Effect of single growth factor and growth factor combinations on differentiation of neural stem cells

Abstract

Objective: The effects on neural proliferation and differentiation of neural stem cells (NSC) of basic fibroblast growth factor-2 (bFGF), insulin growth factor-I (IGF-I), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) were assessed. Also, following combinations of various factors were investigated : bFGF+IGF-I, bFGF+BDNF, bFGF+NGF, IGF-I+BDNF, IGF-I+NGF, and BDNF+NGF.

Methods: Isolated NSC of Fisher 344 rats were cultured with individual growth factors, combinations of factors, and no growth factor (control) for 14 days. A proportion of neurons was analyzed using beta-tubulin III and NeuN as neural markers.

Results: Neural differentiations in the presence of individual growth factors for beta-tubulin III-positive cells were : BDNF, 35.3%; IGF-I, 30.9%; bFGF, 18.1%; and NGF, 15.1%, and for NeuN-positive cells was : BDNF, 34.3%; bFGF, 32.2%; IGF-1, 26.6%; and NGF, 24.9%. However, neural differentiations in the absence of growth factor was only 2.6% for beta-tubulin III and 3.1% for NeuN. For beta-tubulin III-positive cells, neural differentiations were evident for the growth factor combinations as follows : bFGF+IGF-I, 73.1%; bFGF+NGF, 65.4%; bFGF+BDNF, 58.7%; BDNF+IGF-I, 52.2%; NGF+IGF-I, 40.6%; and BDNF+NGF, 40.0%. For NeuN-positive cells : bFGF+IGF-I, 81.9%; bFGF+NGF, 63.5%; bFGF+BDNF, 62.8%; NGF+IGF-I, 62.3%; BDNF+NGF, 56.3%; and BDNF+IGF-I, 46.0%. Significant differences in neural differentiation were evident for single growth factor and combination of growth factors respectively (p<0.05).

Conclusion: Combinations of growth factors have an additive effect on neural differentiation. The most prominent neural differentiation results from growth factor combinations involving bFGF and IGF-I. These findings suggest that the combination of a mitogenic action of bFGF and postmitotic differentiation action of IGF-I synergistically affects neural proliferation and NSC differentiation.

Keywords: Combination of growth factors; Neuron; Single growth factor; Synergistic effect.

Fig. 1

Immunocyotofluorescence for expressions of neuron specific markers (β-tubulin III, NeuN) in the neurogenic stimulation with brain-derived neurotrophic factor (A, E), basic fibroblast growth factor (B, F), insulin growth factor-I (C, G) and nerve growth factor (D, H) for 14 days respectively. Immunocytofluorescence micrographs show β-tubulin III-positive cells (A, B, C, D) and NeuN-positive cells (E, F, G, H). Magnification, 200X.

Fig. 2

Neuronal outgrowth and survival of neural stem cells in response to brain-derived neurotrophic factor, insulin growth factor-I, basic fibroblast growth factor, and nerve growth factor. Neurogenically stimulated cells were cultured for neural cell analysis with β-tubulin III and NeuN for 14 days. Significant differences in neural differentiation with single growth factor were apparent (β-tubulin III, p=0.032; NeuN , p= 0.039).

Fig. 3

Immunocyotofluorescence determined expression of neuron specific markers (β-tubulin III, NeuN) in the 14 day neurogenic stimulation by basic fibroblast growth factor (bFGF)+insulin growth factor-I (A, D), bFGF +brain-derived neurotrophic factor (BDNF) (B, E), and BDNF +nerve growth factor (C, F). Immunocytofluorescence micrographs demonstrating β-tubulin III-positive cells (A-C) and NeuN-positive cells (D-F). Magnification, 200X.

Fig. 4

Neuronal outgrowth and survival of neural stem cells in response to basic fibroblast growth factor (bFGF) +insulin growth factor-I (IGF-I), bFGF +brain-derived neurotrophic factor (BDNF), bFGF +nerve growth factor (NGF), IGF-I +BDNF, IGF-I +NGF, and BDNF +NGF. Neurogenically stimulated cells were cultured for neural cell analysis with β-tubulin III and NeuN for 14 days. Significant differences in neuronal positive cells were apparent with each group (β-tubulin III, p=0.023; NeuN, p=0.018).

Fig. 5

Schematic pathway for neurogenesis by neural stem cells in vitro. Basic fibroblast growth factor induce neural stem cells to proliferate. Insulin growth factor-I, brain-derived neurotrophic factor, and nerve growth factor act to induce the differentiation of postmitotic neuronal precursors.