In vitro differentiation of rabbit bone marrow mesenchymal stem cells into Schwann cells
Induction effect of platelet-rich plasma*☆

Changsuo Xia, Changrong Ding, Yingzhen Wang, Kang Sun, Cailong Zhang, Shaoqi Tian

Department of Orthopaedics, Affiliated Hospital of Qingdao University Medical College, Qingdao  266003, Shandong Province, China

Changsuo Xia☆, Doctor, Associate professor, Department of Orthopaedics, Affiliated Hospital of Qingdao University Medical College, Qingdao  266003, Shandong Province, China

Corresponding author: Changsuo Xia, Doctor, Associate professor, Department of Orthopaedics, Affiliated Hospital of Qingdao University Medical College, Qingdao  266003, Shandong Province, China   
xcs009@163.com

Abstract
BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) differentiate into Schwann cells via specific inducers. However, the induction and culture procedures are complicated. Various growth factors have been used for induction and culture, and are likely affected by their environment.
OBJECTIVE: To explore the differentiation feasibility of platelet-rich plasma-induced rabbit BMSCs, supplemented with various growth factors, into Schwann-like cells in vitro, and to examine the sec-retory function of Schwann-like cells. 
DESIGN, TIME AND SETTING: This comparison study was performed at the Experimental Animal Center, Affiliated Hospital of Qingdao University Medical College, China in October 2008.
MATERIALS: Platelet-rich plasma and BMSCs were respectively obtained from the femoral vein and bone marrow of 2-month old New Zealand rabbits. The rabbit nerve growth factor ELISA kit and rabbit nerve growth factor PCR kit (Jingmei Biotech, China), as well as anti-rabbit S-100 im-munofluorescence staining kit (Boster, China) were used in the present study.
METHODS: BMSCs at passage three were harvested and were induced by 1 mmol/L β-mercaptoethanol and Dulbecco’s-modified eagle's medium complete medium containing 35 ng/mL retinoic acid and 10% fetal bovine serum for the combination induction group, followed by incubation in complete medium supplemented with platelet-rich plasma. BMSCs in the single induction group were induced by β-mercaptoethanol and retinoic acid, followed by incubation in L-DMEM complete medium. BMSCs in the control group were incubated in L-DMEM complete medium.
MAIN OUTCOME MEASURES: Cell growth in each group was observed under an inverted micro-scope. Following induction, S-100 protein expression was identified by immunofluorescence staining. Nerve growth factor protein concentrations in BMSC supernatant were subsequently measured by ELISA. In addition, nerve growth factor mRNA expression in the BMSCs was determined by reverse transcription-polymerase chain reaction.
RESULTS: Overall BMSC morphology was similar to Schwann cells in the combination induction group, and morphology of the majority of BMSCs was similar to Schwann cells in the single induction group 9 days after induction. In the control group, significant changes in cell morphology were not observed 9 days after induction. S-100 protein expression was detected in the combination induction and single induction groups following induction. At days 7, 9, and 11, the expression rate of S-100-positive cells was greater in the combination induction group (P < 0.05). At days 4, 7, 9, and 11, nerve growth factor levels were significantly greater in BMSC supernatant in the combination induction and single induction groups compared with the control group (P < 0.05). At days 7, 9, and 11, nerve growth factor levels were significantly greater in the combination induction group com-pared with the single induction group (P < 0.05). At day 11, nerve growth factor mRNA expression in BMSCs was significantly greater in the combination induction group compared with the single in-duction group (P < 0.05).
CONCLUSION: Culture conditions containing platelet-rich plasma elevated the differentiation effi-ciency of BMSCs into Schwann-like cells, which subsequently secreted nerve growth factor following induction.
Key Words: mesenchymal stem cells; Schwann-like cells; induction; differentiation
 

