Extracts of muscle biopsies from patients with spinal muscular atrophies inhibit neurite outgrowth from spinal neurons

Preparations derived from embryonic and neonatal chick muscle enhance neurite outgrowth when added to cultures of embryonic chick spinal neurons. In the presence of soluble extracts of biopsied muscle from 15 of 20 patients with spinal muscular atrophy (SMA), the in vitro neurite-promoting activity of neonatal chick muscle was inhibited. There was no comparable inhibition using extracts from 20 age-matched pathologic or morphologically normal controls. The neurite-promoting activity in media conditioned by embryonic myotubes was not inhibited by extracts of the SMA group.     

Increase of neurite-promoting activity for spinal neurons in muscles of 'paralysé' mice and tenotomised rats

Various lines of evidence from in vivo and in vitro experiments suggest that muscle-derived factors may enhance the survival and axonal outgrowth of spinal neurons. Our previous results using cultures of embryonic spinal neurons showed that denervation of skeletal muscle increased levels of a neurite-promoting activity in soluble muscle extracts. Here, two other experimental models in which muscle activity was also lowered were investigated. The mutant mouse 'paralysé' exhibits a spontaneous regression of motor nerve endings, with concomitant paralysis. In 'paralysé' mutant muscle extracts, specific neurite-promoting activity was up to 10-fold higher than in extracts prepared from control littermates. Tenotomy is known to retard the regression of polyneuronal motor innervation in skeletal muscle from neonatal rats. Three days after operation, levels of neurite-promoting activity were increased 2-fold with respect to total protein. These results suggest that skeletal muscle activity might regulate the synthesis of molecules affecting nerve growth.     

Neurotrophic effects of FPF-1070 (Cerebrolysin®) on cultured neurons from chicken embryo dorsal root ganglia, ciliary ganglia, and sympathetic trunks

We examined the effect of FPF-1070 (Cerebrolysin) on neurite outgrowth in explant cultures of dorsal root ganglia (DRG), sympathetic trunks (ST), and ciliary ganglia (CG) from 10- to 11-day chicken embryos. FPF-1070 significantly promoted neurite outgrowth in DRG and ST neurons at all concentrations examined, in comparison with phosphate buffered saline-treated negative controls; however, this effect on neurite outgrowth was not as significant as that observed for nerve growth factor-treated positive controls on DRG and ST neurons. Additionally, FPF-1070 exhibited an inverted U relationship between concentration and effectiveness in DRG and ST neurons. In contrast, FPF-1070 did not affect neurite outgrowth in CG neurons although ciliary neurotrophic factor-treated positive controls showed striking neurite outgrowth. Our results demonstrate that FPF-1070 has different neurotrophic effects depending on the subpopulation of neurons. This study clarifies a role for neurotrophic activity in the mechanism of action of FPF-1070.     

Alpha 2-macroglobulin is an astroglia-derived neurite-promoting factor for cultured neurons from rat central nervous system.

The neurite promoting factors in the astroglial conditioned medium (As-CM) were characterized by using primary cultures of embryonic rat neocortical neurons. The factors in the As-CM bind to lectins such as wheat germ agglutinin (WGA), suggesting that they contain sugar moieties. When the WGA-bound fractions were applied on a Superose 6 column, the activity was recovered mainly in two fractions, peak I and peak II. The peak II fraction was further purified by Mono Q anion exchange chromatography. A single protein band of 180 kDa was detected in the final Mono Q fraction by sodium dodecylsulfate polyacrylamide gel electrophoresis. The molecular weight coincided with that of alpha 2-macroglobulin (alpha 2M). Western blotting showed that the single protein band was reacted with anti-alpha 2M antibody but not with anti-fibronectin and anti-laminin antisera. The neurite-promoting activity of the Mono Q fraction was inhibited by anti-alpha 2M antibody. Furthermore, commercially available alpha 2M also promotes neurite outgrowth in our assay system. These results strongly suggested that alpha 2M is one of the neurite-promoting factors in the As-CM.     

