Culture conditions affect proliferative responsiveness of olfactory ensheathing glia to neuregulins

Olfactory ensheathing glia (OEG) have been used to improve outcome after experimental spinal cord injury and are being trialed clinically. Their rapid proliferation in vitro is essential to optimize clinical application, with neuregulins (NRG) being potential mitogens. We examined the effects of NRG-1beta, NRG-2alpha, and NRG3 on proliferation of p75-immunopurified adult OEG. OEG were grown in serum-containing medium with added bovine pituitary extract and forskolin (added mitogens) or in serum-containing medium (no added mitogens). Cultures were switched to chemically defined medium (no added mitogens or serum), NRG added and OEG proliferation assayed using BrdU. OEG grown initially with added mitogens were not responsive to added NRGs and pre-exposure to forskolin and pituitary extract increased basal proliferation rates so that OEG no longer responded to added NRG. However, NRG promoted proliferation but only if cells were initially grown in mitogen-free medium. Primary OEG express ErbB2, ErbB3, and small levels of ErbB4 receptors; functional blocking indicates that ErbB2 and ErbB3 are the main NRG receptors utilized in the presence of NRG-1beta. The long-term stimulation of OEG proliferation by initial culture conditions raises the possibility of manipulating OEG before therapeutic transplantation.     

嗅鞘细胞对神经干细胞增殖与分化的影响

BACKGROUND： Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors. OBJECTIVE： To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells. DESIGN, TIME AND SETTING： Cytology was performed at the Department of Neurology, Tongji Medical College, Huazhong University of Science and Technology, China, from September 2007 to October 2008. MATERIALS： Mouse anti-nestin polyclonal antibody （Chemicon, USA）, mouse anti-glial fibrillary acidic protein （GFAP） IgG1, mouse anti-2＇, 3＇-cyclic nucleotide 3＇-phosphodiesterase （CNPase） IgG1, mouse anti-Tubulin Class-Ill IgG1 （Neo Markers, USA）, Avidin-labeled Cy3 （KPL, USA）, and goat anti-mouse IgGl： fluorescein isothiocyanate （FITC） （Serotec, UK） were used in this study. METHODS：Tissues were isolated from the embryonic olfactory bulb and subependymal region of Wistar rats. Serum-free DMEM/F12 culture media was used for co-culture experiments. Neural stem cells were incubated in serum-free or 5% fetal bovine serum-containing DMEM/F12 as controls. MAIN OUTCOME MEASURES： After 7 days of co-culture, neural stem cells and olfactory ensheathing cells underwent immunofluorescent staining for nestin, tubulin, glial fibrillary acidic protein, and CNPase. RESULTS： Olfactory ensheathing cells promoted proliferation and differentiation of neural stem cells into neuron-like cells, astrocytes and oligodendrocytes. The proportion of neuron-like cells was 78.2%, but the proportion of neurons in 5% fetal bovine serum DMEM/F12 was 48.3%. In the serum-free DMEM/F12, neural stem cells contracted, unevenly adhered to the glassware wall, or underwent apoptosis at 7 days. CONCLUSION： Olfactory ensheathing cells promote differentiation of neural stem cells mainly into neuron-like cells, and accelerate proliferation of neural stem cells. The outcome is better compared with serum-free medium or medium containing 5% fetal bovine serum.     

Effects of olfactory ensheathing cells on the proliferation and differentiation of neural stem cells

BACKGROUND:Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors. OBJECTIVE:To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells. DESIGN,TIME AND SETTING:Cytology was performed at the Department of Neurology,Tongji Medical College,Huazhong University of Science and Technology,China,from September 2007 to October 2008. MATERIALS:Mouse anti-nestin polyclonal antibo...     

