Trichostatin A Induces Both Cell Division Arrest and Neural-linage Differentiation of the Mesenchymal Stem Cells Possibly by Triggering Wnt/β-Catenin Signaling

Research Article

J Stem Cell Res Transplant. 2014;1(2): 1009.

Trichostatin A Induces Both Cell Division Arrest and Neural-linage Differentiation of the Mesenchymal Stem Cells Possibly by Triggering Wnt/β-Catenin Signaling

Bei-Yu Chen1, Meng-Meng Liang2†, Mo-Han Dong3†, Jie-Qiong Zhang3, Jing-Jie Wang4, Mo Li1, Xi Wang2, Zhuo-Jing Luo1* and Liang-Wei Chen2*

1Department of Orthopedics, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, China

2Institute of Neurosciences, The Fourth Military Medical University, Xi’an 710032, China

3Department of Military & Prevention Medicine, Fourth Military Medical University, Xi’an 710032, China

4Department of Gastroenterology, Tangdou Hospital, The Fourth Military Medical University, Xi’an 710038, China

These authors have equal contribution to this work

*Corresponding author: Liang-Wei Chen, PhD, Institute of Neurosciences, The Fourth Military Medical University, Xi’an, 710032, P.R. China

*Corresponding author: Z.J. LUO, Department of Orthopedics, Xijing Hospital, The Fourth Military Medical University, China

Received: August 02, 2014; Accepted: September 27, 2014; Published: September 29, 2014


Introduction: The bone marrow-derived mesenchymal stem cells (BM-MSC), with active proliferation and pluripotent differentiation abilities, present an ideal source of cell transplant therapy. It remains obscure, but, how to promote their neural-linage differentiation efficiently and controlling moderate proliferation for clinical application in neurological diseases.

Methods: By in vitro culture of rat BM-MSC, regulation of a histone deacetylase inhibitor trichostatin A (TSA) in both cell proliferation and neural-linage cell commitment, and possible involvement of Wnt/β-catenin signaling pathway in TSA-inducing biological effect were examined in this study.

Results: By cell count and flowcytometry, TSA exhibited cell division or cycle arrest of BM-MSC at doses above 10ng/ml by increased percentage of G1/S phase and apoptotic cells. Immunocytochemistry and immunoblot revealed promotion effect of TSA on neuronal cell differentiation with appearance of neuron-like ones and increased nestin and Tuj-1 expression in BM-MSC treated with TSA at dose of 100 and 500ng/ml. Enhanced glial cell differentiation was also observed by upregulation of NG2, glial fibrillary acidic protein and CNPase expression in BM-MSC with TSA treatment dose-dependently. Furthermore, significant activation of Wnt/β-catenin signaling was detected in TSA-treated BM-MSC, while Wnt signaling inhibitor IWR1 impeded above TSA-induced effects on BM-MSC.

Conclusion: This study has provided new evidence that TSA could induce neural-linage differentiation and cell division arrest of BM-MSC dose-dependently by triggering Wnt/β-catenin signaling activation, suggesting that TSA may be applied as a candidate drug in manipulation of MSC therapy for the treatment of various neurological disorders.

Keywords: Bone marrow-derived mesenchymal stem cells; Trichostatin A; Histone deacetylase; Wnt/β-catenin signaling; Cell transplant therapy


BM-MSC: Bone Marrow-derived Mesenchymal Stem Cells; GFAP: Glial Fibrillary Acidic Protein; TSA: Trichostatin A


Growing studies have shown that the bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells (BM-MSC) may present an ideal source for tissue engineering or cell transplant therapies in human beings [1,2]. While wide heterogeneity was recognized in BM-MSC depending on their origins, the International Society for Cellular Therapy in 2006 proposed three minimal criteria for cultured human MSC definition [3]: i) when maintained in standard culture conditions, MSC must be plastic-adherent. ii) MSC must express CD73, CD90 and CD105, and lack expression of CD34, CD45, CD14 or CD11b, CD79 or CD19 and HLA-DR surface molecules, and iii) MSC must in vitro differentiate to osteoblasts, adipocytes and chondroblasts. Besides, BM-MSC can also be committed into cell lineages such as neural precursors, cardiomyocytes, liver cells, and possible other cell types. In fact, fast increasing of MSC knowledge and techniques has already led to some clinical trials for therapeutic manipulation of bone and cartilage diseases [4]. However, neural cell fate commitment or induced-differentiation mechanism of BM-MSC still remains a big clinical obstacle regarding their translational purpose for neurological disorders. Inspiringly, it is expected that effective production of MSC-derived functional neural-linage cells shall further extend their therapeutic application in various neurodegenerative diseases and nerve injury events [4].