Synthesis of a growth-associated protein by embryonic rat cerebrocortical neurons in vitro

Proteins synthesized by embryonic rat cortical cultures were studied under conditions that were either permissive or nonpermissive to neurite outgrowth. Freshly dissected cortex from embryonic day 17 rat pups was mechanically dissociated and plated on poly(L-lysine) substrate in the presence of (1) serum-free media, which allowed neuronal survival but no outgrowth; (2) serum, which allowed survival of both neurons and glia as well as neurite outgrowth; or (3) a hormone-supplemented defined media, which allowed preferential survival and outgrowth of neurons. In addition, postnatal tissue was cultured as a source of glia. Cultures were pulse-labeled with 35S-methionine 48 hr after plating and the protein synthesis patterns examined by 2-dimensional gel electrophoresis followed by fluorography. The expression of an acidic 50 kDa protein, associated with the particulate fraction of cells, was found to be a prominent correlate of neurite outgrowth. This protein was synthesized in serum- or hormone-treated embryonic cultures showing neurite outgrowth but was undetectable in embryonic cultures without outgrowth or in postnatal glial cultures. By virtue of its migration position on 2-dimensional gels, its presence in a light membrane fraction, and its cleavage products after Staphylococcus aureus protease treatment, the 50 kDa protein appears to be identical to an acidic 43-49 kDa protein that has been identified in several developing and regenerating neural pathways, as well as to the B-50 phosphoprotein. These findings lend support for a critical role of this protein in neural development and demonstrate the feasibility of using primary CNS cell cultures to study its biosynthesis and function.     

α2-Macroglobulin is an astroglia-derived neurite-promoting factor for cultured neurons from rat central nervous system

The neurite promoting factors in the astroglial conditioned medium (As-CM) were characterized by using primary cultures of embryonic rat neocortical neurons. The factors in the As-CM bind to lectins such as wheat germ agglutinin (WGA), suggesting that they contain sugar moieties. When the WGA-bound fractions were applied on a Superose 6 column, the activity was recovered mainly in two fractions, peak I and peak II. The peak II fraction was further purified by Mono Q anion exchange chromatography. A single protein band of 180 kDa was detected in the final Mono Q fraction by sodium dodesylsulfate polyacrylamide gel electrophoresis. The molecular weight coincided with that ofα₂-macroglobulin (α₂M). Western blotting showed that the single protein band was reacted with anti-α₂M antibody but not with anti-fibronectin and anti-laminin antisera. The neurite-promoting activity of the Mono Q fraction was inhibited by anti-α₂M antibody. Furthermore, commercially available α₂M also promotes neurite outgrowth in our assay system. These results strongly suggested that α₂M is one of the neurite-promoting factors in the As-CM.     

Fibroblast growth factor treatment produces differential effects on survival and neurite outgrowth from identified bulbospinal neurons in vitro

The in vivo application of appropriate trophic factors may enhance regeneration of bulbospinal projections after spinal cord injury. Currently, little is known about the sensitivities of specific bulbospinal neuron populations to the many identified trophic factors. We devised novel in vitro assays to study trophic effects on the survival and neurite outgrowth of identified bulbospinal neurons. Carbocyanine dye crystals implanted into the cervical spinal cord of embryonic day (E)5 chick embryos retrogradely labeled developing bulbospinal neurons. On E8, dissociated cultures containing labeled bulbospinal neurons were prepared. Fibroblast growth factor (FGF)-2 (but not FGF-1) promoted the survival of bulbospinal neurons. FGF receptor expression was widespread in the E8 brainstem, but not detected in young bulbospinal neurons, suggesting that nonneuronal cells mediated the FGF-stimulated survival response. Astrocytes synthesize a variety of trophic factors, and astrocyte-conditioned medium (ACM) also promoted the survival of bulbospinal neurons. As might be expected, FGF-2 function blocking antibodies did not suppress ACM-promoted survival, nor did an ELISA detect FGF-2 in ACM. This suggests that nonneuronal cells synthesize other factors in response to exogenous FGF-2 which promote the survival of bulbospinal neurons. Focusing on vestibulospinal neurons, dissociated (survival assay) or explant (neurite outgrowth assay) cultures were prepared. FGF-2 promoted both survival and neurite outgrowth of identified vestibulospinal neurons. Interestingly, FGF-1 promoted neurite outgrowth but not survival; the converse was true of FGF-9. Thus, differential effects of specific growth factors on survival or neurite outgrowth of bulbospinal neurons were distinguished.     