壳聚糖与大鼠嗅鞘细胞的生物相容性

背景：众多研究证实,嗅鞘细胞移植和生物导管在修复中枢神经系统损伤中发挥了显著的作用,课题组前期研究已证实了壳聚糖在大鼠体内神经系统具有良好的生物相容性。 目的：观察壳聚糖与大鼠嗅鞘细胞的生物相容性。 设计、时间及地点：体外细胞学对比观察,于2005-12/2006-05在武汉协和医院泌尿外科实验室完成。 材料：将壳聚糖粉溶于10 g/L的乙酸溶液中,配制成10 g/L的壳聚糖溶液;显微剥离成年大鼠嗅球表面层,按常规方法行原代细胞培养。 方法：实验分为3组,每组10孔。壳聚糖组将制备好的壳聚糖溶液0.1 mL加入到培养板中,50 ℃烘箱至成膜干燥,加入40 g/L氢氧化钠溶液中和乙酸,双蒸水漂洗,晾干即可。多聚赖氨酸组按常规方法赖氨酸包被培养板。未包被组不用任何材料包被。将原代培养的嗅鞘细胞分别接种至壳聚糖包被、多聚赖氨酸包被和未包被的培养板上进行培养。 主要观察指标：于培养第3,5天显微镜下观察细胞形态学变化,MTT比色实验检测细胞存活和增殖状况。 结果：嗅鞘细胞可在壳聚糖膜上贴壁生长,细胞形态及数量与未包被组相比差异无显著性意义,但与多聚赖氨酸组相比细胞数量偏低。 结论：嗅鞘细胞与壳聚糖有良好的生物相容性。     

Transplantation of olfactory ensheathing cells promotes axonal regeneration and functional recovery of peripheral nerve lesion in rats

Introduction: Olfactory ensheathing cells (OECs) hold promise for cell therapy because they may promote regeneration of the central nervous system. However, OECs have been less studied after peripheral nerve injury (PNI). The purpose of this investigation was to determine the effect of OEC transplantation on a severe sciatic nerve (SN) lesion. Methods: OECs were injected in rats after section and 2‐cm resection of the SN. Results: Three months after therapy, muscle strength and morphometric studies showed complete restoration of the contractile properties of the gastrocnemius and complete repair of the SN. Immunohistochemistry and RT‐PCR studies indicated an increase in the presence of neurotrophic factors. Interestingly, tracking of green fluorescent protein (GFP)‐positive OECs showed that no OECs were present in the SN. Discussion: Our results demonstrate that, after severe PNI, OECs have remarkable potential for nerve regeneration by creating a favorable microenvironment. Muscle Nerve, 2011     

Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa

Olfactory ensheathing cells (OEC) have the ability to promote regeneration in the nervous system. Hence, they hold promise for cell therapy. Most of the experimental studies have investigated the role of OECs taken from olfactory bulb (OB). However, for a clinical human application, olfactory mucosa (OM) seems to be the only acceptable source for OECs. Many studies have compared the distinct ability of OECs from OB and OM to improve functional nerve regeneration after lesion of the nervous system. Nevertheless, the two populations of OECs may differ in several points, which might affect all fate after transplantation in vivo. We report here the first study which compares gene expression profiling between these two populations of OECs. It appears that OB‐OECs and OM‐OECs display distinct gene expression pattern, which suggest that they may be implicated in different physiological processes. Notably, OM‐OECs overexpress genes characteristic of wound healing and regulation of extra cellular matrix. In contrast, OB‐OECs gene profile suggests a prominent role in nervous system development. Hence, OB‐OECs and OM‐OECs fundamentally differ in their gene expression pattern, which may represent a crucial point for future clinical application. © 2010 Wiley‐Liss, Inc.     

Identification of growth factors that promote long-term proliferation of olfactory ensheathing cells and modulate their antigenic phenotype

