, olfactory ensheathing cells （OECs） utilized in basic and clinical research arise primarily from the olfactory bulb mucosa. However, little is known regarding culture, purification, and biological properties of OECs . OBJECTIVE： To isolate and culture OECs utilized modified, differential attachment in combination with neurotrophic factor 3 （NT3） and low concentration serum to explore an optimal in vitro culture method for OECs.DESIGN, TIME AND SETTING： Single-sample observation was performed at the Medical Experimental Center of Stomatology College, Xi＇an Jiaotong University between March 2006 and December 2007. MATERIALS： Twelve samples from aborted embryos, 4-6 months, were used to isolate OECs; rabbit-anti-human p75NTR and glial fibrillary acidic protein （GFAP） antibody were provided by Sigma, USA. METHODS： The differential time was six hours. This was repeated twice, based on Nash＇s differential attachment. Attached OECs were cultured in DMEM-F12 culture medium containing 10% fetal bovine serum （FBS） or 2.5% FBS and NT3. MAIN OUTCOME MEASURES： OEC morphology was observed, and p75NTR and GFAP immunocyto-chemistry was used for identification and purity detection. RESULTS： Some cells attached after three days in culture. Several cells possessed short neurites with good refractivity. Some shuttle-shaped fibroblasts could be seen. On day six, more cells attached, exhibiting a three-dimensional appearance. Many cells appeared dipolar or tripolar, with slender neurites, and fibroblasts were clustered. On day nine, the number of dipolar or tripolar cell bodies with slender neurites was increased, and fibroblasts were clustered. On day 15, fibroblasts occupied the majority of the bottom of the culture bottle, with several OECs surviving at the upper layer. OECs were positive for P75NTR and GFAP expression, as identified by an immunocytologically stained brown cell body and neurites. However, fibroblasts were P75NTR and GFAP-negative. On day 9, OEC purity reached 81%, and the number of proliferating fibroblasts significantly increased. By the end of day 12, OEC purity was reduced to 56%. CONCLUSION： Modified differential attachment, in combination with low concentration serum and NT3, removes fibroblasts and reduces OEC loss. This is an appropriate method for the isolation and culture of human fetal olfactory mucosa-derived OECs.

Involuntary muscle spasm expressed as motor evoked potential after olfactory mucosa autograft in patients with chronic spinal cord injury and complete paraplegia

Object: The efficacy of olfactory mucosa autograft (OMA) for chronic spinal cord injury has been reported. New activity in response to voluntary effort has been documented by electromyography (EMG), but the emergence of motor evoked potential (MEP) reflecting electrophysiological conductivity in the central nervous system, including the corticospinal pathway, after OMA, and the best indications for OMA, have not been clarified. Here, we report the emergence of MEPs after OMA and offer recom-mendations for appropriate indications based on the presence of involuntary muscle spasm (IMS). We used analysis of MEP to examine the efficacy of OMA for patients with complete paraplegia due to chronic spinal cord injury. To clarify the indications for OMA, we investigated the association of IMS and efficacy of OMA. Methods: Four patients, 3 men and 1 woman, were enrolled. The mean age of the cases was 30.3 ± 9.5 years (range, 19 to 40 years). All 4 cases were American Spinal Injury Association (ASISA) grade A. The mean duration from injury to OMA was 95.8 ± 68.2 months (range, 17 to 300 months). Samples of olfactory mucosa were removed, cut into smaller pieces, and grafted into the sites of spinal cord lesions after laminectomy. Postoperative subcutaneous fluid collection, postoperative meningitis, postoperative nosebleed, postoperative infection in the nasal cavity, impaired olfaction, neoplastic tissue overgrowth at the autograft site, new sensory disturbance, and involuntary muscle spasm were investigated as safety issues. Improvements in ASIA grade, variations in ASIA scores, EMG, SSEP, and improved urological function were evaluated as efficacy indicators. Results: There were no serious adverse events in this series. In 2 of the 4 cases, an improvement in motor function below the level of injury was recognized. In one, the motor score was 50 until 16 weeks after surgery, and it increased to 52 from 20 weeks after surgery. In the other, the motor score was 50 until 20 weeks after surgery, and it increased to 52 at 24 weeks after surgery with a further increase to 54 at 48 weeks after surgery. The emergence of MEP was recognized in the latter case at 96 weeks after surgery. The other 2 cases had no improvement in ASIA motor score. Both of these cases who showed improvements in the ASIA motor scores exhibited relative IMS compared with those who had no ASIA motor score recovery. Conclusions: We recognized the emergence of MEPs in a case with complete paraplegia due to chronic spinal cord injury after OMA. IMS might be a candidate of indication of OMA.

