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

Ji W, Zhang X, Ji L, Wang K, Qiu Y - Mol Med Rep (2015)

Bottom Line: 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 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.These results indicate that BDNF and NT‑3 exert a synergistic effect, which may promote the neuronal differentiation of ADSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.

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.

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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.
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f3-mmr-12-04-4981: 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.

Mentions: ADSCs were transfected with lentiviruses at an MOI of 1, 10 and 100 and the number of fluorescent cells gradually increased with the increasing MOI. When cells were transfected with a MOI of 100, the fluorescent intensity was significantly increased in the Lenti-BDNF-GFP transfected group (Fig. 3A), the Lenti-NT-3-RFP transfected group (Fig. 3B), and the Lenti-BDNF-GFP and Lenti-NT-3-GFP co-transfected group (Fig. 3C), concurrently a cell cytotoxic effect was detected. The optimum MOI was determined to be 100, due to the higher transfection efficiency and slightly reduced cytopathogenicity. As determined by flow cytom-etry, the percentage of GFP-positive cells was 91.3% in the Lenti-BDNF-GFP transfected group (Fig. 3D), the percentage of RFP-positive cells was 77.5% in the Lenti-NT-3-RFP transfected group (Fig. 3E), and the percentage of GFP- and RFP-positive cells was 82.2 and 67.0%, respectively in the Lenti-BDNF-GFP and Lenti-NT-3-RFP co-transfected group (Fig. 3F).


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

Ji W, Zhang X, Ji L, Wang K, Qiu Y - Mol Med Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581787&req=5

f3-mmr-12-04-4981: 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.
Mentions: ADSCs were transfected with lentiviruses at an MOI of 1, 10 and 100 and the number of fluorescent cells gradually increased with the increasing MOI. When cells were transfected with a MOI of 100, the fluorescent intensity was significantly increased in the Lenti-BDNF-GFP transfected group (Fig. 3A), the Lenti-NT-3-RFP transfected group (Fig. 3B), and the Lenti-BDNF-GFP and Lenti-NT-3-GFP co-transfected group (Fig. 3C), concurrently a cell cytotoxic effect was detected. The optimum MOI was determined to be 100, due to the higher transfection efficiency and slightly reduced cytopathogenicity. As determined by flow cytom-etry, the percentage of GFP-positive cells was 91.3% in the Lenti-BDNF-GFP transfected group (Fig. 3D), the percentage of RFP-positive cells was 77.5% in the Lenti-NT-3-RFP transfected group (Fig. 3E), and the percentage of GFP- and RFP-positive cells was 82.2 and 67.0%, respectively in the Lenti-BDNF-GFP and Lenti-NT-3-RFP co-transfected group (Fig. 3F).

Bottom Line: 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 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.These results indicate that BDNF and NT‑3 exert a synergistic effect, which may promote the neuronal differentiation of ADSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.

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.

Show MeSH
Related in: MedlinePlus