Abstract

BackgroundIn order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different in vitro conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT1 and MT2 receptors are expressed in NSCs.ResultsRT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT1 receptor mRNA was detected in NSCs maintained for two days in culture, but the MT2 receptor was not seen. An immature MT1 receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 – 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT1 receptor is expressed in both neural (β-tubulin III positive) and glial (GFAP positive) progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours.ConclusionsThe phenotypic characteristics of C17.2 cells suggest that they are a heterogeneous population of NSCs including both neural and glial progenitors, as observed under the cell culture conditions used in this study. These NSCs have an intrinsic ability to express neurotrophic factors, with an apparent suppression of GDNF expression after several days in culture. The detection of melatonin receptors in neural stem/progenitor cells suggests involvement of this pleiotropic hormone in mammalian neurodevelopment. Moreover, the ability of melatonin to induce GDNF expression in C17.2 cells supports a functional role for the MT1 receptor expressed in these NSCs. In view of the potency of GDNF in promoting the survival of dopaminergic neurons, these novel findings have implications for the utilization of melatonin in neuroprotective strategies, especially in Parkinson's disease.

Highlights

  • In order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics

  • Effects of culture conditions on neurotrophic factor and cell-specific marker mRNA expression in C17.2 NSCs Following two days in culture, C17.2 cells remain in an undifferentiated state, as indicated by their flat and rounded appearance (Fig. 1A) and high expression of the stem cell/progenitor cell marker, nestin (Fig. 1C,1E,1G)

  • A similar strong expression of brainderived neurotrophic factor (BDNF) and nerve growth factor (NGF) was observed in differentiating cells after seven days, but glial cell line-derived neurotrophic factor (GDNF) mRNA was relatively lower in cells maintained in 1% fetal bovine serum (FBS) or 10% FBS + 5% horse serum (Fig. 1F,1H)

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Summary

Introduction

In order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Transplanted C17.2 NSCs, genetically modified to express glial cell line-derived neurotrophic factor (GDNF), were found to engraft in the 6-hydroxydopamine-lesioned mouse striatum and to express therapeutic levels of this neurotrophin, with consequent protection of dopaminergic neurons in this model of Parkinson's disease [5]. This and other similar approaches are promising, limitations including the stability and regulation of transduced genes await resolution. We report that C17.2 NSCs exhibit heterogeneous phenotypes and express neurotrophic factors and melatonin MT1 receptors

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