Bone morphogenetic protein-7 induced bone marrow stromal cells differentiate into neuron-like cells＊☆

Kuanxin Li, Yuling Zhang, , Weishan Wang, Bin He, Jianhua Sun, Jinbo Dong, Chenhui Shi

1First Department of Orthopedics, First Affiliated Hospital of Shihezi University Medical College, Shihezi 832008, Xinjiang Uygur Autonomous Region, China

2Department of Scientific Education, First Affiliated Hospital of Shihezi University Medical College, Shihezi 832008, Xinjiang Uygur Autonomous Region, China

lNTRODUCTlON

Bone marrow stromal cells (BMSCs) are non-hematopoietic cells in the bone marrow.

BMSCs can differentiate into nerve cells,bone cells, adipocytes and cardiac muscle cells under certain experimental conditions[1-5]. Bone morphogenetic protein-7 (BMP-7) is considered to function as an inducer for the differentiation of BMSCs into osteoblasts and chondrocytes.

In addition, BMP-7 can move new neurons to the granule cell layer where they can differentiate into mature neurons[6-9].

Previous studies have focused on promoting the differentiation of BMSCs into neuron-like cells, and have examined the efficacy of obtaining neuronal cells that function similarly to normal neurons in vivo[10].

BMSCs have been found to differentiate into different types of nerve cells, such as neurons and astrocytes[11-14]. However, the underlying mechanisms of this differentiation are currently unclear. As such, the present study sought to verify the inducer role of BMP-7 on BMSCs differentiating into neuron-like cells.

RESULTS

Culture and identification of rat BMSCs(Figure 1)

Figure 1 Identification of rat bone marrow stromal cell phenotypes(immunofluorescence staining, × 200).Three types of antigens on the cellular surface (A-C: CD29, CD44, CD90)expressed in the cytoplasm (arrows).

To identify the expression of BMSC phenotypes, we used indirect immunofluorescence staining to purify highly expressed mesenchymal stem cells. The results revealed that CD29, CD44 and CD90 positive cells appeared in the cytoplasm of BMSCs at passage 3, with expression rate accounting for 95% of cells. BMSCs at passage 3 achieved a natural purification of 95%,exhibiting a high level of purity.

Positive rate of BMP-7 induced BMSCs differentiating into neuron-like cells

The results revealed that BMSCs were induced to differentiate into neuron-like cells, with BMP-7 acting as a major inducer. Neuron-specific markers were identified using immunohistochemistry and PCR. The subcultured mesenchymal stem cells were divided into seven groups:BMP-7 25, 50, 75, 100, 125, 150 ng/mL groups: BMP-7(25, 50, 75, 100, 125, 150 ng/mL) + 20 ng/mL basic fibroblast growth factor + 2% B27 + serum-free standard culture medium (DMEM/F12 medium + penicillin 100 U/mL + streptomycin 100 U/mL). Control group: His protein (50 ng/mL) + 2% B27 + serum-free standard culture medium, with five holes in each group. All seven groups were induced for 3 days, and 1, 2, 3 weeks.After a 1-week BMP-7 induction period, the highest positive rate of neuron-like cells was observed in the BMP-7 50 ng/mL group (P ＜ 0.01; Table 1). Accordingly,BMP-7 50 ng/mL was applied for the following experiments.

Table 1 Positive rate of BMSCs differentiating into neuron-like cells after being induced by different concentrations of BMP-7

BMSCs differentiated into neuron-like cells after induction by 50 ng/mL BMP-7

After BMSCs were induced with 50 ng/mL of BMP-7 for 3 days, inverted microscopic observations revealed neuron-like cellular morphological changes, including enhanced soma refraction, and the protrusion of a large number of thin processes. The number of neuron-like cells increased and the processes became longer over the induction time. After 1-week induction period, bipolar or multipolar neuron-like cells were visible, cell processes were abundant and apparent, and several processes had extended branches and become connected into a network. Cells in the control group showed no change in morphology and exhibited elongated spindle swirling growth (Figure 2). The number of neuron-like cells started to decrease at 2 weeks.

Figure 2 Morphology of neuron-like cells after induction of 50 ng/mL bone morphogenetic protein-7 (BMP-7) for 1 week (× 200). 50 ng/mL was the optimal inducing dose,associated with neuron-like cells achieving a peak positive rate of proliferation and differentiation, while the positive rate was lower in other groups.