INTRODUCTION
  
Schwann cells play an important role in peripheral nerve regeneration[1-2]. Schwann cells divide and proliferate following Wallerian degeneration in the distal end of damaged nerves, participate in clearance of degenerative axonal and myelin debris, organize into typical bands of Bungner, and initiate growth of the regenerated axon[1-2]. Moreover, Schwann cells express and secrete various active substances, such as nerve growth factor (NGF), as well as inducing, stimulating, and regulating axonal regeneration and myelin formation, and ultimately enhancing reinnervation[1-2].
Schwann cells significantly promote neural regeneration following implantation in an injury site[3]. However, the number of autologous Schwann cells is limited, and allogeneic Schwann cell implantation induces immunological reactions. This greatly limits the clinical application of Schwann cells.
Schwann cells are the glial cells of the peripheral nervous system. Rat bone marrow mesenchymal stem cells (BMSCs) differentiate into Schwann cells in vitro, but induction and culture methods remain complicated[4-6]. Various growth factors are required for cell proliferation and differentiation[7-8]. Rat BMSCs have been shown to differentiate into Schwann-like cells following in vitro culture with specific inducers[9]. Following exposure to β-mercaptoethanol and retinoic acid, BMSCs have been incubated in medium supplemented with platelet-derived growth factor, transforming growth factor, and basic fibroblast growth factor to increase induction efficiency[10]. However, the various growth factors used in these induction and culture methods are most likely affected by the surrounding environment. Therefore, this study used autologous platelet-rich plasma. Autologous platelet-rich plasma contains various growth factors at a high concentration, with activity lasting 5–8 days[11-12] that supplements the insufficiency of exogenous growth factor. This study was designed to determine whether platelet-rich plasma promotes BMSC differentiation into Schwann cells following induction.

MATERIALS AND METHODS

Design
A comparison in vitro study.
Time and setting
Experiments were performed at the Experimental Animal Center, Affiliated Hospital of Qingdao University Medical College, China in October 2008.
Materials
A total of 18 healthy, clean, New Zealand rabbits, aged 2 months, of both genders and weighing 3.5–4.5 kg, were supplied by the Qingdao Animal Experimental Center, China (license No. SCXK(Lu)2008-0007). Protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of the People’s Republic of China[13].
Reagents and equipment are as follows:

Methods
BMSCs culture and determination
Rabbits were anesthetized with 3.0% pentobarbital sodium (1.0 mL/kg) via the ear vein. A 10-mL syringe containing 3 000 U/mL heparin sodium (1 mL) with a No. 16 cannula was inserted into the marrow cavity to obtain approximately 5 mL bone marrow. In accordance with previously described methods[6], BMSCs were harvested, incubated, and subcultured. The third BMSC passage was utilized for this study.
Extraction of platelet-rich plasma
Platelet-rich plasma was prepared according to previously described methods[14]. A total of 5 mL femoral vein blood was collected and centrifuged at 1 000 r/min for 15 minutes. Blood plasma and platelets were collected from the upper layer and centrifuged at       3 000 r/min for 8 minutes. Blood plasma was removed from the upper layer. The remaining liquid was mixed with activator (thrombin and 10% CaCl2 at a ratio of 9: 1), shaken, and incubated at 4 °C overnight. The samples were then centrifuged at 1 000 r/min for 10 minutes. The supernatant (platelet-rich plasma) was collected for further use.
Induction method and grouping
The third BMSC passage was incubated in 48-well plates and assigned to three groups, with 18 wells per group.
BMSCs in the combination induction group (105/mL) were induced by 1 mmol/L β-mercaptoethanol for 24 hours. Following medium removal, the cells were washed twice in phosphate buffered saline (PBS), and incubated in DMEM (low glucose) complete medium supplemented with 10% fetal bovine serum and 35 ng/mL retinoic acid for 3 days. Following medium removal, the cells were washed twice in PBS, and then incubated in medium containing platelet-rich plasma. Each 1-mL medium sample contained 0.2 mL platelet-rich plasma. During this process, the medium was changed twice.
BMSCs in the single induction group were induced by β-mercaptoethanol and retinoic acid, and then incubated in DMEM complete medium (low glucose).
BMSCs in the control group did not undergo sequential induction, but were rather incubated in DMEM complete medium (low glucose). 
Cell growth was observed under an inverted microscope.
S-100 immunofluorescence staining
In accordance with previously described methods[15], the cell concentration was adjusted to 105/mL at days 4, 7, 9, and 11 in each group. Subsequently, the cells were incubated for 3 days, and then washed three times in PBS, 5 minutes each. The cells were fixed in 4% paraformaldehyde at 4 °C for 5–10 minutes, washed three times in PBS for 5 minutes, incubated in 1% TritonX-100 for 20 minutes at 37 °C, and washed once in PBS. The cells were then mounted on slides with neutral gum, blocked with normal goat serum for 5 minutes, washed once in PBS, and then incubated with goat anti-rabbit S-100 monoclonal antibody (1: 30) at  4 °C overnight, rinsed three times in PBS for 5 minutes. The cells were then incubated in FITC-labeled rat anti-goat IgG (1: 40) at 37 °C for 20 minutes in the dark, and rinsed three times in PBS in the dark for 5 minutes. Fluorescence microscopy revealed positive cells with green fluorescence. A total of 10 visual fields were randomly selected to quantify positive cells. The S-100-positive expression rate was calculated according to the number of S-100-positive cells/total cell number.
Detection of NGF protein concentration by ELISA
According to previously described methods[16-17], at days 4, 7, 9, and 11, a total of 1 mL cell supernatant from each group was collected prior to medium replacement. The supernatant was placed in an Eppendorf tube and centrifuged at 1 000 r/min for 5 minutes, and then stored in an additional Eppendorf tube at –20 °C. A total of 1 000, 500, 250, 125, 62.5, 31.3, and 15.6 pg/mL (sample dilution of an equal volume served as a zero standard) standard preparations (each 0.1 mL) were added sequentially to a 24-well plate. Subsequently, 0.1 mL cell supernatant was added, with triplicate parallel wells for each sample. The samples were incubated at 37 °C for 120 minutes. The liquid was removed from the plate prior to addition of biotinylated goat anti-rabbit NGF antibody (1: 40) (0.1 mL/well) and incubation for 60 minutes at   37 °C. The wells were rinsed three times with 0.01 mol/L PBS, and allowed to soak for 1 minute each time. The avidin-biotin complex working solution (0.1 mL/well) was added and incubated for 30 minutes at 37 °C. The wells were then washed five times with 0.01 mol/L PBS for 2 minutes each time. Subsequently, 3, 3’, 5, 5’-tetramethylbenzidine colored solution (0.1 mL/well) was added for 25 minutes at 37 °C in the dark. The reaction was terminated by the addition of 3, 3’, 5, 5’-tetramethylbenzidine stopping solution (0.1 mL/well). Absorbance values were measured at 450 nm with a microplate reader. The 3, 3’, 5, 5’-tetramethylbenzidine- blank wells served as controls. Absorbance difference values were calculated according to absorbance of all standard preparation and samples – absorbance of zero standard. All data were loaded into CurveExpert 1.3 software (Hixson, TN, USA) to calculate sample concentration.
Detection of NGF mRNA expression using RT-PCR
According to previously described methods[18-19], cells were harvested from each group on day 11. NGF mRNA expression was measured by RT-PCR. NGF and β-actin primer sequences are shown in Table 1.

RNA was extracted from the cells using Trizol reagent[19]. A total of 1 μL RNA was used from each group for reverse transcription and cDNA synthesis, followed by exponential amplification. The cycle profile was as follows: denaturation at 95 °C for 5 minutes; 32 cycles of 94 °C for 30 seconds, 54 °C for 40 seconds, and 72 °C for 30 seconds, followed by 7 minutes at  72 °C. NGF mRNA products were analyzed by 1.5% agarose gel electrophoresis. The gray values of the electrophoresis strip were analyzed using GEL pro3.0 software (Media Cybernetics, Silver Spring, MD, USA). Mean value was obtained after six measurements. β-actin was considered an internal reference.
Main outcome measures
Cell growth in each group was observed under an inverted microscope. S-100 protein expression was identified using immunofluorescence staining following induction. NGF levels in BMSCs supernatant were measured utilizing ELISA following induction. NGF mRNA expression in BMSCs was determined by RT-PCR following induction.
Statistical analysis
All data were analyzed using SPSS 12.0 software (SPSS, Chicago, IL, USA) by Changsuo Xia, and were expressed as Mean ± SD. A completely randomized analysis of variance was used to compare differences among groups. Student-Newman-Keuls method was used for pairwise comparison. A value of P < 0.05 was considered statistically significant.

