Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells (DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium (DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor (NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2 (MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction (qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors (NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCs-CM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls; however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Chinese Library Classification No. R459.9; R364; R622     

Parallel expression of neurotrophic factors and their receptors in chronic inflammatory demyelinating polyneuropathy

The mRNA levels of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and interleukin-6 (IL-6) were examined in sural nerves of 22 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). The mRNAs for NGF, GDNF, LIF, and IL-6 were upregulated, whereas CNTF mRNA was downregulated significantly in the nerves. The NGF, GDNF, and CNTF, but not LIF mRNA expressions were parallel to those of the cognate receptors, suggesting that these cognate soluble receptors effectively present these factors to maintain and regenerate the axons. Furthermore, IL-6 mRNA expression was significantly parallel to both binding and signal-transducing receptor expression, implying a role of the IL-6 signal for non-neuronal cells in CIDP. These findings indicate that multiple neurotrophic growth factors and cytokines are expressed cooperatively with their concomitant receptors in the nerve lesions of CIDP and play an important role particularly in nerve repair.     

Laminin and growth factor receptor activation stimulates differential growth responses in subpopulations of adult DRG neurons

Neurons in the adult rat dorsal root ganglion (DRG) can be classified into at least three separate subpopulations based on morphologic and phenotypic differences. In this study we have focused on the growth response of these specific subpopulations in vitro with respect to laminin (LN) and growth factor receptor activation. Using a cell selection approach we show that LN-induced neurite growth occurs in the absence of added trophic factors only in heavy-chain neurofilament-positive and calcitonin gene-related peptide-positive DRG neurons [nerve growth factor (NGF)-responsive population]. In contrast, LN alone is not sufficient to stimulate significant neurite growth from lectin Griffonia simplicifolia IB4-positive neurons (IB4+ve), although it is still required to elicit a growth response from these cells in the presence of glial-derived neurotrophic factor (GDNF, e.g. neurite growth occurred only when cells were plated on LN in the presence of GDNF). By using chemical inhibitors we demonstrate that only the phosphatidylinositol 3 kinase (PI 3-K)/Akt pathway is required for neurite growth from the NGF-responsive cell population. However, both the PI 3-K/Akt and MEK/mitogen-activated protein kinase signaling pathways are required for neurite growth from the IB4+ve cell population. Thus, we have identified specific signaling events and environmental requirements associated with neurite growth for different subpopulations of adult DRG neurons, pointing to potential therapeutic targets while identifying an inability for any one treatment alone to repair peripheral nerve damage.     

Neuronal differentiation elicited by glial cell line-derived neurotrophic factor and ciliary neurotrophic factor in adrenal chromaffin cell line tsAM5D immortalized with temperature-sensitive SV40 T-antigen

To understand the characteristics of tsAM5D cells immortalized with the temperature-sensitive simian virus 40 large T-antigen, we first examined the responsiveness of the cells to ligands of the glial cell line-derived neurotrophic factor (GDNF) family. tsAM5D cells proliferated at the permissive temperature of 33 degrees C in response to either GDNF or neurturin, but not persephin or artemin. At the nonpermissive temperature of 39 degrees C, GDNF or neurturin caused tsAM5D cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, ciliary neurotrophic factor (CNTF) did not affect the GDNF-mediated cell proliferation at 33 degrees C but promoted the survival and differentiation of GDNF-treated cells at 39 degrees C. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of various neuronal marker genes, indicating that the cells had undergone neuronal differentiation. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39 degrees C became dependent solely on nerve growth factor (NGF) for their survival and neurite outgrowth. Moreover, upon treatment with GDNF plus CNTF, the dopaminergic phenotype was suppressed by the temperature shift. Thus, we demonstrated that tsAM5D cells had the capacity to differentiate terminally into neuron-like cells in response to GDNF plus CNTF when the oncogene was inactivated by the temperature shift. This cell line provides a useful model system for studying the role of a variety of signaling molecules for GDNF/CNTF-induced neuronal differentiation.     

Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors

Neurite-promoting activity in feeding medium conditioned by rat astrocytes and Schwann cells in culture was examined. The conditioned medium (CM) from both types of glial cultures stimulated extensive neurite outgrowth from embryonic chick dorsal root ganglia (DRG) as well as pheochromocytoma (PC12) cells. Both the DRG and PC12 cells also produce neurite outgrowth in the presence of nerve growth factor (NGF). With the DRG, the neurite growth rates observed with the glial cell CM were identical to growth rates seen with NGF. Although anti-NGF antibody did not inhibit the neurite outgrowth produced by either of the glial CM, a nerve growth factor radioreceptor assay did detect an NGF-like molecule in both CM. Since the extensive neurite outgrowth stimulated by the glial CM was not mimicked by pure laminin alone, we conclude that the glial neurite promoting factors are distinct from laminin.     

