A Novel Approach for Mucosal and Bulbar Olfactory Ensheathing Cell Isolation Based on the Non-Adherent Subculture Technique (original) (raw)
Related papers
Primary Olfactory Ensheathing Cell Culture from Human Olfactory Mucosa Specimen
BIO-PROTOCOL, 2017
The human olfactory mucosa is located in the middle and superior turbinates, and the septum of nasal cavity. Olfactory mucosa plays an important role in detection of odours and it is also the only nervous tissue that is exposed to the external environment. This property leads to easy access to the olfactory mucosa for achieving various researches. The lamina propria of olfactory mucosa consists of olfactory ensheathing cells (OECs) that cover the nerve fibers of olfactory. Here we describe a protocol for isolation of OECs from biopsy of human olfactory mucosa.
In vitro Maintenance of Olfactory Mucosa: with Enriched Olfactory Ensheathing Cells
Journal of Stem Cell Research & Therapy, 2013
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.
The role of olfactory ensheating cells in regenerative medicine: review of the literature
Romanian Journal of Rhinology, 2015
Olfactory ensheathing cells (OECs) join olfactory axons in their entrance to the central nervous system, representing a unique population of glial cells with functions in olfactory neurogenesis, axonal growth and olfactory bulb formation. Olfactory ensheathing cells have a great potential to induce repair for neural injuries, in central nervous system and peripheral nervous system, existing numerous experimental and clinical studies lately, reporting beneficial effects in anatomical and functional recovery. Studies are also conducted in order to establish possible pro-regenerative effects of the OECs, their potential in tissue repair and ability to modulate the immune system. The aim of this paper was to review the properties of olfactory ensheathing cells and their potential therapeutic role in regenerative medicine.
Ensheathing cell cultures from the olfactory bulb and mucosa
Glia, 2004
Transplantation of cells cultured from the nerve layers of the adult rat olfactory bulb has been shown to repair CNS tract injuries. The precise cellular composition of the culture appears to be important for this effect. Comparison was made of tissue cultured from the adult rat olfactory mucosa with that from the olfactory bulb. Both yielded mixtures of p75 immunoreactive cells and fibronectin immunoreactive cells. In sequential observations over 21 days in culture, the population of p75-expressing cells was maintained and continued to proliferate for longer in the samples from the olfactory mucosa. For derivation of cells for transplantation, the mucosa can be accessed without the need for intracranial surgery.
Scientific Reports
Human olfactory mucosa cells (hoMcs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. to date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive "offthe-shelf" alternative. the aim of this study was to generate a candidate cell line from late-adherent hoMcs, thought to contain the oec subset. primary late-adherent hoMcs were transduced with a c-MyceR tAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MyceR tAM-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75 ntR , S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. therefore, c-MyceR tAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells. Spinal cord injury (SCI) is a devastating condition affecting 250,000 to 500,000 people a year worldwide 1. The spinal cord is part of the central nervous system (CNS), a tissue with limited regenerative capacity due to the complexity of an unfavourable microenvironment for the re-establishment of neuronal connections 2-5. In contrast with the peripheral nervous system (PNS), the CNS regenerates slowly due to the formation of "glial scars" that are thought to be biochemical and mechanical barriers for axonal regrowth 6-9. In the 1980s, studies focused on the ability of olfactory neurons to regenerate and form connections after surgical lesions throughout life 7,10-12. Such unique regenerative properties have been attributed to the anatomical boundaries of the olfactory system between the PNS and CNS, as well as the presence of cell types such as neural stem cells (NSCs), olfactory ensheathing cells (OECs), and mesenchymal stem cells (MSCs) 13-15. The olfactory mucosa is an accessible source of cells for transplantation as biopsies can be safely taken without invasive intracranial surgery unlike the olfactory bulb 16,17. Moreover, human olfactory mucosa cells (hOMCs) have been successfully transplanted in clinical trials 18-21. This work has shown few adverse effects from immunological and microbiological perspectives and autologous hOMC transplantation is generally considered safe 22. However, an autologous process is difficult to standardise due to large variability in cell composition, thus justifying the need to assess an allogeneic or universal "off-the-shelf " approach. Allogeneic cell therapy aligns with
Acta medica Iranica, 2018
Olfactory ensheathing cells-based therapy for spinal cord injury (SCI) repair has been a possible treatment for clinical study because of their safety in autologous transplantation and potential regenerative capability. However, there are contradictory reports on the results after transplantation in animal models. The purpose of this research was to investigate the effect of acute transplantation of human mucosa-derived olfactory ensheathing cells (OECs) on the repair of the spinal cord. Human olfactory ensheathing cells were isolated from the human mucosa and cultured under supplemented neuronal cell culture medium. They were characterized by immunocytochemistry for olfactory ensheathing cell markers. We induced spinal cord injury at T8-T9 of rats by aneurysm clips and simultaneously injected two million OECs into subarachnoid space of spinal cord. Sensory and motor behaviors were recorded by tail-flick reflex (TFR) and BBB scores, respectively every week for seven weeks after inju...
