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.     

Bone marrow stromal cells promote neurite outgrowth of spinal motor neurons by means of neurotrophic factors in vitro

Transplantation of bone marrow stromal cells (BMSCs) into spinal cord injury models has shown significant neural function recovery; however, the underlying mechanisms have not been fully understood. In the present study we examined the effect of BMSCs on neurite outgrowth of spinal motor neuron using an in vitro co-culture system. The ventral horn of the spinal grey matter was harvested from neonatal Sprague–Dawley rats, cultured with BMSCs, and immunostained for neurofilament-200 (NF-200). Neurite outgrowth of spinal motor neurons was measured using Image J software. ELISA was used to quantify neurotrophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in culture media, and antibodies or exogenous neurotrophic factors were used to block or mimic the effect of BMSCs on neurite outgrowth, respectively. The results showed that neurite outgrowth significantly increased in spinal motor neurons after co-cultured with BMSCs, while the secretion level of BDNF, GDNF and NGF was dramatically elevated in co-culture. However, the neurite outgrowth-promoting effect of BMSCs was found to significantly reduced using antibodies to BDNF, GDNF and NGF. In addition, a fraction of BMSCs was found to exhibit NF-200 immunoreactivity. These results indicated that BMSCs could promote neurite outgrowth of motor neurons by means of neurotrophic factors. The findings of the present study provided new cues for the treatment of spinal cord injury.     

Neurotrophic Actions of Bone Marrow Stromal Cells on Primary Culture of Dorsal Root Ganglion Tissues and Neurons

Application of adult bone marrow stromal cells (BMSCs) provides therapeutic benefits to the treatment of neurological insults. The aim of this study was to explore the potential of nonhematopoietic BMSCs to produce soluble factors and stimulate signaling pathways in neurons that mediate trophic effects. A combination of enzyme-linked immunosorbent assay and two-dimensional gel electrophoresis coupled with mass spectrometry showed that the BMSCs released into the culture medium an array of soluble factors such as nerve growth factor, brain-derived neurotrophic factor, basic fibroblast growth factor, and ciliary neurotrophic factor, which have been shown to exhibit potent neurotrophic effects on neural cells. Immunochemistry, cell viability assay, and quantitative real-time RT-PCR collectively showed that neurite outgrowth and neurogenesis in cultured rat dorsal root ganglion (DRG) explants and neurons were enhanced after they were cocultured with rat BMSCs. Western blot analysis revealed that BMSC-conditioned medium activated phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase and/or phosphoinositide 3-kinase/serine/threonine kinase (PI3K/Akt) in primary culture of rat DRG neurons, which suggested that BMSCs trigger endogenous survival signaling pathways in neurons through their secreted soluble factors. Our data help to elucidate the mechanisms by which BMSCs function as a cell therapy agent in peripheral nerve regeneration.     

Neuroprotective utility and neurotrophic action of neurturin in postnatal motor neurons: comparison with GDNF and persephin.

Neurturin and persephin are recently discovered homologs of glial cell line-derived neurotrophic factor (GDNF). Here, we report that neurturin, like GDNF, increases the choline acetyltransferase activity of normal postnatal motor neurons, induces neurite outgrowth in spinal cord, and potently protects motor neurons from chronic glutamate-mediated degeneration. Persephin, in contrast, does not appear to have neurotrophic or neurite-promoting effects on mature motor neurons and may instead worsen the glutamate injury of motor neurons. This pattern in the TGF-beta family suggests certain receptor specificities, requiring at least the Ret/GFRalpha-1 receptor complex. The results predict potential benefit of neurturin, but not persephin, in the treatment of motor neuron disorders and spinal cord diseases.     

Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro

Different subpopulations of adult primary sensory neurons in the dorsal root ganglia express receptors for different trophic factors, and are therefore potentially responsive to distinct trophic signals. We have compared the effect of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and NT-3, and of glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth in dissociated cultures of sensory neurons from the lumbar ganglia of young adult rats, and attempted to establish subset-specific effects of these trophic factors. We analysed three parameters of neurite growth (percentage of process-bearing neurons, length of longest neurite and total neurite length), which may correlate with particular types of axon growth in vivo, and may therefore respond differently to trophic factor presence. Our results showed that percentage of process-bearing neurons and total neurite length were influenced by trophic factors, whilst the length of the longest neurite was trophic factor independent. Only NGF and GDNF were found to enhance significantly the proportion of process-bearing neurons in vitro. GDNF was more effective than NGF on small, IB4- neurons, which are known to develop GDNF responsiveness early in postnatal development. NGF, and to a much lesser extent GDNF, enhanced the total length of the neurites produced by neurons in culture. BDNF exerted an inhibitory effect on growth, and both BDNF and NT-3 could partially block some of the growth-promoting effects of NGF on specific neuronal subpopulations.     