The growth of neurites from explants of brachial spinal cord exposed to different components of wing bud mesenchyme

The extension of peripheral axons from the brachial spinal cord into the embryonic chick wing bud suggests that the target premuscle cell masses may act as the source of an adhesion gradient which establishes selective nerve pathways. Wing premuscle cell masses were explanted from different stage embryos and tested against age-matched brachial spinal cord for their ability to promote directional neurite outgrowth. It was found that target premuscle which does not contain myotubes could still elicit directional outgrowth in vitro. In contrast, skin and precartilage were unable to promote neuritic outgrowth significantly. Serum-free conditioned media were prepared from stages 20-38 premuscle and tested against age-matched spinal cord explants. There was an increase in the effects of conditioned media on neuritic outgrowth up to stage 35; conditioned media from older-stage premuscles had less effect than that of stage 35 premuscles. These results were shown to be dependent on the maturation of the premuscles and not on that of the spinal cord. When the premuscle conditioned media were preincubated over polylysine substrata, the ability to induce neuritic outgrowth was abolished from media derived from premuscle at stage 27 and older. Conditioned media derived from premuscle at stage 27 or older contain a polylysine-binding neurite-promoting factor which is present in greater amounts in more differentiated muscle. The time of first detection of neurite-promoting factors in stage 27 premuscle conditioned media correlates with the in vivo stages at which muscle-specific nerves branch from the main nerve trunks.     

Neurotrophic activity of S-100 beta in cultures of dorsal root ganglia from embryonic chick and fetal rat

We report here that S-100 beta, a protein with neurotrophic activity on central nervous system neurons, stimulates neuritic outgrowth from cultures of dorsal root ganglia (DRG). S-100 beta elicited neurites from explant and dissociated cell cultures of embryonic chick DRG, and the extent of the response varied with the age of the embryo. Specificity was demonstrated by the observation that incubation of S-100 beta with antibodies directed against S-100 beta reduced the neurite outgrowth, whereas incubation of S-100 beta with normal rabbit serum had little effect. S-100 beta also stimulated the area of neuritic outgrowth from organotypic cultures of fetal rat DRG, showing that the activity of the protein is not restricted to a particular species or culture condition. A mutant S-100 beta lacking neurotrophic activity on cerebral cortex neurons was unable to effectively stimulate neurite outgrowth from DRG cultures. These studies suggest that S-100 beta may play a role in neuronal growth and/or maintenance in the peripheral nervous system.     

Central and peripheral neurite outgrowth differs in preference for heparin-binding versus integrin-binding sequences.

Neurons of the CNS and PNS differ in their response to fibronectin (FN) and proteolytic fragments of FN. The 33 kDa C-terminal cell and heparin-binding fragment of FN, in particular, is a strong promoter of CNS neurite outgrowth. To define further the neurite-promoting activity of the 33 kDa fragment, and to investigate further the differences between PNS and CNS responses to FN and the 33 kDa fragment, we contrasted neurite outgrowth by CNS and PNS neurons on three synthetic peptides representing sequences from this fragment of FN: two heparin-binding peptides, FN-C/H I and FN-C/H II (McCarthy et al., 1990), and an integrin-binding peptide, CS1 (Humphries et al., 1987). Spinal cord (SC) neurons, from the CNS, differed from dorsal root ganglion (DRG) neurons, from the PNS, with respect to substratum preference for heparin-binding versus integrin-binding peptides. SC neurite outgrowth was greatest on the heparin-binding peptide FN-C/H II, while DRG neurite outgrowth was greatest on the a4 beta 1 integrin-binding peptide CS1. To test whether the difference in substratum preference was due to differences in the molecular mechanism by which SC and DRG neurons interact with the 33 kDa fragment of FN, anti-beta 1 integrin antibodies and/or soluble heparin were added to the cultures as potential inhibitors of integrin-mediated or proteoglycan-mediated interactions with FN. SC neurite outgrowth was much more sensitive to the effect of heparin than anti-beta 1 integrin, indicating SC neurite outgrowth may involve predominantly a heparin-sensitive mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental regulation of chick embryo retina neurite extension by extrinsic factors

