Basic research
Co-culture with Schwann cells is an effective way for adipose-derived stem cells neural transdifferentiation

Introduction

Schwann cells (SCs) play a pivotal role in the regeneration and development of peripheral nerves [1, 2]; however, it is not easy to acquire sufficient and highly purified SCs for clinical application without additional morbidity to the nerve donor site, so other strategies are desired.
Adipose-derived stem cells (ADSCs) are isolated from adipose tissue derived from embryonic mesoderm. They are capable of self-renewal and can differentiate along several mesenchymal tissue lineages, including adipocytes, osteoblasts, myo­cytes, chondrocytes, endothelial cells and cardiomyocytes [3, 4]. Liposuction is a common and safe surgical procedure, while subcutaneous adipose tissue is abundant, readily accessible, and relatively expendable, so enough ADSCs can be obtained with minimal risk [5]. Recently it has been proved that ADSCs may also be induced into neural-like cells or a Schwann cell phenotype with the presence of some growth factors in vitro [6-8]. Last year, Zurita et al. [9] reported that bone marrow stromal cells (BMSCs) can realize neural transdifferentiation only by being co-cultured with Schwann cells (SCs) indirectly, and in our former studies we have confirmed that ADSCs could behave so too [10]. So ADSCs may be an ideal alternative cell source for SCs.
Although co-culture seems a simple and possible way to acquire sufficient SCs, we still need to know whether the neural induction efficiency of co-culture is as high as that of other strategies, such as induction by chemicals or chemicals combined with some growth factors. To investigate this problem, we adopted three protocols for neural induction of ADSCs, aiming to compare their neural induction efficiency.