Transplantation of bone marrow mesenchymal stem cells reduces lesion volume and induces axonal regrowth of injured spinal cord

It has been demonstrated that transplantation of bone marrow mesenchymal stem cells (BMSCs) improves recovery of injured spinal cord in animal models. However, the mechanism of how BMSCs promote repair of injured spinal cord remains under investigation. The present study investigated the neural differentiation of BMSCs, the lesion volume and axonal regrowth of injured spinal cord after transplantation. Seven days after spinal cord injury, 3 × 10⁵ BMSCs or PBS (control) was delivered into the injury epicenter of the spinal cord. At 8 weeks after spinal cord injury, transplantation of BMSCs reduced the volume of cavity and increased spared white matter as compared to the control. BMSCs did not express the cell marker of neurons, astrocytes and oligodendrocytes in injured spinal cord. Transmission electron microscopic examination displayed an increase in the number of axons in BMSC rats. The effect of BMSCs on growth of neuronal process was further investigated by using a coculture system. The length and the number of neurites from spinal neurons significantly increased when they cocultured with BMSCs. PCR and immunochemical analysis showed that BMSCs expressed brain‐derived neurotrophic factor (BDNF) and glia cell line‐derived neurotrophic factor (GDNF). These findings demonstrate that transplantation of BMSCs reduces lesion volume and promotes axonal regrowth of injured spinal cord.     

BDNF，NT—3对体外培养的胚鼠脊髓AChE和NADPH—d阳性神经元生长发育的影响

利用AChE和NADPH d酶组织化学染色法研究了脑源性神经营养因子 (brain derivedneurotrophicfac tor ,BDNF)和神经营养因子 3(neurotrophin 3,NT 3)对离体培养的胚胎大鼠脊髓胆碱能神经元和一氧化氮能神经元生长发育的影响。结果显示 :BDNF处理组和NT 3处理组AChE阳性神经元数和NADPH d阳性神经元数均显著高于对照组 (P <0 .0 5 )。BDNF组AChE阳性神经元和NADPH d阳性神经元胞体平均直径、每细胞突起数和最长突起长度均显著高于对照组 (P <0 .0 5 )。NT 3组NADPH d阳性神经元的生长发育与对照组无明显差异 ,仅AChE阳性神经元的每细胞突起数和最长突起长度显著高于对照组 (P <0 .0 5 ) ,对胞体发育无影响。结果提示 :BDNF ,NT 3促进脊髓神经元的存活和生长发育 ,二者的作用具有选择性和特异性。     

IGF-1 and BDNF promote chick bulbospinal neurite outgrowth in vitro

Injured neurons in the CNS do not experience significant functional regeneration and so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on numerous factors, one of which is the decreased availability of neurotrophic factors. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play a pivotal role in motor dysfunction after spinal cord injury, relatively little is known about the trophic sensitivity of these populations. This study explores the response of bulbospinal populations to various trophic factors. Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and spinal projection neurons from the raphe nuclei. Nerve growth factor (NGF), neurotrophin-4 (NT-4) and glial derived neurotrophic factor (GDNF) did not effect process outgrowth in vestibulospinal neurons. At the developmental stages used in this study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology.     

Neurotrophic properties of olfactory ensheathing glia

Olfactory ensheathing cells (OEC) constitute a specialized population of glia that accompany primary olfactory axons and have been reported to facilitate axonal regeneration after spinal cord injury in vivo. In the present report we describe OEC neurotrophic factor expression and neurotrophic properties of OECs in vitro. Investigation of the rat olfactory system during development and adulthood by radioactive in situ hybridization revealed positive labeling in the olfactory nerve layer for the neurotrophic molecules S-100beta, CNTF, BMP-7/OP-1, and artemin, as well as for the neurotrophic factor receptors RET and TrkC. Ribonuclease protection assay of cultured OEC revealed expression of NGF, BDNF, GDNF, and CNTF mRNA, while NT3 and NT4 mRNA were not detectable. In vitro bioassays of neurotrophic activity involved coculturing of adult OEC with embryonic chick ganglia and demonstrated increased neurite outgrowth from sympathetic, ciliary, and Remak's ganglia. However, when culturing the ganglia with OEC-conditioned medium, neurite outgrowth was not stimulated to any detectable extent. Our results suggest that the neurotrophic properties of OEC may involve secretion of neurotrophic molecules but that cellular interactions are crucial.     