Developmental changes in the control of neurite extension by extracellular factors can be examined by utilizing cultures of neurons from various aged embryos. Several conditioned media and tissue extracts were added to cultures of chick embryo retinal neurons on collagen substrates for 1, 3 and 5 days in vitro. Neurite outgrowth, measured as the percentage of neurons with neurites and the length of neurites, was promoted by optic tectal extract and cornea conditioned medium in retina neurons from younger ages (6- to 12-day embryos), but not from older ages (14- and 16-day embryos). The promotion of neurite outgrowth by optic tectal extracts may be mediated by a promotion of glial cell growth. The developmental changes in neurite extension may be due to either an altered sensitivity to the neurite promoting factors or by an altered intrinsic ability of retinal neurons to extend processes.     

A glia-derived nexin promotes neurite outgrowth in cultured chick sympathetic neurons

A glia-derived neurite-promoting factor has been purified from medium conditioned by C6 rat glioma cells. It induces neurite outgrowth in cultured mouse neuroblastoma cells and inhibits granule cell migration in explants of mouse cerebellum. This factor is a potent serine protease inhibitor which has recently been shown to belong to the protease nexin family. It has therefore been called glia-derived nexin (GDN). We report here that GDN also promotes neurite outgrowth in dissociated chick superior cervical ganglion neurons grown in serum-free medium. In these neurons, the presence of nerve growth factor is not required for the stimulatory effect of GDN in the initial phase of neurite outgrowth. These experiments demonstrate that a glia-derived protein with protease inhibitory activity can modulate neurite outgrowth in cultured chick sympathetic neurons.     

Fibroblast Growth Factor Treatment Produces Differential Effects on Survival and Neurite Outgrowth from Identified Bulbospinal Neurons in Vitro

The in vivo application of appropriate trophic factors may enhance regeneration of bulbospinal projections after spinal cord injury. Currently, little is known about the sensitivities of specific bulbospinal neuron populations to the many identified trophic factors. We devised novel in vitro assays to study trophic effects on the survival and neurite outgrowth of identified bulbospinal neurons. Carbocyanine dye crystals implanted into the cervical spinal cord of embryonic day (E)5 chick embryos retrogradely labeled developing bulbospinal neurons. On E8, dissociated cultures containing labeled bulbospinal neurons were prepared. Fibroblast growth factor (FGF)-2 (but not FGF-1) promoted the survival of bulbospinal neurons. FGF receptor expression was widespread in the E8 brainstem, but not detected in young bulbospinal neurons, suggesting that nonneuronal cells mediated the FGF-stimulated survival response. Astrocytes synthesize a variety of trophic factors, and astrocyte-conditioned medium (ACM) also promoted the survival of bulbospinal neurons. As might be expected, FGF-2 function blocking antibodies did not suppress ACM-promoted survival, nor did an ELISA detect FGF-2 in ACM. This suggests that nonneuronal cells synthesize other factors in response to exogenous FGF-2 which promote the survival of bulbospinal neurons. Focusing on vestibulospinal neurons, dissociated (survival assay) or explant (neurite outgrowth assay) cultures were prepared. FGF-2 promoted both survival and neurite outgrowth of identified vestibulospinal neurons. Interestingly, FGF-1 promoted neurite outgrowth but not survival; the converse was true of FGF-9. Thus, differential effects of specific growth factors on survival or neurite outgrowth of bulbospinal neurons were distinguished.     

