Remyelination of spinal cord axons by olfactory ensheathing cells and Schwann cells derived from a transgenic rat expressing alkaline phosphatase marker gene

Transplantation of cell suspensions containing olfactory ensheathing cells (OECs) has been reported to remyelinate demyelinated axons in the spinal cord with a Schwann cell (SC)-like pattern of myelination. However, questions have been raised recently as to whether OECs can form SC-like myelin. To address this issue we prepared SCs and OECs from transgenic rats in which a marker gene, human placental alkaline phosphatase (hPAP), is linked to the ubiquitously active promoter of the R26 gene. SCs were prepared from the sciatic nerve and OECs from the outer nerve-fiber layer of the olfactory bulb. Positive S100 and p75 immunostaining indicated that >95% of cells in culture displayed either SC or OEC phenotypes. Suspensions of either SCs or OECs were transplanted into an X-irradiation/ethidium bromide demyelinating lesion in the spinal cord. We observed extensive SC-like remyelination following either SC or OEC transplantation 3 weeks after injection of the cells. Alkaline phosphatase (ALP) chromagen reaction product was associated clearly with the myelin-forming cells. Thus, cell suspensions that are enriched in either SCs or OECs result in peripheral-like myelin when transplanted in vivo.     

Anti-β1 integrin antibody inhibits schwann cell meylination

Schwann cells (SCs) co-cultured with sensory neurons require ascorbate supplementation for basal lamina assembly and differentiation into myelinating cells. The ascorbate requirement can be bypased by adding a purifed basal lamina component, laminin, to SC/neuron cocultures. We have examined the role of laminin receptors, Namely, the β1 subfamily of integrins, in the process of myelination. We demonstrate by immunostaining or immunoprecipitation that undifferentiated SCs in contact with axons express large amounts of the β1 subunit in association with the α1 or α6 subunit. In co-cultures of myelinating SCs, α1β1 is no longer present, α6β1 is still present but at reduced levels, and α6β4 is expressed at much higher levels than in co-cultures of undifferentiated SCs. Immunogold labelling at the electron microscope level suggested that β1 integrins are randomly distributed on undifferentiated SCs, become localized to the SC surface contacting basal lamina in differentiating SCs before the onset of myelination, and are not detected on myelinating SCs. Fab fragments of β1 function-blocking antibody block both attachment of isolated SCs to laminin and formation of myelin sheaths by SCs co-cultured with neurons in ascorbate-supplemented medium. SCs unable to myelinate in the presence of the anti-β1 antibody assemble patchy basal lamina that is only loosely attached to the cell surface and in some cases appears to be detaching from the membrane. In contrast, an α1β1 function-blocking antibody only partially blocks attachment of isolated SCs to laminin but has no inhibitory effect on SC myelination. These results are consistent with the hypothesis that a member of the β1 subfamily of integrins other than α1β1 binds laminin present in basal lamina to the SC surface and transduces signals that are critical for initiation of SC differentiation into a myelinating cell. 1994 John Wiley & Sons, Inc.     

The Effects of Co-transplantation of Olfactory Ensheathing Cells and Schwann Cells on Local Inflammation Environment in the Contused Spinal Cord of Rats

Inflammatory response following spinal cord injury (SCI) is important in regulation of the repair process. Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are important donor cells for repairing SCI in different animal models. However, synergistic or complementary effects of co-transplantation of both cells for this purpose have not been extensively investigated. In the present study, we investigated the effects of co-transplantation of OECs and SCs on expression of pro- or anti-inflammatory factor and polarization of macrophages in the injured spinal cord of rats. Mixed cell suspensions containing OECs and SCs were transplanted into the injured site at 7 days after contusion at the vertebral T10 level. Compared with the DMEM, SC, or OEC group, the co-transplantation group had a more extensive distribution of the grafted cells and significantly reduced number of astrocytes, microglia/macrophage infiltration, and expression of chemokines (CCL2 and CCL3) at the injured site. The co-transplantation group also significantly increased arginase⁺/CD206⁺ macrophages (IL-4) and decreased iNOS⁺/CD16/32⁺ macrophages (IFN-γ), which was followed by higher IL-10 and IL-13 and lower IL-6 and TNF-α in their expression levels, a smaller cystic cavity area, and improved motor functions. These results indicate that OEC and SC co-transplantation could promote the shift of the macrophage phenotype from M(IFN-γ) to M(IL-4), reduce inflammatory cell infiltration in the injured site, and regulate inflammatory factors and chemokine expression, which provide a better immune environment for SCI repair.     

Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system

ABSTRACT: BACKGROUND: Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC), satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2) is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS). RESULTS: Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL) region of the lumbar spinal cord (LSC) in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM) resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly. CONCLUSION: We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.     