Olfactory ensheathing cells can develop into distinct subtypes in culture after incubation in serum-free medium conditioned by astrocytes, which have Schwann cell-like and astrocyte-like properties. It has not been possible so far to modulate and grow large numbers of these olfactory ensheathing cell subtypes. In this study, we have shown that astrocyte-conditioned medium, although promoting differentiation of the two olfactory ensheathing cell types, is growth-restrictive after 14 days, probably due to the upregulation of p16 and p27. Growth arrest can be overridden and cells maintained for a further 11 weeks, by a mitogen mix of fibroblast growth factor 2, forskolin, and heregulin (olfactory mitogen medium) combined with astrocyte-conditioned medium. In the absence of astrocyte-conditioned medium, combinations of the same factors can also override growth arrest but to a lesser extent. Olfactory mitogen medium combined with astrocyte-conditioned medium upregulates O4 and low-affinity nerve growth factor receptor expression on olfactory ensheathing cells, leading to a 100% Schwann cell--like phenotype. If cells are maintained in olfactory mitogen medium alone, or if they are treated with forskolin or fibroblast growth factor 2 diluted in serum-free medium, O4 and low-affinity nerve growth factor receptor expression remains at 100%, but there is also an increase in expression of E-NCAM, the astrocyte-like marker. Medium containing serum also overrides growth arrest, but for only 4 weeks, during which time most differentiation-specific markers disappear. These studies have allowed us to define conditions to modulate the olfactory ensheathing cell phenotype.     

Calponin is expressed by fibroblasts and meningeal cells but not olfactory ensheathing cells in the adult peripheral olfactory system

Olfactory ensheathing cells (OECs), the principal glial cells of the peripheral olfactory system, have many phenotypic similarities with Schwann cells of the peripheral nervous system. This makes reliably distinguishing these two cells types difficult, especially following transplantation into areas of injury in the central nervous system. In an attempt to identify markers by which these two cells types can be distinguished, a recent proteomic analysis of fetal OECs and adult Schwann cells identified the actin-binding protein calponin as a potential marker expressed by OECs but not Schwann cells. Since many studies designed with the translational goal of autologous transplantation in mind have used adult OECs, this study examined the expression of calponin by adult OECs, both in vivo within the peripheral olfactory system and in vitro. Calponin colocalized with strongly fibronectin positive fibroblasts in the olfactory mucosa (OM) and meningeal cells in the olfactory bulb (OB) but not with S100beta or neuropeptide-Y positive OECs. In tissue culture, calponin was strongly expressed by fibronectin-expressing fibroblasts from OM, sciatic nerve and skin and by meningeal cells from the OB, but not by p75(NTR)- and S100beta-expressing OECs. These data, supported by Western blotting, indicate that calponin can not be used to distinguish adult OECs and Schwann cells.     

Olfactory ensheathing cells: Attractant of neural progenitor migration to olfactory bulb

Olfactory ensheathing cells (OECs) are the glial cells that derive from the olfactory placode, envelop olfactory axons in the course of migration from the olfactory epithelium to the olfactory bulb and reside primarily in the olfactory nerve layer. OECs transplantation as a promising experimental therapy for axonal injuries has been intensively studied; however, little is known about their roles in olfactory bulb development. In this study, we examined the effects of OECs on the migration of neural progenitors in rostral migratory stream (RMS). Initially, the neurosphere migration assay showed that OEC‐conditioned medium promoted progenitors to migrate from RMS neurospheres in a concentration dependent manner. Moreover, co‐culturing OECs nearby the RMS explants led to asymmetric migration of explants in different developing stages. However, OECs could influence the migration in a distance not further than 1.5 mm. Finally, slice assay that mimic the circumstance in vivo revealed that OECs had a chemoattractive activity on RMS neural progenitors. Together, these results demonstrate that OECs attract neural progenitors in RMS through the release of diffusible factors and it is likely that OECs mainly influence radial migration in the olfactory bulb but not tangential migration of the RMS invivo during development. This suggests a previously unknown function for OECs in olfactory development and a novel mechanism underlying the targeting of RMS cells. © 2010 Wiley‐Liss, Inc.     