Heparan sulfate proteoglycan and laminin mediate two different types of neurite outgrowth

Spinal cord neurons cultured in vitro have been shown to respond to changes in their environment by means of 2 different types of neurite outgrowth: (1) neurite elongation and (2) emergence and branching of newly formed neurites. Culture of spinal cord neurons with heparan sulfate proteoglycan (HSPG) medium resulted in a 3-fold increase in neurite elongation compared to the control. Extensive branching was seen when neurons were cultured in laminin-supplemented culture medium. HSPG-induced elongation and laminin-induced branching of neurites were blocked by specific anti-HSPG and antilaminin sera, respectively. Furthermore, laminin antibodies did not inhibit neurite elongation and HSPG antibodies did not block neurite branching. Conditioned medium from primary embryonic rat muscle cultures (MCM) mimicked the effects of both HSPG and laminin on neurite outgrowth. Immunoprecipitation with anti-HSPG and antilaminin antibodies demonstrated that MCM contains these 2 basal lamina components. Our observations suggest that HSPG and laminin might be highly effective molecules for promoting neurite outgrowth of rat spinal cord neurons in vitro.     

Identification of an ectodomain within the LAR protein tyrosine phosphatase receptor that binds homophilically and activates signalling pathways promoting neurite outgrowth

Elucidation of mechanisms by which receptor protein tyrosine phosphatases (PTPs) regulate neurite outgrowth will require characterization of ligand-receptor interactions and identification of ligand-induced signalling components mediating neurite outgrowth. The first identified ligand of the leucocyte common antigen-related (LAR) receptor PTP consists of a 99-residue ectodomain isoform, termed LARFN5C, which undergoes homophilic binding to LAR and promotes neurite outgrowth. We employed peptide mapping of LARFN5C to identify an active neurite-promoting domain of LAR. A peptide mimetic consisting of 37 residues (L59) and corresponding to the fifth LAR fibronectin type III (FNIII) domain prevented LARFN5C homophilic binding, demonstrated homophilic binding to itself and promoted neurite outgrowth of mouse E16-17 hippocampal neurons and of dorsal root ganglia explants. Response to L59 was partially lost when using neurons derived from LAR-deficient (-/-) mice or neurons treated with LAR siRNA, consistent with homophilic interaction of L59 with LAR. L59 neurite-promoting activity was decreased in the presence of inhibitors of Src, Trk, PLCgamma, PKC, PI3K and MAPK. L59 activated Src (a known substrate of LAR), FAK and TrkB and also activated downstream signalling intermediates including PKC, ERK, AKT and CREB. BDNF augmented the maximal neurite-promoting activity of L59, a finding consistent with the presence of shared and distinct signalling pathways activated by L59 with BDNF and L59 with TrkB. These studies are the first to identify an ectodomain of LAR (located within the fifth FNIII domain) capable of promoting neurite outgrowth and point to novel approaches for promotion of neurite outgrowth.     

Nerve growth factor in medium conditioned by embryonic chicken heart cells

The present report demonstrates that embryonic chicken heart cells in culture release different nerve growth promoting factors to their culture medium, one which is biologically and immunologically similar to mouse gland beta NGF. Serum-free heart cell conditioned medium thus promoted neurite outgrowth from sympathetic and ciliary ganglia and supported survival of dissociated ciliary neurons. The addition of affinity purified antibodies against mouse beta NGF does substantially but not completely inhibit the fibre outgrowth from sympathetic ganglia, but does not to any extent diminish the effects on the parasympathetic neurons. The chicken NGF recovered from polyacrylamide gels after electrophoresis greatly enhanced sympathetic fibre outgrowth, an activity completely suppressive by anti-beta NGF antibodies. We conclude that a chicken NGF is being produced by the embryonic heart cells in culture, and that this factor may be produced also in the embryo to fulfill a role in heart innervation.     