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.     

Neurotrophic effects of BDNF on embryonic gonadotropin-releasing hormone (GnRH) neurons

Secretion of gonadotropin-releasing hormone (GnRH) at the median eminence is the essential activator of the reproductive axis. The mechanisms by which embryonic GnRH neurons migrate from the olfactory placode to the preoptic area and then elaborate neurites that course through the hypothalamus to terminate at the median eminence are largely unknown. We investigated the hypothesis that GnRH neurite outgrowth is promoted by brain-derived neurotrophic factor (BDNF) because GnRH neurites course through BDNF-rich areas of the forebrain during their development. Confocal microscopy revealed that most (86%) cultured embryonic GnRH cells tagged with a green fluorescent protein reporter were immunoreactive for TrkB. In primary cultures of E12.5 olfactory tissue, treatment with BDNF induced a dose-dependent increase in neurite outgrowth, but had no discernible effect on branching. BDNF induced phosphorylation of Ca(2+)/cAMP response element-binding protein (pCREB) in both GnRH and non-GnRH cells in these cultures. This was not associated with phosphorylation of ERK in GnRH-immunoreactive cells, though BDNF treatment did stimulate pERK in neighbouring non-GnRH cells. Promotion of neurite outgrowth is unlikely therefore to result from activation of the Ras-MAPK/ERK pathway. We conclude that the developing GnRH secretory system is directly sensitive to BDNF and that this polypeptide functions as a neurotrophic factor for GnRH neurons.     

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.     

The biological effects of cell-delivered brain-derived neurotrophic factor on cultured spiral ganglion cells

The benefit achieved by the use of cochlear implants depends among other factors on the number of surviving spiral ganglion cells (SGCs). Neurotrophic factors, especially brain-derived neurotrophic factor (BDNF), have a protective effect on spiral ganglions. Coating of the cochlear implant electrode with BDNF-producing cells may provide long-term delivery of the factor. Therefore, the hypothesis that BDNF-producing fibroblasts can enhance cell survival of cultured SGCs was tested. Lentiviral infection of fibroblasts resulted in BDNF production. Conditioned medium obtained from infected fibroblasts was used for the cultivation of SGCs. As a result, improved survival and neurite outgrowth was observed on SGCs. Our results demonstrate that lentivirally infected fibroblasts produce BDNF that has neurotrophic effects on spiral ganglions.     

Ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneurons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ng/ml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease. © 1996 Wiley-Liss, Inc.     

Hydrophobic dipeptide Leu-Ile protects against neuronal death by inducing brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis

We investigated whether certain hydrophobic dipeptides, Leu-Ile, Leu-Pro, and Pro-Ile, which partially resemble the site on FK506 that binds to immunophilin, could stimulate glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) synthesis in cultured neurons and found only Leu-Ile to be an active dipeptide. Leu-Ile protected against the death of mesencephalic neurons from wild-type mice but not from mice lacking the BDNF or GDNF gene. Next, we examined the effects of i.p. or i.c.v. administration of Leu-Ile on BDNF and GDNF contents. Both types of administration increased the contents of BDNF and GDNF in the striatum of mice. Also, peripheral administration of Leu-Ile inhibited dopaminergic (DA) denervation caused by unilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum of mice. The number of rotations following a methamphetamine challenge was lower in the Leu-Ile-treated group than in the nontreated group. Next, we compared the calcineurin activity and immunosuppressant activity of Leu-Ile with those of FK506. Leu-Ile was not inhibitory toward calcineurin cellular activity in cultured neuronal cells. Furthermore, Leu-Ile did not suppress concanavalin A (ConA)-induced synthesis/secretion of interleukin-2 by cultured spleen cells, suggesting that the immunosuppressant activity of Leu-Ile may be negligible when used as a therapeutic tool for neurodegenerative diseases.     