Obviously, it is critical that efficiency of neural cell inducement and moderate control of cell proliferation are secured for establishment of a practical and safe MSC therapy [2,4]. Though a number of substances, neurotrophic or growth factors were found to exhibit certain promoting effect on neural cell differentiation, tumorigenicity of pluripotent stem cells still remains one most concern issue in cell transplantation [4]. Indeed, currently, there are still lacked of well-defined factors, drugs or chemicals with such both natural properties, i.e. neural cell fate commitment-promoting benefit and tumorigenicity-blocking effects in BM-MSC [4]. Recently, evidences indicated that certain epigenetic factors were actively involved in regulating differentiation of embryonic or adult stem cells [5-8]. For instance, Trichostatin A (TSA), a histone deacetylation inhibitor previously used for an anti-cancer agent, could sufficiently induce neuronal cell fate from the embryonic neural stem cells [9]. Moreover, only these embryonic neural stem cells with TSA treatment could definitely differentiate into functional neurons that were characterized with active potential and induced spikes, and underling mystery or inducing mechanism attracts a great attention [9]. In this study, interestingly, we demonstrated that TSA could effectively induce both neural-linage cell commitment and cell division or cycle arrest of rat BM-MSC, possibly through triggering Wnt/β-catenin signaling activation by using cell culture, flowcytometry, immunocytochemistry and western blot methods.

Materials and Methods

A total of twenty adult female Sprague-Dawley (SD) rats were used in the present study and supplied from the Animal Center of the Fourth Military Medical University of China. All animal experiments followed were carried out in according with the National Institute of Health guide for the care and use of Laboratory animals (NIH Publications No. 80-23) revised 1996, approved by the Committee of Animal Use for Research and Education of the Fourth Military Medical University, and all efforts were made to minimize animal suffering and reduce the number of animals used.

For cell culture preparation of BM-MSC, bone marrow mesenchymal cells were isolated from the femur bone of rats, which were sacrificed by animal decapitation. The bone marrow were washed out in PBS at room temperature, collected in glass tube and allowed to precipitation for a while. The cell suspension was seeded in dish in 10ml of MEM-α medium (Invitrogen) supplemented with 15% fetal bovine serum (Biosera, UK) and allowed to grow for 4-6 days until cell clones appeared. Cell pellets were collected, re-suspended and seeded in density 0.5-1×106 in T25 flasks (Corning) in MEM growth medium. After culture in a humidified 5% CO2/95% air incubator at 37°C about 5-7 days and cells grew fast in attached growth mode. They were dissociated by using accutase digestion and subjected to 5-6 passages each in about 3-5 days. After immunostaining confirmation of specific biomarkers (CD90+/CD45¯), BM-MSC cultured in 5-6 passages were used for following TSA experiments.

TSA treatment was performed to detect its influence on cell proliferation and cell differentiation of BM-MSC in vitro. Cell cultures of MSC were prepared in these groups, i.e. pre-treat, control, TSA-1ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, 500ng/ml, 1000ng/ml and 2000ng/ml working dilutions. TSA (Sigma-aldrich, St Louis, MO, 78552), which was dissolved in DMSO and diluted with PBS, was added in cell culture medium, incubated with DMEM/F12 (Gibco) medium supplemented with 2% B27 (Invitrogen) without bovine serum and continued for 3 days and 7 days. Cell survival and cell growth ware observed firstly under phase-contrast microscopy. MSC cell samples of control, TSA-10ng/ml, 100ng/ml and 500ng/ml group of 3d or 7d time-points were freshly collected for flowcytometry and western blot, or the cultured cells were terminated by fixation with 4% paraformaldehyde for 10 min for immunocytochemical detection of morphology and growth state of differentiated MSC.


Flowcytometry was performed to detect cell cycle, cell viability and apoptosis of cultured BM-MSC in a standard protocol. Fresh cell samples were collected and centrifuged for 5min and supernatants were discarded. Cell pellets were suspended and incubated with 1-2ml accutase for 10 min at 37°C. After gentle repeated beating, cell suspension was re-centrifugated for 5min at 1000r, and supernatant was abandoned. The cells were re-suspended in 1ml PBS supplemented with 2ml dehydrated alcohol, shaken for a moment, sealed and kept at 4°C overnight. The prepared cell samples of BM-MSC were measured under a flowcytometer for cell cycle, cell survival, necrosis and apoptosis. For data presentation, percentages of defined cells like G1, S, and G2 phase, survival, necrosis, early apoptosis or late apoptosis were quantified among control, TSA-10ng/ml, 100ng/ml and 500ng/ ml groups.