Exogenous Schwann Cells Migrate,Remyelinate and Promote Clinical Recovery in Experimental Auto-Immune Encephalomyelitis

Schwann cell (SC) transplantation is currently being discussed as a strategy that may promote functional recovery in patients with multiple sclerosis (MS) and other inflammatory demyelinating diseases of the central nervous system (CNS). However this assumes they will not only survive but also remyelinate demyelinated axons in the chronically inflamed CNS. To address this question we investigated the fate of transplanted SCs in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in the Dark Agouti rat; an animal model that reproduces the complex inflammatory demyelinating immunopathology of MS. We now report that SCs expressing green fluorescent protein (GFP-SCs) allografted after disease onset not only survive but also migrate to remyelinate lesions in the inflamed CNS. GFP-SCs were detected more frequently in the parenchyma after direct injection into the spinal cord, than via intra-thecal delivery into the cerebrospinal fluid. In both cases the transplanted cells intermingled with astrocytes in demyelinated lesions, aligned with axons and by twenty one days post transplantation had formed Pzero protein immunoreactive internodes. Strikingly, GFP-SCs transplantation was associated with marked decrease in clinical disease severity in terms of mortality; all GFP-SCs transplanted animals survived whilst 80% of controls died within 40 days of disease.     

Adenosine: an activity-dependent axonal signal regulating MAP kinase and proliferation in developing Schwann cells

Nonsynaptic release of ATP from electrically stimulated dorsal root gangion (DRG) axons inhibits Schwann cell (SC) proliferation and arrests SC development at the premyelinating stage, but the specific types of purinergic receptor(s) and intracellular signaling pathways involved in this form of neuron-glia communication are not known. Recent research shows that adenosine is a neuron-glial transmitter between axons and myelinating glia of the CNS. The present study investigates the possibility that adenosine might have a similar function in communicating between axons and premyelinating SCs. Using a combination of pharmacological and molecular approaches, we found that mouse SCs in culture express functional adenosine receptors and ATP receptors, a far more complex array of purinergic receptors than thought previously. Adenosine, but not ATP, activates ERK/MAPK through stimulation of cAMP-linked A2(A) adenosine receptors. Both ATP and adenosine inhibit proliferation of SCs induced by platelet-derived growth factor (PDGF), via mechanisms that are partly independent. In contrast to ATP, adenosine failed to inhibit the differentiation of SCs to the O4+ stage. This indicates that, in addition to ATP, adenosine is an activity-dependent signaling molecule between axons and premyelinating Schwann cells, but that electrical activity, acting through adenosine, has opposite effects on the differentiation of myelinating glia in the PNS and CNS.     

Sema4D Knockdown in Oligodendrocytes Promotes Functional Recovery After Spinal Cord Injury

Semaphorin4D (Sema4D) belongs to Semaphorins family and is secreted and membrane-bound protein. Its function on angiogenesis and axon regeneration makes it an ideal therapeutic target for spinal cord injury (SCI). Here we examined Sema4D expression profile by real-time PCR and western blot and found Sema4D was upregulated after SCI. In vitro study showed Sema4D was not only expressed in oligodendrocytes but also in endothelial cells (ECs). Hypoxia can mimic Sema4D upregulation in both cell lines. Moreover, overexpression of Sema4D through lentivirus in ECs promoted tube formation. However, Sema4D overexpression in oligodendrocytes precursor cells (OPCs) inhibited neuron myelination in neuron-oligodendrocyte co-culture system. Therefore, Sema4D knockdown in OPCs was applied in SCI rats. The results indicated that Sema4D knockdown significantly promoted functional recovery with blood–brain barrier score. Taken together, our data suggest that specific Sema4D knockdown in oligodendrocytes without disturbing its angiogenesis effect can be a beneficial strategy for SCI treatment.     

Human schwann cells in vitro. II. Myelination of sensory axons following extensive purification and heregulin-induced expansion