Neurotrophic effects of 7,8-dihydroxycoumarin in primary cultured rat cortical neurons

Objective Neuronal loss in the central nervous system is central to the occurrence of neurodegenerative diseases. Pharmaceutical companies have devoted much effort to developing new drugs against such diseases, since there are currently no effective drugs for neurodegenerative disease treatment. Promoting the capacity for nerve regeneration is an ideal treatment target. The present study aimed to investigate the neurotrophic effects of 7,8-dihydroxycoumarin (DHC) or daphnetin in primary cultured rat cortical neurons. Methods Cortical neurons were identified by microtubule-associated protein 2 (MAP2) immunostaining. Morphological observation was used to measure the average length of neurite outgrowth. MTT and lactate dehydrogenase assays were used to assess neuronal survival. The mRNA expression of MAP2 and brain-derived neurotrophic factor (BDNF) was measured by RT-PCR. Results MAP2 immunostaining showed that most of the cultured cells were neurons. Compared with the vehicle control group, DHC promoted neurite outgrowth and prolonged neuronal survival time at concentrations ranging from 2 to 8 μmol/L. Expression of both BDNF mRNA and MAP2 mRNAwas increased in the groups treated with 2, 4 and 8 μmol/L DHC. Conclusion DHC significantly increases neurite outgrowth and promotes neuronal survival in primary cultured rat cortical neurons. The neurotrophic effects of DHC are probably associated with increased BDNF expression.     

GDNF‐enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons

We previously demonstrated that coadministration of glial cell line‐derived neurotrophic factor (GDNF) with grafts of Schwann cells (SCs) enhanced axonal regeneration and remyelination following spinal cord injury (SCI). However, the cellular target through which GDNF mediates such actions was unclear. Here, we report that GDNF enhanced both the number and caliber of regenerated axons in vivo and increased neurite outgrowth of dorsal root ganglion neurons (DRGN) in vitro, suggesting that GDNF has a direct effect on neurons. In SC‐DRGN coculture, GDNF significantly increased the number of myelin sheaths produced by SCs. GDNF treatment had no effect on the proliferation of isolated SCs but enhanced the proliferation of SCs already in contact with axons. GDNF increased the expression of the 140 kDa neural cell adhesion molecule (NCAM) in isolated SCs but not their expression of the adhesion molecule L1 or the secretion of the neurotrophins NGF, NT3, or BDNF. Overall, these results support the hypothesis that GDNF‐enhanced axonal regeneration and SC myelination is mediated mainly through a direct effect of GDNF on neurons. They also suggest that the combination of GDNF administration and SC transplantation may represent an effective strategy to promote axonal regeneration and myelin formation after injury in the spinal cord. © 2009 Wiley‐Liss, Inc.     

Glial support of CNS neuronal survival, neurite growth and regeneration

In an attempt to identify specific molecular and cellular requirements necessary to support long-term maintenance and differentiation of central neurons we have identified laminin-HSPG and free fibronectin as two major neurite promoting substrate adhesion factors released by immature cerebral astrocytes in serum-free culture. Astrocytes further secrete diffusible neurotrophic protein factor(s) which are permanently required for survival of cultured neurons from various brain regions. However, both the presence of substrate-bound neurite-promoting factors and diffusible neurotrophic activities were not sufficient to support long-term maintenance of central neurons in culture. Cell contact-mediated interactions which appear to be cell type-restricted (e.g. to neurons and astrocytes, but not to fibroblasts) are further required for neuronal stabilization. The implantation of immature astroglial cells into the injured adult CNS should provide a supportive environmental condition for damaged neurons to enhance their recovery and stimulate regenerative responses.     

Developmental regulation of a neurite-promoting factor influencing statoacoustic neurons.

The present study investigated a target-derived, neurite-promoting factor (NPF) released by the developing chick otocyst and its effects on statoacoustic ganglia (SAG). SAG explants cultured in the absence of otocysts produced little neurite outgrowth at all stages of development examined (E4-E13). However, extensive neurite outgrowth was seen when E4-E6 SAG were cultured in the presence of otocysts of the same age. The amount of neurite outgrowth observed in cocultures steadily decreased at later developmental stages. E7-E9 cocultures produced less outgrowth and E10-E13 cocultures produced the least outgrowth compared to E4-E6 cocultures. Additionally, otocysts from older stages were unable to promote outgrowth of E4 SAG. Thus, the level of the factor released by the otocysts declined during development. In contrast, neurite outgrowth was promoted when E10-E15 SAG were cocultured in the presence of younger stage otocysts. Our data indicate that the release of NPF from chick otocysts decreased from E6 to E13, although the ability of SAG neurons to respond to the NPF was maintained throughout development.