Material and methods

All animal experiments described in this report were approved by the Ethical Guideline Committee for Animal Care of West China College of Medical Sciences, Sichuan University.
Isolation and culture of adipose-derived stem cells and Schwann cells
Adipose-derived stem cells were isolated from the subcutaneous adipose tissue of the inguinal region of adult Sprague-Dawley rats. The subcutaneous fat was carefully dissected and minced using sterile scissors. The cut tissue was then enzymatically dissociated for 60 min at 37°C using 0.1% (w/v) collagenase type I (Sigma, UK). The solution was passed through a 75-µm filter to remove undissociated tissue, neutralized by the addition of -Modified Eagle Medium (-MEM; Hyclone, USA) containing 20% (v/v) fetal bovine serum (FBS, Hyclone, USA) and centrifuged at 1000×g for 8 min. The stromal cell pellet was resuspended in -MEM containing 20% (v/v) FBS (Hyclone, USA) with 1% (v/v) penicillin/streptomycin solution. Cultures were maintained at subconfluent levels in a 37°C incubator with 5% CO2 and passaged with trypsin/EDTA (Sigma, UK) when required.
Schwann cells were first isolated from the bilateral sciatic nerves of 1-to-2-day-old Sprague-Dawley rat pups under aseptic conditions and placed into Hank’s balanced salt solution supplemented with 1% (v/v) penicillin/streptomycin. The epineurium and connective tissue were carefully removed on ice under a microscope, and the sciatic nerves were cut into 1 mm fragments, and then dissociated with 0.25% trypsin (Sigma, UK) and 0.1% collagenase (Sigma, UK) for 30 min at 37°C. The solution was neutralized by the addition of -Modified Eagle Medium (-MEM; Hyclone, USA) containing 20% (v/v) fetal bovine serum (FBS, Hyclone, USA) and centrifuged at 1000×g for 8 min. The cell pellet was resuspended in -MEM containing 20% (v/v) FBS (Hyclone, USA) with 1% (v/v) penicillin/streptomycin solution and cultured in a 37°C incubator with 5% CO2. After 24 h, G418 (Gibeco, Germany) was added to the culture to a final concentration of 100 mg/l for another 3 days. This was replaced with -MEM containing 20% FBS, and 3 days later the G418 treatment was repeated. Cells were then maintained in -MEM containing 20% (v/v) FBS (Hyclone, USA) with 2 µM forskolin (Sigma, UK), 20 ng/ml basic fibroblast growth factor (bFGF; PeproTech Ltd., UK) and 1% (v/v) penicillin/streptomycin solution and cultured in a 37°C incubator with 5% CO2. After the cultures reached confluency, they were rinsed three times with PBS and dissociated with 0.25% trypsin and 1 mM EDTA for 5 min at 37°C and subcultured at a density of 5 × 103 cells/cm². The culture medium was changed every 72 h.
Identification of adipose-derived stem cells and Schwann cells
For immunocytochemical assessment of stem cell markers, ADSCs at passage 2 were cultured on glass coverslips for 24 h and then fixed in 4% (w/v) paraformaldehyde for 15 min. Fixative was removed and cells were washed 3 × 2 min in PBS and permeabilised using 0.5% (v/v) Triton X-100 for 20 min, 37°C. The cells were washed 3 × 2 min in PBS and 5% (v/v) normal goat serum blocking solution (Sigma, UK) was added for 20 min at room temperature. Monoclonal STRO-1 antibody (Santa Cruz, CA, USA) was added and incubated at 4°C overnight. Cells were washed 3 × 5 min in PBS and goat anti-mouse FITC-labelled secondary antibody added for 1 h at room temperature before observation under a fluorescence microscope. CD90 and CD34 antibodies (Santa Cruz) were added and incubated at 4°C overnight. Sequentially, the coverslips were incubated with secondary biotinylated antibodies and horseradish peroxide conjugated streptavidin in order to detect the primary antibody. The peroxidase reaction was developed using 3,30-diaminobenzidine tetrahydro­chloride (DAB) as a chromogen, and the coverslips were counterstained with haematoxylin.
Schwann cells were identified in living cultures on the basis of cell soma and nuclear morphology, using phase contrast microscopy. In fixed cultures, they were identified by immunocytochemical labelling for S100 protein with the same procedure described above.
Inductive protocols for adipose-derived stem cells neural transdifferentiation
Growth medium was removed from sub-confluent ADSCs cultures at passage 2 and replaced with medium supplemented with 1 mM b-merca­ptoethanol (Sigma-Aldrich, UK) for 24 h. Then three different protocols were applied to induce ADSCs.
Protocol A
Cells were washed and then cultured in -MEM without FBS but supplemented with 2% dimethyl sulphoxide (DMSO) for 5 h.
Protocol B
Following Protocol A, cells were washed and medium replaced with differentiation medium; cell growth medium supplemented with 10 ng/ml neurotrophic factor (NGF; PeproTech Ltd., UK), 10 ng/ml basic fibroblast growth factor (bFGF; PeproTech Ltd., UK), 14 µM forskolin (Sigma, USA) and 10 ng/ml brain-derived neurotrophic factor (BDNF; PeproTech Ltd., UK). Cells were incubated for 2 weeks under these conditions with fresh medium added approximately every 72 h.
We named the protocol of co-culturing ADSCs and SCs as Protocol C. To co-culture ADSCs and SCs, we use Transwell culture dishes with polycarbonate membrane (Millipore, USA). The newest kind of porous membrane of the insert is optically transparent, and the pore size of membranes in our experiment is 1.0 µm. In our experimental conditions, the pore size is much smaller than the size of the ADSCs body (approximately 80-160 µm), which greatly inhibits the migration of ADSCs into the lower chamber. Schwann cells (6 × 104/cm², in 3 ml culture medium) were cultured in the well, and ADSCs (3 × 104 cells/cm²) were cultured on the permeable membrane support, with co-cultures maintained in -MEM containing 20% (v/v) FBS, so that both types of cells were exposed to the same culture media conditions, without cell contact. The culture medium was changed every 72 h. At 14 day after co-culture, the membranes were cut away from the insert, and mounted on the glass coverslips for study with immunocytochemistry techniques.
Immunocytochemistry
Differentiated ADSCs cultures with protocols A and B were trypsinised and replated on glass coverslips for immunostaining as above. The polycarbonate membrane with co-cultured ADSCs in Protocol C was cut at 14 days and mounted on glass coverslips for immunostaining too. Cells were incubated with mouse anti-glial fibrillary acidic protein (GFAP; Santa Cruz, USA), mouse anti-S100 (Santa Cruz, USA) and mouse anti-nestin (Santa Cruz, USA) overnight at 4°C. Sequentially, the coverslips were incubated with secondary biotinylated antibodies and horseradish peroxide conjugated streptavidin in order to detect the primary antibody. The peroxidase reaction was developed using 3,30-diaminobenzidine tetrahydro­chloride (DAB) as a chromogen, and the coverslips were counterstained with haematoxylin.
RNA isolation and reverse transcription-polymerase chain reaction
Total RNA was extracted from all of the specimens using the Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Complementary strand DNA synthesis and PCR amplification of the target message RNA were performed by TaKaRa RT-PCR kit (TaKaRa, Japan). PCR oligonucleotide primers and annealing temperature are shown in Table I. All of the primers were determined through established GenBank sequences and the amplification of GAPDH was used as a control for assessing PCR efficiency. The products were electrophoresed on 3% agarose gels, stained with Goldview and visualized with Quantity One software (BIO-RAD).
Real-time PCR analyses
To further investigate the RT-PCR results, real-time PCR analyses were conducted for the above three selected genes as shown in Table I. The same total RNA used for RT-PCR analyses was reverse-transcribed into single-stranded cDNA using the TaKaRa RT kit (TaKaRa, Japan) following the manufacturer’s protocol. Then a total sample volume of 20 µl, composed of 10 µl of SYBR Premix Ex TaqTM (2×), 0.4 µl of forward primer (10 µM), 0.4 µl of reverse primer (10 µM), 0.4 µl of ROX Reference Dye (50×), 2 µl of cDNA, and 6.8 µl of dH₂O, was used for the real-time PCR experiments. Each unknown sample was tested in triplicate. Real-time PCR analyses were carried out using Applied Biosystems 7300 Real-Time PCR System, with the following thermal cycle conditions: 95°C for 10 s; and 45 repeated cycles of 95°C for 5 s followed by 60°C for 31 s. At the end of the programme, a melting curve analysis was done. The former PCR products had been analysed using gel electrophoresis to ensure the absence of primer dimers and non-specific PCR product amplification. GAPDH was used as the housekeeping gene for the normalization of the gene expression data. Gene expression relative quantitative differences were analysed according to data derived from the standard curve.