Co-culture conditions are well established in which Schwann cells (SCs) derived from immature or adult rats proliferate and form myelin in response to contact with sensory axons. In a companion article, we report that populations of adult-derived human Schwann cells (HASCs) fail to function under these co-culture conditions. Furthermore, we report progressive atrophy of neurons in co-cultures containing populations of either human fibroblasts. Two factors that might account for the insufficiency of the co-culture system to support HASC differentiation are the failure of many HASCs to proliferate and the influence of contaminating fibroblasts. To minimize fibroblast contamination of neuron-HASC co-cultures, we used fluorescence-activated cell sorting to highly purify HASC populations (to more than 99.8%). To stimulate expansion of the HASC population, a mitogenic mixture of heregulin (HRGβ1 amino acid residues 177-244; 10 nM), cholera toxin (100 ng/mL), and forskolin (1 μM) was used. When these purified and expanded HASCs were co-cultured with embryo-derived rat sensory neurons, neuronal shrinkage did not occur and after 4 to 6 weeks some myelin segments were seen in living co-cultures. This myelin was positively identified as human by immunostaining with a monoclonal antibody specific to the human peripheral myelin protein P₀ (antibody 592). Although this is the first reported observation of myelination by HASCs in tissue culture, it should be noted that myelination occurred more slowly and in much less abundance than in comparable cultures containing adult rat-derived SCs. We anticipate that further refinements of the HASC co-culture system that enhance myelin formation will provide insights into important aspects of human SC biology and provide new opportunities for studies of human peripheral neuropathies. © 1995 John Wiley & Sons, Inc.     

SSeCKS Promotes Tumor Necrosis Factor-α Autocrine via Activating p38 and JNK Pathways in Schwann Cells

Tumor necrosis factor-alpha (TNF-α) derived from activated Schwann cells (SCs) plays a critical role as an inflammatory mediator in the peripheral nervous system disease. TNF-α could act as an autocrine mediator in SC activation. In this study, we found knockdown Src-suppressed protein kinase C substrate (SSeCKS) expression suppressed TNF-α production induced by TNF-α, overexpression of SSeCKS could promoted TNF-α autocrine in SCs. Such effects might be resulted in SSeCKS promoted p38 and JNK activation in SCs treated by TNF-α. Thus present data show that while SCs activation, SSeCKS may plays an important role in the release of inflammatory mediators.     

P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord

The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3–5 mM) or a P2X7R agonist, 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.     

Hedgehog-mediated paracrine interaction between hepatic stellate cells and marrow-derived mesenchymal stem cells

During liver injury, bone marrow-derived mesenchymal stem cells (MSCs) can migrate and differentiate into hepatocytes. Hepatic stellate cell (SC) activation is a pivotal event in the development of liver fibrosis. Therefore, we hypothesized that SCs may play an important role in regulating MSC proliferation and differentiation through the paracrine signaling pathway. We demonstrate that MSCs and SCs both express hedgehog (Hh) pathway components, including its ligands, receptors, and target genes. Transwell co-cultures of SCs and MSCs showed that the SCs produced sonic hedgehog (Shh), which enhanced the proliferation and differentiation of MSCs. These findings demonstrate that SCs indirectly modulate the activity of MSCs in vitro via the Hh pathway, and provide a plausible explanation for the mechanisms of transplanted MSCs in the treatment of liver fibrosis.     

PDGF,NT-3 and IGF-2 in Combination Induced Transdifferentiation of Muscle-Derived Stem Cells into Schwann Cell-Like Cells

Muscle-derived stem cells (MDSCs) are multipotent stem cells with a remarkable long-term self-renewal and regeneration capacity. Here, we show that postnatal MDSCs could be transdifferentiated into Schwann cell-like cells upon the combined treatment of three neurotrophic factors (PDGF, NT-3 and IGF-2). The transdifferentiation of MDSCs was initially induced by Schwann cell (SC) conditioned medium. MDSCs adopted a spindle-like morphology similar to SCs after the transdifferentiation. Immunocytochemistry and immunoblot showed clearly that the SC markers S100, GFAP and p75 were expressed highly only after the transdifferentiation. Flow cytometry assay showed that the portion of S100 expressed cells was more than 60 percent and over one fourth of the transdifferentiated cells expressed all the three SC markers, indicating an efficient transdifferentiation. We then tested neurotrophic factors in the conditioned medium and found it was PDGF, NT-3 and IGF-2 in combination that conducted the transdifferentiation. Our findings demonstrate that it is possible to use specific neurotrophic factors to transdifferentiate MDSCs into Schwann cell-like cells, which might be therapeutically useful for clinical applications.     

Hedgehog signaling regulates myelination in the peripheral nervous system through primary cilia

Myelination is an essential prerequisite for the nervous system to transmit an impulse efficiently by a saltatory conduction. In the peripheral nervous system (PNS), Schwann cells (SCs) engage in myelination. However, a detailed molecular mechanism underlying myelination still remains unclear. In this study, we hypothesized that the primary cilia of SCs are the regulators of Hedgehog (Hh) signaling-mediated myelination. To confirm our hypothesis, we used mouse dorsal root ganglion (DRG)/SC co-cultures, wherein the behavior of SCs could be analyzed by maintaining the interaction of SCs with DRG neurons. Under these conditions, SCs had primary cilia, and Hh signaling molecules accumulated on the primary cilia. When the SCs were stimulated by the addition of desert hedgehog or smoothened agonist, formation of myelin segments on the DRG axons was facilitated. On the contrary, upon administration of cyclopamine, an inhibitor of Hh signaling, myelin segments became comparable to those of controls. Of note, the ratio of SCs harboring primary cilium reached the highest point during the early phase of myelination. Furthermore, the strongest effects of Hh on myelination were encountered during the same stage. These results collectively indicate that Hh signaling regulates myelin formation through primary cilia in the PNS.     

