The isolation and cultivation of bone marrow stem cells and evaluation of differences for neural-like cells differentiation under the induction with neurotrophic factors

Abstract

The bone marrow represents the most common source from which to isolate mesenchymal stem cells (MSCs). They can be obtained directly from patients and successfully induced to form various differentiated cell types. In addition, cell-based transplantation therapies have been proven to be promising strategies for curing disease of the nerve system. Therefore, it was particularly important to establish an easy and feasible method for the isolation, purification, and differentiation of bone marrow stromal cells (BMSCs). The aim of this study was to isolate and characterize putative bone marrow derived MSCs from Sprague-Dawley (SD) rats. Furthermore, differentiation effects were compared between the GDNF-induction group and the BDNF-induction group. Of these, BMSCs were isolated from the SD rats in a traditional manner, and identified based on plastic adherence, morphology, and surface phenotype assays. After induction with GDNF and BDNF, viability of BMSCs was detected by MTT assay and neuronal differentiation of BMSCs was confirmed by using immunofluorescence and Western blotting. Besides, the number of BMSCs that obviously exhibited neuronal morphology was counted and the results were compared between the GDNF-induction group and BDNF-induction groups. Our results indicate that direct adherence was a simple and convenient method for isolation and cultivation of BMSCs. Furthermore, BMSCs can be induced in vitro to differentiate into neuronal cells by using GDNF, which could achieve a more persistent and stable inducing effect than when using BDNF.

Fig. 1

Identification of cultivated cells. The percentage of stained cells for each antibody was CD34: 0.62 %, CD44: 99.69 %, CD45: 0.28 % and CD90: 95.05 %

Fig. 2

Morphology of BMSCs. a 1 day cultivation. The appearance of the cells was polygonal or rod-shaped (green arrows, scale bar  10 μm). b 3 days cultivation. The appearance of the cells was polygon-like or flat-shaped (green arrows, scale bar 10 μm). c 5 days cultivation. The appearance of the cells was almost uniform and spindle-shaped (green arrows, scale bar 10 μm). d 7 days cultivation. Cells are arranged significantly in a swirling pattern (green circle, scale bar 10 μm). (Color figure online)

Fig. 3

In the GDNF-induction group and BDNF-induction group, the anti-NSE staining is shown as green fluorescence. Nuclei are labeled with DAPI, shown as blue fluorescence (left panels). The panels show that the cells, strongly stained for NSE, formed tapered triangular and axon-like structures of neuronal cells (red arrows). Some of the cells, weakly stained for anti-NSE, still maintained the flat-shaped or irregular shape (white arrows). In the control group, nuclei were also labeled with DAPI, shown as blue fluorescence but no staining of anti-NSE was detected (scale bar 10 μm). (Color figure online)

Fig. 4

Immunostaining of BMSCs with anti-MAP-2 antibody 5 days after induction. The expression of MAP-2 was shown as green fluorescence. Red arrows show significant filament structures of cells and white arrows show irregular-shaped or flat-shaped morphology of cells 5 days after induction (for GDNF-induction group and BDNF-induction group, scale bar 50 μm. For control group, scale bar  100 μm). (Color figure online)

Fig. 5

Quantitation of differentiation into neuron-like cells 7 days after induction: a 7 days after induction with GDNF, images show that most of the BMSCs exhibited significant morphological changes including soma retraction, transparency and appearance of filament structures (green arrow), a small number of the cells maintained polygon-like or flat-shaped morphology (red arrow) (scale bar 10 μm) b 7 days after induction with BDNF, images show that most of the BMSCs exhibited filament structures and changes of transparency (green arrow) but most of the cells maintained polygon-like or flat-shaped morphology (red arrow) (scale bar 50 μm). c The result of statical analysis indicated that significant difference existed in the two groups. * p < 0.05. (Color figure online)

Fig. 6

Morphological change and Western blot analysis 9 days after induction. a After 9 days of induction with GDNF, most of the BMSCs formed bipolar or multipolar synaptic structures of neuronal cells (green arrows) (scale bar 50 μm). b After 9 days of induction with BDNF, most of the BMSCs showed obvious cytoplasm retraction or lysis (red arrow) and only a small number of cells exhibited bipolar structures of neuronal cells (green arrows) (scale bar 100 μm). c and d: Expression of NSE among the 3 groups after 3 (c) and 9 (d) days of induction by using Western blot analysis. (Color figure online)

Fig. 7

Co-expression of NSE and MAP-2 9 days after induction. a Image of BMSCs induced by GDNF for 9 days and stained with DAPI (scale bar 50 μm). b Image of BMSCs induced by GDNF for 9 days and stained with anti-NSE (green) (scale bar 50 μm). c Image of BMSCs induced by GDNF for 9 days and stained with anti-MAP-2 (red) (scale bar 50 μm). d The merged images of panels b and c. e Image of BMSCs induced by BDNF for 9 days and stained with anti-NSE (scale bar 50 μm). f Image of BMSCs induced by BDNF for 9 days and stained with anti-MAP-2 (scale bar 50 μm). g Image of BMSCs cultured in the control group for 9 days and stained with NSE (scale bar 50 μm). h Image of BMSCs cultured in the control group for 9 days and stained with MAP-2 (scale bar 50 μm). (Color figure online)

Fig. 8

Comparison of viability of BMSCs among the 3 groups in vitro (*p < 0.05, ^# p > 0.05). (Color figure online)