Bone marrow-derived mesenchymal stem cells differentiate into nerve-like cells in vitro after transfection with brain-derived neurotrophic factor gene

Abstract

Bone marrow-derived mesenchymal stem cells can differentiate into a variety of adult cells. Brain-derived neurotrophic factor (BDNF) is briefly active during differentiation and induces mesenchymal stem cells to differentiate into nerve cells. In this study, we cloned human BDNF to generate a recombinant pcDNA3.1(-)-BDNF vector and transfected the vector into bone marrow-derived mesenchymal stem cells. We selected these cells with Geneticin-418 to obtain BDNF-BMSCs, which were induced with retinoic acid to obtain induced BDNF-BMSCs. The transfected cells displayed the typical morphology and surface antigen profile of fibroblasts and were observed to express clusters of differentiation 29, 44, and 90 (observed in matrix and stromal cells), but not clusters of differentiation 31, 34, and 45 (observed in red blood cells and endothelial cells), via flow cytometry. Enzyme-linked immunosorbent assays showed that transfected bone marrow-derived mesenchymal stem cells secreted more BDNF than non-transfected bone marrow-derived mesenchymal stem cells. Immunocytochemistry and real-time reverse transcription polymerase chain reaction analysis showed that non-induced BDNF-BMSCs maintained a higher proliferative capacity and expressed higher amounts of brain-derived neurotrophic factor, nestin, neuron-specific enolase, and glial fibrillary acid protein than non-transfected bone marrow-derived mesenchymal stem cells. An additional increase was observed in the induced BDNF-BMSCs compared to the non-induced BDNF-BMSCs. This expression profile is characteristic of neurocytes. Our data demonstrate that bone marrow-derived mesenchymal stem cells transfected with the BDNF gene can differentiate into nerve-like cells in vitro, which may enable the generation of sufficient quantities of nerve-like cells for treatment of neuronal diseases.

Figure 1

Coding sequence (CDS) of the BDNF gene and expression of pcDNA3.1(−)-BDNF in HEK293 cells. (A) BDNF gene CDS. The CDS of the BDNF gene can be clearly observed (M: DL2000 DNA Marker, 1: BDNF CDS). (B) Expression of pcDNA3.1(−)-BDNF in HEK293 cells. BDNF was detected by western blot in HEK293 cells transfected with pcDNA3.1(−)-BDNF, but not in HEK293 cells transfected with pcDNA3.1(−) (CON: HEK293 cells transfected with pcDNA3.1(−), BDNF: HEK293 cells transfected with pcDNA3.1(−)-BDNF).

Figure 2

Flow cytometric analysis of third-passage BMSCs. (A–C) BMSCs were positive for CD29, CD44, and CD90 staining. (D–E) BMSCs were negative for CD34, CD45, and CD31 staining.

Figure 3

BMSC morphology and its change after transfection (magnification, ×100). (A) First generation of BMSCs. BMSCs cultured for 12 d were primarily characterized by long spindles and arranged with certain directivity. (B) RA-induced BMSCs. BMSC morphology and density did not markedly change after induction by RA. (C) Transfected BMSCs. Some BMSCs showed deformation after transfection with the human BDNF gene and treatment with G418, with a protruded soma and nerve-like form. (D) Induced BDNF-BMSCs. More BDNF-BMSCs differentiated and were deformed upon induction with retinoic acid (RA), where protruded soma were more apparent, and several cells were reticulated by connecting with one another.

Figure 4

Proliferation in BMSCs of BDNF-BMSCs. (A) Proliferation was determined by MTT assay at 48, 96, and 144 h. Values are presented as the mean ± SD for three independent experiments. (B) Growth curves. BDNF-BMSCs displayed a higher growth rate than BMSCs from days 2 to 7. The points represent the mean ± SD for triplicate experiments. (C) Representative histograms of flow cytometric analysis for cell cycle distribution. The proportion of BDNF-BMSCs in the S and G₂/M phases was higher and that in the G₀/G₁ phases was lower than that in BMSCs. ^# P < 0.05, compared with BMSCs (induced or non-induced). *P < 0.05, compared with non-induced BDNF-BMSCs.

Figure 5

Immunocytochemical analysis of BMSCs and BDNF-BMSCs (brown staining highlighted by arrows, magnification ×200). (A1–A4) Non-induced BMSCs expressed low levels of BDNF, nestin, NSE, and GFAP. (B1–B4) Low levels of BDNF, nestin, NSE, and GFAP were also detected in induced BMSCs. (C1–C4) The number of cells positively co-stained with BDNF, nestin, NSE, and GFAP in non-induced BDNF-BMSCs was significantly higher than that in BMSCs. Over 98% of non-induced BDNF-BMSCs were positively stained for BDNF. (D1–D4) Over 98% of induced BDNF-BMSCs were positively stained for BDNF. More cells positively co-stained for nestin, NSE, and GFAP were detected in induced BDNF-BMSCs than in non-induced BDNF-BMSCs. (E) Rates of positive immunostaining in BMSCs and BDNF-BMSCs, as determined by immunocytochemistry for BDNF, nestin, NSE, and GFAP (%; mean ± SD). ^# P < 0.01 compared with non-induced BMSCs and induced BMSCs; *P < 0.05 compared with non-induced BDNF-BMSCs.

Figure 6

mRNA expression of BMSCs and BDNF-BMSCs. BDNF, nestin, NSE, and GFAP mRNA expression of BMSCs and BDNF-BMSCs determined by RT-PCR (mean ± SD). ^# P < 0.01 compared with non-induced BMSCs and induced BMSCs; *P < 0.05 compared with non-induced BDNF-BMSCs.