Abstract
BackgroundHuman neural precursor cells (hNPC) are candidates for neural transplantation in a wide range of neurological disorders. Recently, much work has been done to determine how the environment for NPC culture in vitro may alter their plasticity. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) are used to expand NPC; however, it is not clear if continuous exposure to mitogens may abrogate their subsequent differentiation. Here we evaluated if short-term removal of FGF-2 and EGF prior to plating may improve hNPC differentiation into neurons.Principal FindingsWe demonstrate that culture of neurospheres in suspension for 2 weeks without EGF-FGF-2 significantly increases neuronal differentiation and neurite extension when compared to cells cultured using standard protocols. In this condition, neurons were preferentially located in the core of the neurospheres instead of the shell. Moreover, after plating, neurons presented radial rather than randomly oriented and longer processes than controls, comprised mostly by neurons with short processes. These changes were followed by alterations in the expression of genes related to cell survival.ConclusionsThese results show that EGF and FGF-2 removal affects NPC fate and plasticity. Taking into account that a three dimensional structure is essential for NPC differentiation, here we evaluated, for the first time, the effects of growth factors removal in whole neurospheres rather than in plated cell culture.
Highlights
Evidence of neurogenesis in the adult brain of birds [1], rodents and primates[2,3,4], and the demonstration of the presence of stem cells in specific brain regions, such as the subventricular zone (SVZ) and the hippocampus [5,6], brought new perspectives for cell therapy and neural regeneration [7]
These results show that Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) removal affects neural precursor cells (NPC) fate and plasticity
To verify basic aspects of neurosphere growth we performed growth rates, BrdU and TUNEL assays, comparing the control (CTR) group and the group cultured without EGF and FGF-2, in mitogen free medium (MFM)
Summary
Evidence of neurogenesis in the adult brain of birds [1], rodents and primates[2,3,4], and the demonstration of the presence of stem cells in specific brain regions, such as the subventricular zone (SVZ) and the hippocampus [5,6], brought new perspectives for cell therapy and neural regeneration [7]. An experimental model to study neural stem cells is the heterogeneous free-floating aggregates of cells, termed neurospheres [9,10,11]. The progenitor cells, in turn, give rise only to other progenitor cells. In this way, only a small fraction of the neurosphere corresponds to genuine stem cells [12]. We use the terminology neural precursor cells (NPC) to describe both cell types within the neurosphere [13,14]. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) are used to expand NPC; it is not clear if continuous exposure to mitogens may abrogate their subsequent differentiation. We evaluated if short-term removal of FGF-2 and EGF prior to plating may improve hNPC differentiation into neurons
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