Effect of Mancozeb Inhalation on the Olfactory Mucosa in Rats

Background: Mancozeb, (ethylene-bis-dithiocarbamate), is an important fungicide useful against a wide range of fungus affecting ornamental plants, crops, and fruits. We aimed to evaluate the changes in the olfactory mucosa due to mancozeb toxicity and, if cytokine is active, IL-6 immunoactivity. Material and Method: In experimental group, the mancozeb (500 mg/kg) was administered with inhalation to 10 male Wistar Albino rats for five days a week. The control group (n = 10) received distilled water with spray at the same time period. The experiment was terminated after three weeks. Samples were placed in 10% formaldehyde for fixation and placed in paraffin, sections of 5 μm were prepared from paraffin blocks and stained with Hematoxylin-Eosin. Interleukin-6 (IL-6) primary antibody were used for immunohistochemical analysis. Result: In the mancozeb group, olfactory epithelial cell degeneration and apoptosis, inflammation, dilatation and congestion in the vessels were observed. IL-6 expression was increased in vascular endothelium and inflammatory cells. Conclusions: Mancozeb was thought that the increase in IL-6 expression due to the increase in cell degeneration signal was thought to affect the development of cell apoptosis and angiogenesis, and that the use of mancozeb might adversely affect the olfactory mechanism.

Transplantation of Olfactory Mucosa as a Scaffold for Axonal Regeneration Following Spinal Cord Contusion in Rats

Object: The inability of the spinal cord to regenerate after SCI is due to the extremely limited regenerative capacity of most central nervous system (CNS) axons, along with the hostile environment of the adult CNS, which does not support axonal growth. It seems that for successful axonal regeneration to take place, a supportive local environment is required after the injury. We have previously reported that transplantation of the olfactory mucosa is effective in restoring functional recovery in rats following spinal cord transaction. In this study, we examined histological features of olfactory mucosa grafts in rats subjected to a spinal cord contusion protocol. Respiratory mucosa was utilized as a control, as we have previously found that respiratory mucosa does not support neuronal generation. Methods: The rats spinal cords were crash-injured by dropping a 10-g metal rod from a height of 7.5 cm, and a couple of weeks later, the injury sites were exposed, and both olfactory and respiratory mucosae were inserted into the posterior sulcuses of the spinal cord. The each number of olfactory and mucosa transplanted rats were five. The Basso, Beattie, and Bresnahan (BBB) score was observed. Immunohistochemical study for neurofilament was performed. Results: Olfactory mucosa transplanted rats following spinal cord injury can support at least partial hind limb motor recovery compared with respiratory mucosa transplanted rats and we identified numerous axons surrounding the transplanted olfactory mucosa cells, and penetrating the olfactory mucosa at the transplant site. Conclusion: Olfactory mucosa might be a suitable scaffold for axonal regeneration.

Efficient RT-QuIC seeding activity for α-synuclein in olfactory mucosa samples of patients with Parkinson’s disease and multiple system atrophy

Background:Parkinson’s disease(PD)is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms.PD is characterized by intraneuronal accumulation of abnormalα-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of eitherα-synuclein,as in the case of Multiple System Atrophy(MSA)or tau,as in the case of Corticobasal Degeneration(CBD)and Progressive Supranuclear Palsy(PSP),in other disease-specific brain regions.Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization ofα-synuclein or tau aggregates in the brain.Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues,including receptor neurons of the olfactory mucosa(OM).Methods:We have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion(RT-QuIC)assay for OM analysis.In particular,by using human recombinantα-synuclein as substrate of reaction,we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induceα-synuclein aggregation,and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP.Results:Our results showed that a significant percentage of MSA and PD samples inducedα-synuclein aggregation with high efficiency,but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect.Notably,the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination.Conclusions:Our study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities forα-synuclein.Additional studies are required for(i)estimating sensitivity and specificity of the technique and for(ii)evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms.RT-QuIC analyses of OM and cerebrospinal fluid(CSF)can be combined with the aim of increasing the overall diagnostic accuracy of these diseases,especially in the early stages.