Microtubule-associated protein 2 (MAP-2),intermediate neurofilament protein (NF-M) and glial fibrillary acidic protein (GFAP) expression in BMSCs after BMP-7 induction

To verify whether the cells differentiating from BMP-7-induced BMSCs were neuron-like cells, we utilized neuron-specific markers for early neuron-like cells (MAP-2 and NF-M) and glial cell marker GFAP monoclonal antibodies. Cells stained brown-yellow by diazoaminobenzene were considered positive cells. The results revealed that MAP-2-stained cells exhibited brown granules at 1 week after induction (Figure 3A).The performance of NF-M was similar to MAP-2 (Figure 3B). GFAP showed no positive expression (Figure 3C).

MAP-2, NF-M and GFAP mRNA expression in BMSCs after BMP-7 induction

MAP-2 mRNA amplified products exhibited target bands at 373 bp (Figure 4). NF-M mRNA products exhibited target bands at 513 bp (Figure 5). GFAP expression was negative, consistent with immunohistochemical staining results (Figure 6).

Figure 3 Microtubule-associated protein 2 (MAP-2, A),intermediate neurofilament protein (NF-M, B) and glial fibrillary acidic protein (C) expression after 1 week induction (immunohistochemistry staining, × 400).Neuron-like cells in the cytoplasm of MAP-2 and NF-M show brown-yellow granules, suggesting positive expression (arrows point to MAP-2 or NF-M positive cells).Control group (D) showed no significant change.

Figure 4 Microtubule-associated protein 2 mRNA expression after induction with bone morphogenetic protein-7 (BMP-7). M: Marker; 1: BMP-7 50 ng/mL for 1 week; 2: BMP-7 50 ng/mL for 2 weeks; 3: BMP-7 50 ng/mL for 3 weeks; 4: control group. Microtubuleassociated protein target band was visible at 373 bp, while the control group showed no bands.

Figure 5 Neurofilament protein mRNA expression after induction with bone morphogenetic protein-7 (BMP-7).M: Marker; 1: BMP-7 50 ng/mL for 1 week; 2: BMP-7 50 ng/mL for 2 weeks; 3: BMP-7 50 ng/mL for 3 weeks;4: control group. Neurofilament protein target band was visible at 513 bp, while the control group showed no bands.

Figure 6 Glial fibrillary acidic protein mRNA expression after induction with bone morphogenetic protein-7(BMP-7). M: Marker; 1: BMP-7 25 ng/mL group; 2: BMP-7 50 ng/mL group; 3: BMP-7 75 ng/mL group; 4: BMP-7 100 ng/mL group; 5: BMP-7 125 ng/mL group; 6: BMP-7 150 ng/mL group; 7: control group. No band was visible at 364 bp in the BMP-7 50 ng/mL group.

DlSCUSSlON

We used three types of nervous system-specific markers in this study: (1) MAP-2, a glycolytic enzyme present in nerve cells and a specific marker of mature neurons[15].(2) NF-M, the unique structure of mature neurons, can maintain neuronal morphology, stabilize neuronal microtubules, and is an important component of protruding cytoplasm. (3) GFAP, a specific marker of glial cells, which can maintain both form and function of glial cells. GFAP expression is a hallmark identifier of the presence of glial cells[16].

The induced bone marrow-derived neurons were identified with immunofluorescence. The results demonstrated that both MAP-2 and NF-M were expressed in the cytoplasm, while no GFAP expression was exhibited. Taken together, these findings indicate the potential of BMP-7 for inducing the differentiation of rat BMSCs into nerve cells, rather than glial cells. Reverse transcription-PCR and immunohistochemistry revealed the similar results. Rat BMSCs induced by BMP-7 were found to differentiate into neuron-like cells, and express nerve cell-specific markers. In addition, the present results are in accordance with previous reports[17-18].