The olfactory bulb and olfactory mucosa obtained from human cadaver donors as a source of olfactory ensheathing cells

During the last decade, olfactory ensheathing cells (OECs) have been successfully applied in multiple experimental approaches aimed to repair damaged mammalian spinal cord. Some of these experiments have consequently been translated into clinical trials. Finding a reliable source of human OECs that is easily accessible and can ensure a sufficient number of cells is a major prerequisite for conducting studies on OEC-mediated spinal cord regeneration. Here, we present a procedure for obtaining olfactory bulbs (OBs) and olfactory mucosa (OM) simultaneously from adult cadaver heart-beating donors for OEC isolation and analyze some of the factors that may condition successful OEC culture. We show that the results of OEC culture from OBs (10 cases) correlated significantly with warm ischemia time (WIT) as well as the initial viability of the isolated cells. Efficient OEC culture was possible when the WIT for the OB was up to 20 min. Brain damage, assessed by determination of S100B serum level, was not related to the success of OEC culture from the OB. Cadaver OM (7 cases) was shown to be a more reliable source of human OECs than the OB. In most of the examined cases the efficacy of culturing OECs from cadaver OM obtained even 180 min after cardiac arrest was comparable to that of living patients. The method of obtaining OBs and OM from cadavers enables the use of an alternative source of primary adult human OECs for further preclinical and clinical studies on their neurotrophic properties.     

Glial cell line-derived neurotrophic factor promotes olfactory ensheathing cells migration

Olfactory ensheathing cells (OECs) are a unique type of macroglia with axonal growth-promoting properties. The migrating ability of OECs in CNS is essential for neural regeneration. However, little is known about the extracellular and intracellular factors that regulate OEC migration. In the present study, we examined the effects of glial cell line-derived neurotrophic factor (GDNF) on OECs migration. Initially, the "scratch" migration assay, Boyden chamber assay, and explant migration assay showed that GDNF could promote OECs migration in vitro. Treatment of OECs with GDNF also induced cytoskeleton reorganization and up-regulated expression of cytoskeleton proteins. GDNF-induced OECs migration was demonstrated depending on GFRalpha-1 and Ret receptor, and activation of JNK and Src signaling cascades. Furthermore, GDNF was found to promote implanted OECs migration in a spinal cord hemisection injury model. Together, we report, to our knowledge for the first time, that GDNF stimulate OECs migration in vitro and in vivo.     

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 im     

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...     

Gap junctional communication and connexin expression in cultured olfactory ensheathing cells

The olfactory ensheathing cell (OEC) is a unique glial cell able to support neurite outgrowth in the CNS throughout life. The OEC has been described as having both Schwann cell-like and astrocyte-like characteristics. The purpose of this study was to compare gap junctional communication and connexin (Cx) expression in cultured olfactory ensheathing cells with both astrocytes and Schwann cells to establish which of these two cells types they most closely resemble. We examined the Cx mRNA profile of OECs, astrocytes, and Schwann cells using primers to Cx26, Cx32, Cx37, Cx43, Cx46, and Cx50. All connexins tested except Cx50 were expressed by all three cell types when initially cultured. However, we observed differences in the levels of expression of Cx32 and Cx26 between astrocytes, Schwann cells, and OECs that became pronounced with time. All three cell types show limited and variable gap junctional communication in culture as assessed by the transfer of microinjected Lucifer yellow. OECs had limited coupling compared with Schwann cells and astrocytes, although the extent of the dye spread through OECs was more comparable to that seen with Schwann cells than astrocytes. Thus, OECs display a profile of Cx expression that more closely resembles the Cx expression of Schwann cells rather than astrocytes.     

Astrocytes, but not olfactory ensheathing cells or Schwann cells, promote myelination of CNS axons in vitro

We have examined the interaction between olfactory ensheathing cells (OECs), Schwann cells (SC), oligodendrocytes, and CNS axons using cultures generated from embryonic rat spinal cord. Oligodendrocyte process extension and myelination in these cultures was poor if the cells were plated on OECs or SCs. Myelin internodes and nodes of Ranvier formed frequently if these cultures were plated onto monolayers of neurosphere-derived astrocytes (NsAs). In the myelinated fibers generated on NsAs, Nav channels, caspr, and neurofascin molecules were correctly assembled at the nodes of Ranvier. The density of neurites, survival, and antigenic differentiation of oligodendrocytes was similar on OEC and NsAs monolayers. However, on OEC monolayers, despite a transient increase in the number of endogenous oligodendrocytes, there was a decrease in oligodendrocyte process extension and axonal ensheathment when compared with cultures plated on NsAs monolayers. To determine if these changes were due to axonal or glial factors, spinal cord oligodendrocytes were plated onto monolayers of OECs, NsAs, and poly-L-lysine in the absence of neurons. In these cultures, process extension and myelin-like membrane formation by oligodendrocytes was improved on monolayers of OEC. This suggests that inhibition of process extension is mediated via cross-talk between OECs and neurites. In cultures containing axons plated on OEC monolayers, oligodendrocyte process formation, axonal ensheathment, and myelination occurred albeit lower if the cultures were supplemented with NsAs conditioned medium. These data suggest OECs can permit neurite extension and oligodendrocyte proliferation, but lack secreted factor(s) and possible cell-cell contact that is necessary for oligodendrocyte process extension and myelination.     