Immunofluorescent staining was performed to demonstrate neural cell markers and Wnt/β-catenin signaling molecules in cultured BM-MSC. After culture for 3d and 7d in 24-well plates, cells were processed for immunocytochemical staining protocol. Briefly, cells were incubated with 10% donkey serum-containing blocking solution for 30min at room temperature, and followed by incubation of primary antibody solution containing 10% donkey serum, 0.3% triton X-100 in PBS at 4°C for 24h, i.e. rabbit anti-nestin (Sigma-aldrich, 1:1000), rabbit anti-β-tubulin III (Tuj-1, Sigma-aldrich, 1:500), rabbit anti-NG2 (Sigma-aldrich, 1:800), mouse anti-glial fibrillary acidic protein (GFAP, Dark, 1:4000) and rabbit anti-CNPase (Sigma-aldrich, 1:500), respectively. After three washes with PBS, cells were followed with incubation of Alexa Fluor-488, or Alexa Fluor-594 conjugated donkey anti-mouse or rabbit IgG (1:500, Molecular Probes) for 4h at room temperature. Finally, nuclear counterstaining with DAPI (Sigma-aldrich, D9564) for 10min was performed to visualize total cultured MSC. After PBS wash, cell samples were mounted with Fluorescence-preserving VECTASHIELD Mounting medium (Vector, H-1000) and examined under epifluorescent microscope and laser scanning confocal microscope (LSCM, Olympus, FV-1000). For control staining experiment, the primary antibody was substituted with normal mouse or rabbit serum. Immunoreactive cells were not detected in controls (data not shown), and specificity of antibodies used was also provided by manufacture’ data sheet.

Western blot

Western blot was performed to quantify expression of several neural cell markers and Wnt/β-catenin signaling molecules in cell culture in a standard protocol. Briefly, protein extracts were prepared from cultured MSC. Fresh cell samples were collected and homogenized at 4°C in 5 volumes of ice-cold lysis buffer [50mM Tris (pH 7.4), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Complete, Roche Diagnostics)], and centrifugation of homogenates for 10 min (12000g) was then performed. After protein concentration was determined by the BCA assay (Beyotime, China), supernatant was mixed with four volumes of protein loading buffer, boiled for 5min at 99°C and stored at 4°C. Total protein (20-30 μg per loading passage) was loaded using loading buffer commercial for electrophoresis on 10-12% SDS-PAGE gels and transferred to the nitrocellulose membranes (Bio-Rad). After these membranes were blocked with 5% skimmed milk (Wandashan, China) in Tris-buffered saline containing 0.05% Tween 20, immunoblotting was performed with primary antibody to nestin, Tuj-1, NG2, CNPase, GFAP, Wnt1, Wnt3a, Wnt5a, β-catenin or phosphor-β-catenin respectively, and followed by secondary antibody incubation. Mouse anti-Wnt1 (Sigma-aldrich, 1:1000), rabbit anti- Wnt3a (abcam, 1:500), mouse anti-Wnt5a (Sigma-aldrich,1:1000), rabbit anti-β-catenin (Sigma-aldrich, 1:1000), rabbit anti-phosphor- β-catenin (Sigma-aldrich, 1:1000), and rabbit anti-β-actin (Sigma-aldrich, 1:800) were used. Visualization and detection of immunoblot bands was performed by an enhanced chemiluminescence (ECL) detection system (CWBio, China), and images were digitally acquired using ChemiScope System (Clinx, China). By using β-actin as internal control, quantitative analysis was carried out and data were presented in ratios.

Wnt signaling inhibition experiment

To confirm role of Wnt/β-catenin signaling pathway in TSA-induced cell differentiation of MSC, a blocking experiment was carried out in cell culture by administration of a specific inhibitor IWR-1, which can induce stabilization of APC/Axin2/GSK3β complex and decrease of cytoplamic free β-catenin level through a direct interaction and works as Wnt signaling inhibitor [10,11]. In TSA+IWR1 group, IWR-1 (Sigma-aldrich, I0161) was dissolved in saline and added in culture medium in 10 μM at 10min before TSA treatment (TSA-100ng/ml plus IWR1), and saline group was used as controls. After maintaining culture for 3d and 7d, differentiation cell markers and changes were examined in BM-MSC of control, TSA-10, 100, 500ng/ml, and TSA-100ng/ml+IWR1-10 μM group by western blot and immunocytochemistry.

Statistic analysis

For data analysis of flowcytometry, immunocytochemistry and western blot, total MSC cells, immunopositive cells and immunoblot density of nestin, Tuj-1, NG2, GFAP, CNPase, Wnt1, Wnt3a, Wnt5a, phosphor-β-catenin and β-catenin were counted or measured and given as mean ± S.E.M. (n=3-5, independent experiments). Differences between means were analyzed by one-way ANOVA (SPSS 18.0) with different markers or signaling molecules as independent factors. When ANOVA showed significant difference among means, the pair-wise comparisons between means were also performed by post hoc testing, and the significance level was set at a P value of 0.05 for all analyses.


Cell division arrest and cell apoptosis of BM-MSC induced by TSA treatment

Influence of TSA treatment on cell survival, apoptosis and cell cycle were first observed in cell culture after BM-MSC in high-purity and uniform were established through 5-6 passages and characterized with specific BM-MSC biomarkers as reported previously [3,4]. Cell morphology and cell density of BM-MSC were observed and compared among these groups, i.e. pre-treat, control, TSA-1ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, 500ng/ml, 1000ng/ml and 2000ng/ml (Figure 1A). Obvious changes in morphology and cell numbers of BM-MSC dose-dependently occurred at doses of above TSA-50ng/ ml working dilution. Cell count data revealed that BM-MSC in single unit field decreased after treatment of TSA dose-dependently and significantly in comparison with that of control (Figure 1B).