We found that BMP-7-induced rat BMSCs exhibited morphological characteristics of nerve cells and expressed neuron-specific markers. The results initially indicated that BMSCs yielded to bone marrow-derived nerve cells. However, a simple sign of neuron-specific marker expression cannot confirm the presence of nerve cells. Shin et al[19]proposed that nerve-like cells should meet several criteria before they can be conclusively considered nerve cells. These criteria include the ability to produce action potentials, the presence of a single polar axon and multiple dendrites, and the ability to communicate with other neurons via synapses.Therefore, an electrophysiological study is necessary to further clarify the function of the induced bone marrow-derived neuron-like cells.

bFGF is an important mitogenic factor, and an inducer for morphogenesis and differentiation. In the current study,the third generation of BMSCs were induced with different concentrations of BMP-7 combined with basic fibroblast growth factor[20-21]. The positive rate and viability of neuron-like cells were found to define the optimal induction conditions. Immunohistochemistry and reverse transcription-PCR detection of induced bone marrow-derived neuron-like cells revealed the expression of neuronal markers MAP-2 and NF-M, while no expression of glial cell marker GFAP was found.

These findings confirmed that the induced cells were neuron-like cells, rather than glial cells. BMP-7 has been found to function as an inducer for BMSCs differentiating into osteoblasts and cartilage cells, but few studies have examined its role in the differentiation of BMSCs into neuron-like cells. The Wnt-1 gene may play a regulatory role during differentiation[18], but the underlying mechanisms remain unclear and require future investigation.

Overall, the present results confirmed that BMP-7 can promote the differentiation of BMSCs into neural cells,producing bone marrow-derived neuron-like cells.

MATERlALS AND METHODS

Design

A comparative study of cytology in vitro.

Time and setting

The experiment was accomplished between January and September 2010 at the Laboratory of Molecular Biology,Shihezi University School of Medicine, China.

Materials

A total of 24 healthy 4-week-old Sprague-Dawley rats(half male, half female) weighing 200-250 g, were purchased from the Experimental Animal Center of Xinjiang Medical University, China, with license No.

SCXK (Xinjiang) 2003-0002. The experimental protocols were in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of the People’s Republic of China[22].

Methods

Isolation, culture and identification of BMSCs

BMSCs were separated using whole bone marrow adherent culture. The femur and tibia of 4-week-old Sprague-Dawley rats were isolated under sterile conditions. Excessive tissues on the bone surface were removed. Samples were placed in L-Dulbecco’s Modified Eagles Medium (DMEM), and the bone marrow cavity was exposed. A hole at the femoral and tibial bone ends was made with a large needle, and the medullary cavity was injected with serum-containing DMEM using a 10-mL syringe. At the other end of the bone, the rinsed bone marrow was collected, cellular suspensions were centrifuged at 1 200 r/min, and the supernatant was discarded. Cells were suspended in L-DMEM medium containing 15% fetal bovine serum, and seeded into culture flasks at 1 × 106cells/mL, at 37°C, in a 5% CO2incubator. The suspended blood cells were removed,and cells were digested with 0.125% trypsin. The third generation of BMSCs were rinsed with PBS and fixed in 4% paraformaldehyde at room temperature, blocked with 10% normal goat serum, mouse anti-human antibody(Shanghai Bluegene Biotech Co., Ltd., China): CD29(1: 100), CD44 (1: 100), CD90 (1: 100) monoclonal antibodies. Blank controls were treated with PBS instead of primary antibody. Samples were incubated with antibodies at 4°C overnight. Fluorescein isothiocyanate-labeled goat anti-mouse IgG (1: 100)antibody (Shanghai Kebo Biotechnology Co., Ltd., China)was added into the medium, in which cells were incubated at room temperature and cemented with glycerol PBS. Cells were excited under a laser resonance confocal microscope (LSM-510, Carl Zeiss,Oberkochen, Germany) at 488 nm, green fluorescence was observed in the cytoplasm with an even distribution,and the nuclei were not stained. Green fluorescence indicated positivity, expressed as fluorescence intensity.

BMP-7 induced BMSCs differentiating into neuron-like cells

Pre-induction: Standard pre-induction medium[23]included phenol red, 4.5 g/L glucose, 3.7 g/L sodium bicarbonate, 0.11 g/L sodium pyruvate, and 2 mg L-glutamate. In addition, basic fibroblast growth factor 20 ng/mL was added. Third-generation BMSCs were collected, digested with trypsin, then counted and inoculated into 24-well plates. The cells were induced in pre-induction medium for 1 week (BMSC differentiation crossed germ layers, so the pre-induced BMSCs were more prone to differentiate into neurons). Each hole contained 500 μL, and each group contained five holes,with 2 × 103cells/well. Cells were cultured at 37°C in 5%CO2.