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.     

v-src genes stimulate neurite outgrowth in pheochromocytoma (PC12) variants unresponsive to neurotrophic factors

The NGF-nonresponsive rat pheochromocytoma PC12 variant nnr5, isolated by Green et al. (J Cell Biol 102:830-843, 1986), responds poorly or not at all to fibroblast growth factor. Transformation of PC-12nnr5 cells with v-src-expressing retroviruses results in vigorous neurite outgrowth, similar to that seen in the parent cell line. Thus though the PC12nnr5 cell line has a greatly impaired ability to respond to neurotrophic factors it still may extend neurites. This data is consistent with a model in which PC12nnr5 cells are unable to propagate intracellular second messengers, and this defect may be related to the expression of c-src gene products.     

Changing responsiveness of developing midbrain dopaminergic neurons for extracellular growth factors

Numerous purified growth factors as well as yet-unidentified neurotrophic activities within mesencephalic glia support the survival of dopaminergic neurons. To further characterize the functional role of these multiple growth factor influences in dopaminergic cell development, various purified growth factors as well as mesencephalic glial-conditioned medium (CM) were screened for effects on dopaminergic cell survival and glial numbers in serum-free low density cultures of the dissociated embryonic day (E) 15 and E17 rat mesencephalon. In E15 mesencephalic cultures, dopaminergic cell survival increased with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), transforming growth factor α (TGFα), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-BB (PDGF-BB), and interleukin-6 (IL-6). bFGF, TGFα, PDGF, and IL-6 also stimulated glial proliferation as demonstrated by autoradiographic labeling for ³H-thymidine. Moreover, CM derived from the mesencephalic glial cell line Mes42 completely prevented the death of E15 dopaminergic neurons within the initial days of cultivation. In E17 mesencephalic cultures, survival-promoting effects on dopaminergic neurons were present with BDNF, GDNF, and bFGF. TGFα, IGF-1, PDGF-BB, and IL-6 stimulated glial proliferation but did not affect dopaminergic cell survival. Similarly, mesencephalic glial-CM completely failed to support the survival of E17 dopaminergic neurons. These observations demonstrate that during embryonic development, dopaminergic cell survival sequentially depends on distinct sets of growth factors. The concomitant loss of sensitivity of developing dopaminergic neurons for mesencephalic glial-CM as well as TGFα, IGF-1, PDGF-BB, and IL-6 further provides evidence that these growth factors indirectly affect early dopaminergic neurons through glial-mediated processes and suggests a crucial role of glia during the initial stages of neuronal development. J. Neurosci. Res. 51:508–516, 1998. © 1998 Wiley-Liss, Inc.     

Dental pulp cells provide neurotrophic support for dopaminergic neurons and differentiate into neurons in vitro; implications for tissue engineering and repair in the nervous system

Glial cell line-derived neurotrophic factor (GDNF) mRNA is highly expressed by dental pulp cells (DPCs) prior to the initiation of dental pulp innervation. We show that radioactively labelled exogenous GDNF is retrogradely transported from neonatal teeth and vibrissae to the trigeminal neurons, indicating that GDNF acts as a classical neurotrophic factor in the trigeminal system. We also show that DPCs from both rats and humans produce nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and GDNF mRNAs in vitro, promote the survival and phenotypic characteristics of embryonic dopaminergic (DA) neurons and protect DA neurons against the neurotoxin 6-hydroxy-dopamine (6-OHDA) in vitro. By using inhibitory antibodies to NGF, BDNF and GDNF, we show that the promotion of DA neuron survival relates to the production and release of neurotrophic proteins by DPCs in vitro. We suggest that in vivo production of neurotrophic factors by DPCs play roles in tooth innervation. However, continued production of neurotrophic factors by the DPCs might have wider implications. We propose that the dental pulp is a viable source of easily attainable cells with possible potential for development of autologous cell transplantation therapies. We also show that a population of neural crest-derived dental pulp cells acquire clear neuronal morphology and protein expression profile in vitro, indicating the presence of a cell population in the dental pulp with neuronal differentiation capacity that might provide additional benefits when grafted into the CNS.     