Statistical analysis

To compare the gene expression relative quantity differences among sample groups A, B and C for genes S100, nestin and GFAP, one-way ANOVA was used for the analyses of real-time PCR gene expression data with software SPSS 11.5, then Student-Newman-Keuls test was adopted to draw the conclusions. P < 0.05 was considered significant.

Results

Immunocytochemical assessment of ADSCs stem cell markers shows positivity for STRO-1 and CD90, but negativity for CD34 (Figure 1A). The results prove the stem cell characterization of our cultured ADSCs and are consistent with the former studies [8, 11]. The morphology of Schwann cells under phase contrast microscopy is typical spindle elongated shape, and immunocytochemical assessment shows positivity for S100 (Figure 1B).
Immunostaining shows that all ADSCs after induction with protocols A, B and C show positivity in different degrees for the protein nestin, a neural progenitor marker, and proteins S-100 and GFAP, glial markers (Figure 2). To confirm immuno­cytochemistry results, RT-PCR was performed and the results give further support to them. Subsequently, we performed real-time-PCR to investigate the gene expression relative quantity differences of the above three genes among sample groups A, B and C. For S100, nestin and GFAP gene expression relative quantities, statistical analyses show no statistical differences between group B and C separately (p > 0.05), but B and C show significant statistical differences compared to A (p < 0.05), which means that the above three gene expression levels are higher in group B and C than those in group A, but almost the same between group B and C (Table II–IV). To make the results clear and direct, we use a bar graph to show the relative quantity differences among the three groups for genes S100, nestin and GFAP (Figure 3).

Discussion

In 2000, Woodbury et al. first induced BMSCs to differentiate into neural phenotype with b-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanisole as the neural induction media in vitro, expressing neuron-specific enolase, NeuN, neurofilament-M, tau, trKA and nestin [12]. However, those expressions cannot last longer than 6 days [12]. Some other authors questioned the above conclusion, suggesting that chemical neural induction results in cellular stress, leading to the physical contraction of cells into a neuron-like morphology [13, 14]. Thereafter, several studies reported that chemicals combining some growth factors, such as PDGF, NGF, bFGF, GGF and BDNF, can induce stable neural transdifferentiation of mesenchymal stem cells (MSCs) [15-20]. Recently, two papers proved that with the presence of certain growth factors, ADSCs could also be induced to differentiate into a Schwann cell phenotype in vitro [8, 11]. But this method is rather expensive and complicated, so Zurita performed direct co-culture of SCs and BMSCs and proved BMSCs neural transdifferentiation [21]. However, the possibility of cell fusion could not be excluded as a cause of apparent BMSCs neural transdifferentiation according to former studies [22-25]. Therefore, Zurita performed indirect co-culture of SCs and BMSCs with Transwell culture dishes, also proving BMSCs neural differentiation even if without intercellular contacts [9]. So we performed indirect co-culture of SCs and ADSCs with Transwell culture dishes. Both immunostaining and RT-PCR results confirmed ADSCs neural differentiation, and we have proved in our former research that S100, nestin and GFAP gene expression relative quantities of ADSCs could maintain a stable and high level at 14 day after being co-cultured with SCs. Meanwhile, we performed Protocol A, neural induction with chemicals only; and Protocol B, neural induction by chemicals combined with growth factors. Because our former studies proved that Protocol B was the most effective among several induction protocols with chemicals combined with growth factors, we chose Protocol B as judgement to co-culture in this experiment.
Real-time PCR results show that there are no statistical differences between sample group B and C for the above three gene expression relative quantities of differentiated ADSCs (p > 0.05), while both B and C have significant statistical differences with sample group A (p < 0.05). So we think that as a simple and economical method, co-culture is as effective as induction by chemicals combined with some growth factors when inducing ADSCs to transdifferentiate into neural-like cells or a Schwann cell phenotype, but induction only by chemicals is not as effective as them. We conclude that co-culturing ADSCs and SCs is an effective and practical way for ADSCs neural transdifferentiation, and thus may be a promising strategy to induce ADSCs neural transdifferentiation in vivo.

Acknowledgments

This study was supported by funds from the National Natural Science Foundation of China (NO.30772448).

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