BMP-7 induction: The culture plate was taken out at 1 week after pre-induction, the liquid within the culture plate was removed. The induction medium was added and cells were incubated in CO2incubator at 37°C. The medium was replenished every 2 days.

Immunohistochemical detection of NF-M, MAP-2 and GFAP expression after BMP-7 induction

The coverslips covered with the cells induced for 24 hours were taken out and rinsed with 37°C PBS (pH 7.2) three times, for 3 minutes each time. Samples were then fixed in cold (-20°C) acetone for 10 minutes. Each coverslip was added with 50 μL peroxidase blocking agent, and incubated at room temperature for 10 minutes, to block endogenous peroxidase activity. Samples were again rinsed with PBS three times, for 2 minutes each time.

After the PBS was removed, culture medium was added with 50 μL 10% goat serum, and cells were incubated at room temperature for another 10 minutes. The diluted neuron-specific antibody was added to the culture medium: mouse anti-human MAP-2 (1: 500; Shanghai Hua Yi Biotechnology Co., Ltd.), NF-M (1:100; Shanghai Kebo Biotechnology Co., Ltd.), and GFAP monoclonal antibody (1: 50; Yansheng (Shanghai) Biochemical Reagent Co., Ltd., China). The tested cells were completely covered and incubated at room temperature for 2 minutes. PBS solution was removed, and 100 μL biotinylated goat anti-mouse antibody (Shanghai Lengton Bioscience Co., Ltd., China) was added into the medium. Cells were then incubated at room temperature for 1 hour. 100 μL horseradish peroxidase-conjugated streptavidin (Lengton) was added into the medium and cells were incubated at room temperature for 10 minutes. 100 μL DAB coloration liquid was added, and cells were observed under an optical microscope (Olympus, Tokyo, Japan). At the interval of each step, cells were rinsed with PBS three times, for 2 minutes each time. Hematoxylin counterstaining was followed by rinsing, dehydrating through graded ethanol,xylene transparency, drying, and neutral gum cementing.

The control group was incubated with PBS instead of primary antibody. Yellow or brown granules in the cytoplasm by MAP-2, NF-M and GFAP staining were considered to indicate positive cells. Ten visual fields at high power (400 ×) were randomly selected with 100 cells in each visual field. Staining results were expressed as the percentage of positive cells.

Reverse transcription-PCR detection of NF-M, MAP-2 and GFAP mRNA expression in neuron-like cells after BMP-7 induction

The MAP-2, NF-M and GFAP mRNA expression was detected with reverse transcription-PCR, using β-actin as an internal reference.

After neuron-like cell density reached 80%, total RNA was extracted with TRIzol (Sigma). The sample was then added with chloroform and centrifuged. The upper aqueous phase was transferred to a new tube,and isopropyl alcohol was added.

Primer sequencing is as follows:

F: Forward; R: reverse.

This was followed by centrifugation and removal of the supernatant. Ethanol was then added, the salt in the precipitations was washed away, and the samples were centrifuged. The samples were then dried, and RNase-free water was added. The total cellular RNA extracted underwent 1.5% agarose gel electrophoresis,and was observed with a gel-imaging system (Bio-Rad,Hercules, CA, USA).

Statistical analysis

Data were analyzed with SPSS 13.0 statistical software(SPSS, Chicago, IL, USA). Data between groups were compared with analyses of variance, and the mean value between groups was compared with an SNK-q test. A level of P ＜ 0.05 was considered to indicate statistically significant differences.

Author contributions:Kuanxin Li provided and integrated data.

Yuling Zhang was responsible for statistical analysis. Jinbo Dong proposed and designed the study. Weishan Wang was responsible for data analysis. Bin He wrote the manuscript.Jianhua Sun provided data support. Chenhui Shi guided the study.

Conflicts of interest:None declared.

Funding:This study was financially sponsored by the Science and Technology Research and Development Program of Shihezi University, No. ZRKX2009YB23.

Ethical approval:The study was approved by the Animal Ethics Committee, First Affiliated Hospital of Shihezi University School of Medicine, China.

Supplementary information:Supplementary data associated with this article can be found, in the online version,by visiting www.nrronline.org, and entering Vol. 6, No. 22,2011 item after selecting the “NRR Current Issue” button on the page.

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