Neurosurgery, 2008
We sought to study the yield of olfactory ensheathing cells from biopsies of the mucosa of the nasal septum. These specialized cells encourage regeneration of nerves of the central nervous system and may be of value for spinal cord and nerve injuries. METHODS: We undertook a prospective observational study of biopsies of nasal mucosa by endonasal dissection of the mucosa of the nasal septum during the approach for routine transsphenoidal surgeries. Samples were cultured in the laboratory, and the yield of olfactory ensheathing cells was compared as to the location, size, and weight of the biopsies and the age of the patients. RESULTS: A better yield of olfactory ensheathing cells was obtained from areas of the septum that were more superior and posterior in position. The yield was not related to the size of the biopsy or the patient's age. CONCLUSION: Septal mucosa is a possible source of olfactory ensheathing cells, although the yield may be smaller than that which may be obtained from mucosa of the lateral nasal cavity and superior turbinate.
Isolation of putative stem cells present in human adult olfactory mucosa
PLOS ONE
The olfactory mucosa (OM) has the unique characteristic of performing an almost continuous and lifelong neurogenesis in response to external injuries, due to the presence of olfactory stem cells that guarantee the maintenance of the olfactory function. The easy accessibility of the OM in humans makes these stem cells feasible candidates for the development of regenerative therapies. In this report we present a detailed characterization of a patient-derived OM, together with a description of cell cultures obtained from the OM. In addition, we present a method for the enrichment and isolation of OM stem cells that might be used for future translational studies dealing with neuronal plasticity, neuroregeneration or disease modeling.
Glia, 2001
Ensheathing cells exclusively enfold olfactory axons. The ability of olfactory axons to reinnervate the adult mammalian olfactory bulb throughout the lifetime of an organism is believed to result from the presence of this unique glial cell in the olfactory system. This theory has been substantiated by research demonstrating the ability of transplanted ensheathing cells to promote axonal regrowth in areas of the central nervous system that are normally nonpermissive. A simple method for purifying ensheathing cells resulting in a large yield of cells is therefore invaluable for transplantation studies. We have developed such a method based on the differing rates of attachment of the various harvested cell types. The greatest percentage of cells (70.4%) that attached during the first step of the separation was determined to be fibroblasts. The remainder of the cells were classified as astrocytes (20.8%) and ensheathing cells (6.8%). The percentage of attached astrocytes (67.6%) was greatly increased during the second purification step while the percentage of fibroblasts decreased greatly (27.9%) and the percentage of ensheathing cells (5.3%) slightly decreased. In the final cultures, 93.2 % of the attached cells were ensheathing cells, while astrocytes (5.9%) and fibroblasts (1.4%) were only minor components. This simple, inexpensive method of purifying ensheathing cells will facilitate their use in central nervous system regeneration research. GLIA 34:81-87,
Purity determining of cultured OECs from olfactory mucosa of rats\' pups
Journal of Basic Research in Medical Sciences
Introduction: Cell transplantation is one of the main strategies for spinal cord injury repair. As OECs of the olfactory mucosa can be obtained by simple biopsy in all individuals without affecting their smell sensation, OECs considered as a promising candidate for autologous transplantation in the nervous system injury, especially for spinal cord repair. Thereby in the current study OECs were cultured from olfactory mucosa of 7 days old rats' pups and their purity was examined by flow-cytometry after simultaneous double staining for p75 and GFAP markers. Materials and methods: 7 days old Wistar rats' pups were deeply anesthetized by ketamine / xylazine (60/6mg/Kg). Then the nasal cavity was opened sagittally and the olfactory mucosa was separated from posterior part of nasal septum and at last OECs were obtained from lamina properia of olfactory mucosa and were cultured. The cultured cells were simultaneously immunolabeled for p75 and GFAP markers and finally purity of cultured cells assessed by flow-cytometry. Results: cultured OECs demonstrated two different morphologies, a spindle shape Schwannlike and an astrocyte-like OECs with flat sheet-like morphology. In addition, simultaneous immunolabeling for p75 and GFAP markers of OECs exhibited OECs were positive for both markers at the same time. The flow-cytometry results displayed that 87.9±2.4% of cells were p75/ GFAP double positive cells,1.05±0.4 only p75 positive and 5.8±1.5% were single positive for GFAP. Conclusion: Purity of cultured OECs in our study is probably more than 87.9% by flowowing to p75+/S100+ and GFAP+/S100+ olfactory ensheathing cells were not counted. Thus the culture procedure of this study seems to be a good protocol for OECs purifying and cell therapy in CNS damages.