The mitosis and immunocytochemistry of olfactory ensheathing cells from nasal olfactory mucosa

Objective： To culture olfactory ensheathing cells （OECs） of rats in vitro and to investigate its morphology, mitosis and immunocytochemistry, and to explore if the OECs could be a new donation for transplantation. Methods： OECs were harvested from olfactory mucosa of Sprague Dawleys rats based on the differing rates of attachment of the various cell types, followed by glial fibrillary acidic protein （GFAP）, nerve growth factor （ NGF）, anti-low affinity receptor for NGF （ NGFRp75 ）, brain-derived neurotrophic factor （BDNF） , neurotrophino3 （ NTo3 ） and S-100 immunocytochemistry. The morphological changes and mitosis were observed under a phase contrast microscope at different culture time. Results - Three morphologically distinct types of cells, bipolar, multipolar and flat morphology were present in the primary culture of adult rat olfactory mucosa. Mitosis was characterized by a retraction of all processes, forming a sphere that divided into spherical daughter cells, the daughter cells sent out their processes. The OECs were immunoreactive for GFAP, NGFRp75, S-100, NGF, BDNF and NT-3. Conclusions： The OECs from nasal olfactory mucosa cultivated in the medium with fetal bovine serum could survive, divide, differentiate, and express the neurotrophin. It may become an accessible source for autologous grafting in spinal cord injury.

Ocular stem cells:a narrative review of current clinical trials

·Stem cells are undifferentiated cells showcasing a remarkable capacity of self-replenishing and differentiating into mature cells.Their ability to proliferate connotes that a designated stem cell source is capable of generating an unrestricted number of mature cells.The ever-increasing comprehension of position,activity,and function of ocular stem cells has led to rapid progress and incessant improvement of possible procedures and therapies.A narrative review was conducted to summarize the current evidence on clinical trials and respective literature,regarding current evolution in the field of ocular regenerative medicine.We tried to ascertain the safety of experimental and clinical procedures,their effectiveness,and the ethical repercussion of their use.

Olfactory swab sampling optimization for α-synuclein aggregate detection in patients with Parkinson’s disease

Background: In patients with Parkinson’s disease (PD), real-time quaking-induced conversion (RT-QuIC) detection of pathological α-synuclein (α-syn) in olfactory mucosa (OM) is not as accurate as in other α-synucleinopathies. It is unknown whether these variable results might be related to a different distribution of pathological α-syn in OM. Thus, we investigated whether nasal swab (NS) performed in areas with a different coverage by olfactory neuroepithelium, such as agger nasi (AN) and middle turbinate (MT), might affect the detection of pathological α-syn. Methods: NS was performed in 66 patients with PD and 29 non-PD between September 2018 and April 2021. In 43 patients, cerebrospinal fluid (CSF) was also obtained and all samples were analyzed by RT-QuIC for α-syn. Results: In the first round, 72 OM samples were collected by NS, from AN (NSAN) or from MT (NSMT), and 35 resulted positive for α-syn RT-QuIC, including 27/32 (84%) from AN, 5/11 (45%) from MT, and 3/29 (10%) belonging to the non-PD patients. Furthermore, 23 additional PD patients underwent NS at both AN and MT, and RT-QuIC revealed α-syn positive in 18/23 (78%) NSAN samples and in 10/23 (44%) NSMT samples. Immunocytochemistry of NS prepara-tions showed a higher representation of olfactory neural cells in NSAN compared to NSMT. We also observed α-syn and phospho-α-syn deposits in NS from PD patients but not in controls. Finally, RT-QuIC was positive in 22/24 CSF samples from PD patients (92%) and in 1/19 non-PD. Conclusion: In PD patients, RT-QuIC sensitivity is significantly increased (from 45% to 84%) when NS is performed at AN, indicating that α-syn aggregates are preferentially detected in olfactory areas with higher concentration of olfactory neurons. Although RT-QuIC analysis of CSF showed a higher diagnostic accuracy compared to NS, due to the non-invasiveness, NS might be considered as an ancillary procedure for PD diagnosis.