Effects of brain‑derived neurotrophic factor and neurotrophin‑3 on the neuronal differentiation of rat adipose‑derived stem cells

Abstract

Tissue engineering is a promising method that may be used to treat spinal cord injury (SCI). The underlying repair mechanism of tissue engineering involves the stable secretion of neurotrophins from seed cells, which eventually differentiate into neurons; therefore, the selection of appropriate seed cells, which stably secrete neurotrophins that easily differentiate into neurons requires investigation. Adipose‑derived stem cells (ADSCs), which are adult SCs, are advantageous due to convenience sampling and easy expansion; therefore, ADSCs are currently the most popular type of seed cell. Brain‑derived neurotrophic factor (BDNF) and neurotrophin‑3 (NT‑3) possess superior properties, when compared with other neurotrophic factors, in the maintenance of neuronal survival and promotion of SC differentiation into neurons. The present study used two lentiviruses, which specifically express BDNF and NT‑3 [Lenti‑BDNF‑green fluorescent protein (GFP), Lenti‑NT‑3‑red fluorescent protein (RFP)], to transfect third‑generation ADSCs. Three types of seed cell were obtained: i) Seed cells overexpressing BDNF (ADSC/Lenti‑BDNF‑GFP); ii) seed cells overexpressing NT‑3 (ADSC/Lenti‑NT‑3‑RFP); and iii) seed cells overexpressing BDNF and NT‑3 (ADSC/Lenti‑BDNF‑GFP and NT‑3‑RFP). The transfected cells were then induced to differentiate into neurons and were divided into a further four groups: i) The BDNF and NT‑3 co‑overexpression group; ii) the BDNF overexpression group; iii) the NT‑3 overexpression group; and iv) the control group, which consisted of untransfected ADSCs. The results of the present study demonstrate that BDNF and NT‑3 expression was higher 10 days after induction, as detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting. Neuron‑specific enolase is a neuronal marker, the expression of which was highest in the BDNF and NT‑3 co‑overexpression group, followed by the BDNF overexpression group and then by the NT‑3 overexpression group. The lowest expression levels of NSE were detected in the control group, as determined by RT‑qPCR, western blotting and immunofluorescent staining. These results indicate that BDNF and NT‑3 exert a synergistic effect, which may promote the neuronal differentiation of ADSCs. The present study provides a solid theoretical foundation for future experiments regarding the use of tissue engineering technology for the treatment of SCI.

Figure 1

Observation of rat ADSC morphology (magnification, ×100). (A) Rat ADSC morphology 10 h after inoculation. Primary culture after (B) 72 h and (C) 8 days. (D) Third-generation ADSC. ADSC, adipose-derived stem cell.

Figure 2

Staining of adipose-derived stem cells following osteogenic induction (magnification, ×100). (A and B) Alkaline phosphatase staining 2 weeks after osteogenic induction in the (A) experimental and (B) control groups. (C and D) Alizarin red staining 4 weeks after osteogenic induction in the (C) experimental and (D) control groups.

Figure 3

Determination of lentiviral transfection efficiency. (A–C) Fluorescence intensity of adipose-derived stem cells in the (A) Lenti-BDNF-GFP transfected group (magnification, ×200), (B) Lenti-NT-3-RFP transfected group (magnification, ×200) and the (C) Lenti-BDNF-GFP and Lenti-NT-3-RFP co-transfected group (magnification, ×100). Number of (D) GFP-positive cells in the Lenti-BDNF-GFP transfected group, (E) RFP-positive cells in the Lenti-NT-3-RFP transfected group and (F) GFP- and RFP-positive cells in the Lenti-BDNF-GFP and Lenti-NT-3-RFP co-transfected group, as determined by flow cytometry. RFP, red fluorescent protein; GFP, green fluorescent protein; BDNF, brain-derived neurotophic factor; NT-3, neurotrophin-3.

Figure 4

NSE immunofluorescent staining. (A) NSE-positive cells from each group (magnification, ×200). (B) The percentage of NSE-positive cells in each group. Data are presented as the mean ±standard error. NSE, neuron-specific enolase; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3.

Figure 5

BDNF, NT-3 and NSE mRNA expression levels, as determined by reverse transcription-quantitative polymerase chain reaction 10 days after induction. Data are presented as the mean ±standard error. BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; NSE, neuron-specific enolase.

Figure 6

BDNF, NT-3 and NSE protein expression levels were examined by (A) western blotting 10 days after induction. (B) Quantification of western blotting. Data are presented as the mean ±standard error. BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; NSE, neuron-specific enolase.