The Expression of Nerve Growth Factor Receptor on Schwann Cells and the Effect of These Cells on Regeneration of Axons in Traumatically Injured Human Spinal Cord

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Insulin-like growth factor-I (IGF-I) and IGF binding protein-5 in Schwann cell differentiation

Schwann cells (SCs) are the myelin producing cells of the peripheral nervous system. During development, SCs cease proliferation and differentiate into either a myelin-forming or non-myelin forming mature phenotype. We are interested in the role of insulin-like growth factor-I (IGF-I) in SC development. We have shown previously SCs proliferate in response to IGF-I in vitro. In the current study, we investigated the role of IGF-I in SC differentiation. SC differentiation was determined by morphological criteria and expression of myelin proteins. Addition of 1 mM 8-bromo cyclic AMP (cAMP) or growth on Matrigel matrix decreased proliferation and induced differentiation of SCs. IGF-I enhanced both cAMP and Matrigel matrix-induced SC differentiation, as assessed by both morphological criteria and myelin gene expression. Cultured SCs also express IGF binding protein-5 (IGFBP-5), which can modulate the actions of IGF-I. We examined the expression of IGFBP-5 during SC differentiation. Both cAMP and Matrigel matrix treatment enhanced IGFBP-5 protein expression and cAMP increased IGFBP-5 gene expression five fold. These findings suggest IGF-I potentiates SC differentiation. The concomitant up-regulation of IGFBP-5 may play a role in targeting IGF-I to SCs and thus increase local IGF-I bioavailability. J. Cell. Physiol. 171:161–167, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of 9-O-Acetyl GD3 Ganglioside in Mycobacterium leprae Infection of Schwann Cells

Mycobacterium leprae (ML), the etiologic agent of leprosy, mainly affects the skin and peripheral nerves, leading to demyelization and loss of axonal conductance. Schwann cells (SCs) are the main cell population infected by ML in the nerves, and infection triggers changes in the SC phenotype from a myelinated to a nonmyelinated state. In the present study, we show that expression of 9-O-acetyl GD3, a ganglioside involved in cellular anti-apoptotic signaling and nerve regeneration, increases in SCs following infection with ML. Observation by confocal microscopy together with coimmunoprecipitation suggested that this ganglioside participates in ML attachment and internalization by SC. Immunoblockage of 9-O-acetyl GD3 in vitro significantly reduced adhesion of ML to SC surfaces. Finally, we show that activation of the MAPK (ERK 1/2) pathway and SC proliferation, two known effects of ML on SCs that result in demyelization, are significantly reduced when the 9-O-acetyl GD3 ganglioside is immunoblocked. Taken together, these data suggest the involvement of 9-O-acetyl GD3 in ML infection on SCs.     

Krüppel-Like Factor 6 Rendered Rat Schwann Cell More Sensitive to Apoptosis via Upregulating FAS Expression

Krüppel-like factor 6 (KLF6) is a tumor suppressor gene and play a role in the regulation of cell proliferation and apoptosis. After the peripheral nerve injury (PNI), the microenvironment created by surrounding Schwann cells (SCs) is a critical determinant of its regenerative potential. In this study, we examined the effects of KLF6 on SCs responses during PNI. Both KLF6 mRNA and protein expression levels were upregulated in the injured sciatic nerve, and immunofluorescence results showed that many KLF6-positive cells simultaneously expressed the SC markers S-100 and p75NTR. The apoptosis inducers TNFα and cisplatin upregulated KLF6 expression in primary cultured SCs and the SC line RSC96. Although KLF6 overexpression exacerbated cisplatin- and TNFα-induced apoptosis, expression levels of the apoptosis regulators Bcl2 and Bax were not significantly affected in either KLF6-overexpressing or KLF6-depleted RSC96 cells. Realtime PCR arrays and qRT-PCR demonstrated that KLF6 overexpression upregulated four pro-apoptotic genes, FAS, TNF, TNFSF12, and PYCARD, and inhibited expression of the anti-apoptotic IL10 gene expression. Further analysis revealed that FAS protein expression was positively correlated with KLF6 expression in SCs. These data suggest that KLF6 upregulation may render SCs more vulnerable to apoptosis after injury via upregulating FAS expression.     

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.