Influence of the culture substratum on the expression of choline acetyltransferase activity in purified motoneurons from rat embryos

Motoneurons from E14 rat embryos have been retrogradely labelled with a carbocyanine derivative and purified approximately 20-fold by density gradient centrifugation. For certain experiments, motoneurons have been purified to near homogeneity by cell sorting. These neurons were then cultured at low density on various substrata. Laminin stimulated neurite outgrowth of the identified motoneurons and stimulated choline acetyltransferase (ChAT) development by 2-fold; these cultures could not be maintained for more than 4 days. Motoneurons could be cultured for at least 7 days on monolayers of both living and lysed astrocytes from newborn cortex. The cells survived equally well on both substrata but neurite outgrowth was less elaborated on lysed astrocytes on which motoneurons displayed a mono- or bipolar, rather than a multipolar, morphology. Furthermore, ChAT developed at a 2-fold higher level on lysed, rather than living, newborn astrocytes. Such a difference was not observed using astrocytes from E14 rat embryos. In the latter case, both living and lysed astrocytes supported ChAT development at the same rate as lysed newborn astrocytes. Consequently, living astrocytes from newborn rat cortex are relatively less permissive for ChAT expression. On the other hand, both living and lysed astrocytes from newborn cortex induced ChAT expression by sympathetic neurons to the same extent. We propose that, in addition to retrograde factors from muscle origin, cell surface molecules expressed by embryonic astrocytes stimulate ChAT expression by motoneurons. Such molecules would be present in newborn astrocytes in an intracellular form and could be unmasked by cell lysis. Different cell surface molecules may induce ChAT activity in sympathetic neurons.     

Bone mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml(-1), respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.     

Effects of Cerebrolysin on the outgrowth and protection of processes of cultured brain neurons

Summary. Cerebrolysin (Cere, EBEWE Arzneimittel, Austria), a peptidergic drug produced by a standardised enzymatic breakdown of porcine brain proteins, consists of a mixture of 75% free amino acids and 25% low molecular weight peptides (<10 k DA). Cerebrolysin was shown to protect against MAP2 loss in primary embryonic chick neuronal cultures after brief histotoxic hypoxia and in a rat model of acute brain ischemia. Since MAP2 is involved in processes like neuronal growth, plasticity and dendritic branching, we address the question whether Cere is protecting processes against degeneration in a chronic low serum (2% FCS) cell stress model and whether the spontaneous outgrowth of axon-like processes is influenced. This was accomplished by quantification of the neurite lengths of embryonic chicken telencephalon neurons after 4 and 8 days. Additionally, time-laps video microscopy was performed to study a possible influence of Cere on the growth cone behaviour of axon-like processes. To distinguish between effects caused by the peptide fraction and the effects related to free amino acids, we used an artificial amino acid solution (AA-mix). Results demonstrate a process outgrowth promoting effect of the AA-mix and Cere after 4 DIV. After 8 days neuronal network degeneration occurred in the AA-mix treated cultures, whereas Cere treated cultures still presented a well differentiated neuronal network. Dying neurons could release factors possibly impeding neurite outgrowth and Cere was shown to increase the viability of chicken cortical neurons. Neither the addition of BDNF nor serum supplementation (5% and 10% FCS) could protect the neuronal network against degeneration after 8 DIV, although these treatments were shown to ameliorate the viability of chicken telencephalon neurons. This result together with the finding obtained using the artificial amino acid solution points to the peptide fraction of Cere to be responsible for the protection of processes against degeneration. Time-laps studies of Cere treated cultures revealed a significant decrease of the velocities characterising random growth cone movements, which is thought to be responsible for an increase in the length of axon-like processes after 4 DIV. Received September 21, 2000; accepted November 22, 2000     

Bone mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

Abstract

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml^?1, respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.