The glycoprotein fibulin-3 regulates morphology and motility of olfactory ensheathing cells in vitro

The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. A specialized glial cell within the olfactory nerve, called olfactory ensheathing cell (OEC), and their associated extracellular matrix are thought to play an important role during regenerative events in this system. To gain insight into novel molecules that could mediate the OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments were conducted which revealed high expression of the glycoprotein fibulin-3 in OECs. This observation was confirmed with quantitative PCR. In vivo, the distribution of all members of the fibulin family, fibulin-3 included, was localized to the lamina propria underneath the olfactory epithelium, in close association within olfactory nerve bundles. To manipulate fibulin-3 gene expression in cultured OECs, lentiviral vector constructs were designed to either transgenically express or knock-down fibulin-3. Experimental data showed that increased levels of fibulin-3 induced profound morphological changes in cultured OECs, impeded with their migratory abilities and also suppressed OEC-mediated neurite outgrowth. Knock-down of fibulin-3 levels resulted in reduced OEC proliferation. In conclusion, the data provide novel insights into a putative role for fibulin-3 in the regulation of cell migration and neurite outgrowth within the primary olfactory pathway.     

Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose

Several studies have demonstrated the potential of olfactory ensheathing cells for the repair of central and peripheral nerve injury. However, the majority of these studies have been performed with olfactory ensheathing cells derived from the olfactory bulbs, situated inside the skull. A more clinically relevant source of olfactory ensheathing cells is the olfactory mucosa, located in the nose. To be successful, an autologous transplant of nasal ensheathing glia would require a large number of purified cells. To address this issue, we have focused our research on three neurotrophic factors, namely nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3). We show here that their respective receptors, TrkA, TrkB, TrkC, as well as p75(NTR) (the low affinity NGF receptor), are expressed in vitro by the nasal ensheathing cells; the three neurotrophins promote purification and proliferation of these glial cells, with an optimal concentration of 50 ng/ml; and human ensheathing cells can be easily biopsied and highly purified using a serum-free medium supplemented with NT3. This technique opens the door for clinical trials in which nasal ensheathing cells will be autotransplanted in humans suffering from nerve injury.     

嗅鞘细胞诱导神经干细胞分化后神经元电生理特性的研究

目的探索大鼠嗅鞘细胞对神经干细胞(NSC)分化的影响,以及分化后神经元电生理特性。方法取新生鼠大脑皮质,原代培养大鼠NSC。NSC分为实验组和对照组,实验组将无血清培养的NSC中加入嗅鞘细胞条件培养液,对照组单纯无血清培养NSC。光镜下观察细胞分化情况,免疫组化法分别检测巢蛋白(nestin)、神经生长因子受体(NGFRp75)、神经丝蛋白(NF200)和胶质纤维酸性蛋白(GFAP)的表达,膜片钳检测神经元电生理特性。结果实验组嗅鞘细胞主要诱导NSC分化为神经元,少量分化为胶质细胞。对照组NSC逐渐萎缩,最终死亡。分化后的神经元记录到快速激活、快速失活能被河豚毒素特异阻断的钠电流,以及慢激活、慢失活能被四乙铵特异阻断的延迟整流性钾电流。结论嗅鞘细胞能诱导NSC分化成神经元,分化后的神经元具有活跃的电生理特性。