RESULTS

Cell morphology
Four days after induction of β-mercaptoethanol and retinoic acid, the majority of cells in the combination induction and single induction groups did not present with typical BMSC morphology. The cells were small and thin, with long processes.
At day 9, cells in the combination induction group did not exhibit typical BMSC morphology, but presented with abundant processes, which were similar to Schwann cells. The morphology of the majority of cells was similar to Schwann cells in the single induction group. No significant difference in cell morphology was detected in the control group (Figure 1).

Results from immunofluorescence staining following differentiation of BMSCs into Schwann cells
Cells in the combination and single induction groups expressed S-100 protein following treatment with β-mercaptoethanol and retinoic acid (Figure 2, Table 2). The S-100 expression rate was gradually increased over time in the combination and single induction groups. Cells were negative for S-100 protein in the control group.

The S-100 expression rate was similar between the combination and single induction groups following 4 days in culture (P > 0.05). The S-100 expression rate was significantly increased in the combination induction group at days 7, 9, and 11 following culture (P < 0.05) (Table 2).

NGF levels in cell supernatant following induction
NGF concentrations were significantly greater in BMSC supernatant from the combination and single induction groups, compared with the control group (P < 0.05).
At day 4, there was no significant difference in NGF concentrations between the combination and single in-duction groups (P > 0.05).
At days 7, 9, and 11, NGF levels were significantly greater in the combination induction group compared with the single induction group (P < 0.05) (Table 3).

NGF mRNA expression in cells following induction
At day 11, a NGF mRNA band was observed in the combination and single induction groups, but the NGF mRNA band was not detected in the control group (Figure 3). The relative expression intensity of NGF mRNA was significantly greater in the combination induction group (0.527 ± 0.035) compared with the single induction group (0.351 ± 0.029, P < 0.05). 

DISCUSSION

Platelet-rich plasma is typically isolated following centrifugation blood, and contains high levels of various growth factors, such as platelet-derived growth factor, transforming growth factor, vascular endothelial growth factor, epidermal growth factor, and insulin-like growth factor[20-21]. Activity of these growth factors could last for 5–8 days, and these growth factors play important roles in regulating cell proliferation and differentiation[22-23]. Results from the present study demonstrated that BMSCs treated with platelet-rich plasma significantly increased the Schwann cell differentiation efficiency following exposure to β-mercaptoethanol and retinoic acid. This suggested that growth factors in platelet-rich plasma might play a role in BMSC differentiation into Schwann cells.
Many methods have been utilized to prepare platelet-rich plasma. This study used double centrifugation, which was based on the difference in sedimentation coefficient of blood components. Erythrocytes were initially precipitated and discarded in the first centrifugation. Studies have shown that platelets with a low sedimentation coefficient can be precipitated, resulting in high concentrations of platelets[12]. The biological effects of platelet-rich plasma might be dependent on various high-concentration growth factors, which have synergistic effects and affect cell chemotaxis, proliferation, and differentiation[22-23]. However, further studies are needed to control and regulate concentrations of various growth factors in platelet-rich plasma. Someya et al[24] observed the morphology of Schwann-like cells differentiated from BMSCs and measured S-100 expression. However, that study failed to measure the function of Schwann cells. In the present study, NGF was used to observe the function of differentiated cells, which demonstrated that the resulting cells secreted NGF and expressed NGF mRNA. Moreover, the addition of platelet-rich plasma increased the induction efficiency and cell secretory function following induction.
In summary, rabbit BMSCs were induced to differentiate into NGF-secreting Schwann-like cells. Furthermore, platelet-rich plasma significantly increased the differen-tiation efficiency of BMSCs into Schwann cells during in vitro culture.

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 (Edited by Yang SA/Qiu Y/Song LP)