Int J Stem Cells.  2020 Nov;13(3):386-393. 10.15283/ijsc20038.

In Vitro Study of Adipose-Derived Mesenchymal Stem Cells Transduced with Lentiviral Vector Carrying the Brain-Derived Neurotrophic Factor Gene

• Nan D
• Dou X
• Qi Y
• Zhang W
• He G
• Zhang X

• KMID: 2508912
• DOI: http://doi.org/10.15283/ijsc20038

Abstract

Brain-derived neurotrophic factor (BDNF) exerts its survival-promoting effects on photoreceptors and retinal ganglion cells, however, delivery systems with little-to-no side effect are needed to sustain its controlled release and long-term efficacy. Our previous studies demonstrated that adipose-derived stem cells (ADSCs) are ideal delivery systems for gene therapy; moreover, ADSCs present unique properties like migration to damaged tissue sites, immunomodulation and anti-inflammation. Herein, we propose to employ ADSCs as the BDNF gene delivery vehicle. Different Analyses like flow cytometry, differentiation and cell proliferation assays etc demonstrated that BDNF were successfully transduced into ADSCs and the stemness of ADSCs was maintained even with the transduction. Real Time PCR and Western blot were used to measure mRNA and protein expressions of the BDNF-transduced ADSCs. The results demonstrated that the BDNF expression level of the lentiviral-BDNF transduced ADSCs is significantly increased and, moreover, enhanced the expression of other neurotrophic and downstream signaling factors. The data suggest that ADSCs are a reliable delivery vehicle for BDNF and could be used for the treatment of various diseases.

Keyword

Brain-derived neurotrophic factor; Adipose-derived mesenchymal stem cells; Lentiviral transduction; Drug delivery; Neural protection

Figure

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  Fig. 1 Cell morphology and growth profiles of ADSCs and BDNF-ADSCs.

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  Fig. 2 (A) Cell surface marker profiles of ADSCs and BDNF-ADSCs. Murine ADSCs and BDNF-ADSCs were stained with stem cells’ surface markers CD29, Sca1; hematopoietic markers CD34 and CD45; phagocytic lineage marker CD11b; and endothelial marker CD31 and analyzed by flow cytometry. Green lines represent ADSCs stained with CD29-FITC, CD31-FITC, CD34-FITC, Scal-1-FITC; Red lines show ADSCs stained with CD45-PE, CD11b-PE; Blue lines represent BDNF-ADSCs stained with the same antibody as ADSCs for compariso, and purple area shows the respective IgG isotype control for each antibody. (B) Cell size based on forward scatter signal of flow cytometry (p=0.13, t-test, n=6).

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  Fig. 3 Qualitative and quantitative adipogenic and osteogenic differen-tiation of ADSCs and BDNF-ADSCs. Cells were incubated in adipogenic or osteogenic differentiation media for 21 days and stained with Oil Red O and Alizarin Red respectively. The inserts in osteogenensis of ADSCs panels emphasize the calcium nodus produced by the ADSCs. ****means much statistically signifi-cant (p<0.0001, n=3).

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  Fig. 4 Colony forming unit assays for ADSCs and BDNF-ADSCs. A total of 100 cells were plated on 10 cm cell culture plates and incubated for 14 days. Cells were stained with 3% crystal violet, and colonies 2 mm or larger in diameter were counted. ***means much statistically significant (p<0.0001, n=3).

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  Fig. 5 Real-time PCR analysis of cytokine and chemokine profiles of BDNF-ADSCs vs ADSCs. ns means the expression levels are not statistically significant compared to those of ADSCs (p>0.05, n=3). ***means the expression levels are highly statistical significant compared to those of ADSCs (p<0.001, n=3).

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  Fig. 6 Western-Blot analyses for murine protein expression of NGF, BDNF, bFGF and CNTF in BDNF-ADSCs and ADSCs. *means statistically significant (p<0.05, n=3), and **means much statistically significant (p<0.01, n=3).

Reference

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