Gene transfer of glial cell-derived neurotrophic factor and cardiotrophin-1 protects PC12 cells from injury: involvement of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase pathways

Gene therapy for neurodegenerative diseases may utilize the expression of neurotrophic factors because of their potential to promote survival and regeneration of injured neuronal cells. Increasing numbers of these factors are being considered for gene transfer, but their specificity and efficacy in neuroprotection are greatly variable. The major aims of this study were to carry out gene transfer of various neurotrophic factors and investigate their mechanisms of action as well as their protective effects on the viability of rat pheochromocytoma (PC12) cells. We used glutamate, S-nitroso-N-acetyl-DL-penicillamine (SNAP), and staurosporine to induce excitatory damage, oxidative stress, and apoptosis, respectively, because these mechanisms are thought to participate in various disease processes leading to degeneration of cells. We utilized adenovirus vectors for efficient gene transfer of trophic factors (glial-cell derived neurotrophic factor [GDNF] and cardiotrophin-1 [CT-1]) or calbindin-D28k. We found that GDNF and CT-1 gene transfers were equally effective in saving PC12 cells from injury, but calbindin expression did not show any beneficial effects. GDNF gene transfer was much more efficient in protecting PC12 cells from damage than direct GDNF administration. The protection by GDNF expression against staurosporine was mediated through both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MAPK kinase; MEK) pathways, but only the MEK pathway was involved in the protection against SNAP. In contrast, the protective effect of GDNF against glutamate toxicity was independent of these RET-dependent signal transduction pathways.     

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.     

Pathology-related differential expression regulation of NGF, GDNF, CNTF, and IL-6 mRNAs in human vasculitic neuropathy

The mRNA levels of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and interleukin-6 (IL-6) were examined in sural nerves of 22 patients with acute necrotizing vasculitic neuropathies. NGF, GDNF, and IL-6 mRNAs were upregulated and CNTF mRNA was downregulated in the lesioned nerves, but their up- and down-regulation levels were not correlated with each other, showing that these mRNAs were independently expressed. The expression of NGF and CNTF mRNAs was clearly correlated with the degree of infiltrated macrophages and T cells, and myelinated fiber density, respectively. These findings indicate that these neurotrophic factors are differentially expressed temporally and spatially in the vasculitic nerve lesion by an underlying pathology-related process.     

Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination

Glial cell line-derived neurotrophic factor (GDNF) is the prototypical member of a growth factor family that signals via the cognate receptors ret and GDNF-receptor alpha-1. The latter receptors are expressed on a variety of neurons that project into the spinal cord, including supraspinal neurons, dorsal root ganglia, and local neurons. Although effects of GDNF on neuronal survival in the brain have previously been reported, GDNF effects on injured axons of the adult spinal cord have not been investigated. Using an ex vivo gene delivery approach that provides both trophic support and a cellular substrate for axonal growth, we implanted primary fibroblasts genetically modified to secrete GDNF into complete and partial mid-thoracic spinal cord transection sites. Compared to recipients of control grafts expressing a reporter gene, GDNF-expressing grafts promoted significant regeneration of several spinal systems, including dorsal column sensory, regionally projecting propriospinal, and local motor axons. Local GDNF expression also induced Schwann cell migration to the lesion site, leading to remyelination of regenerating axons. Thus, GDNF exerts tropic effects on adult spinal axons and Schwann cells that contribute to axon growth after injury.     

Effects of polychlorinated biphenyls (PCBs) on expression of neurotrophic factors in C6 glial cells in culture

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that affect nervous system function. Glial cells are among the first lines of defense in the nervous system and are involved in activities, including production of neurotrophic factors, which maintain an environment optimally suited for neuronal function. In this study, we investigated the effects of a commercial mixture of PCBs, Aroclor 1254 (A1254), on neurotrophic factor secretion by C6 cells in culture. C6 cells were exposed to medium containing 10 ppm A1254, 0.1% dimethyl sulfoxide (DMSO=vehicle), or normal culture medium. Glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) protein were measured by enzyme-linked immunosorbant assay. GDNF mRNA was measured by real-time RT-PCR. The role of protein kinase C (PKC) signaling in A1254 effects was investigated using bisindolylmaleimide, a PKC antagonist. Exposure to A1254 increased NGF (8.8x10(-5)+/-8.2x10(-6)pg NGF/cell) and GDNF (1.0x10(-4)+/-6.7x10(-6)pg GDNF/cell) secretion compared to DMSO treated controls (5.0x10(-5)+/-7.5x10(-6)pg NGF/cell and 6.2x10(-5)+/-3.1x10(-6)pg GDNF/cell). The effect of A1254 was long-lived, as GDNF secretion was elevated following 5 days of exposure (4.1x10(-5)+/-1.7x10(-6)pg GDNF/cell in A1254 exposed cells vs. 2.9x10(-5)+/-2.3x10(-6)pg GDNF/cell in DMSO exposed cells). GDNF mRNA expression was also elevated following exposure to A1254 (1.14+/-0.07 gene expression units in A1254 exposed cells vs. 0.8+/-0.07 gene expression units in DMSO exposed cells). Bisindolylmaleimide was able to block the effects of A1254 on GDNF secretion. Thus, one potential mechanism by which PCBs may alter nervous system function is via disruption of neurotrophic factor expression by glial cells. The observation that neurotrophic factor expression was increased following exposure to PCB may suggest that glial cells increase expression of neuroprotective genes following exposure to potentially damaging agents such as PCBs.