J Mol Hist (2014) 45:707–714 DOI 10.1007/s10735-014-9594-z

ORIGINAL PAPER

The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells Xiao Liu • Zhengzheng Zhang • Xianliang Yan He Liu • Licai Zhang • Aiming Yao • Chengcheng Guo • Xiaoyun Liu • Tie Xu



Received: 15 May 2014 / Accepted: 26 August 2014 / Published online: 2 September 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK

Xiao Liu, Zhengzheng Zhang and Xianliang Yan have contributed equally to this work. X. Liu  A. Yao  C. Guo Emergency Center, The Affiliated Hospital of Xuzhou Medical College, First Aid and Relief Medical Department of Xuzhou Medical College, Xuzhou 221002, China Z. Zhang Obstetrics and Gynecology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China X. Yan Emergency Center, Qilu Hospital of Shandong University, Jinan 250012, China H. Liu  L. Zhang Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221004, China X. Liu The Central Laboratory, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China T. Xu (&) Emergency Center, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China e-mail: [email protected]

signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10 lg/l) and basic fibroblastic growth factor (bFGF, 10 lg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10 lM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20 ± 8.677 (positive ratio 0.2140 ± 0.0119) to 109.20 ± 8.430 (positive ratio 0.3193 ± 0.0161) per visual field in the presence of Y-27632, while GFAPpositive cell number increased from 96.30 ± 8.486 (positive ratio 0.18 ± 0.0152) to 107.50 ± 8.683 (positive ratio 0.27 ± 0.0115) (P \ 0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74 ± 0.05 vs. 1.03 ± 0.06; GFAP: 0.64 ± 0.08 vs. 0.97 ± 0.05, both P \ 0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases. Keywords ROCK  Neuron  Neuroglial cells  Neurodegenerative diseases Abbreviations BMSCs Bone-marrow mesenchymal stem cells EGF Epidermal growth factor bFGF Basic fibroblastic growth factor NSE Neuron-specific enolase GFAP Glial fibrillary acidic protein

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Introduction Multipotent stem cells have been isolated from numerous adult tissues, including the mammalian central nervous system (Estes et al. 2010; Li et al. 2008; Parekkadan and Milwid 2010; Ramiya et al. 2000). These adult stem cells circumvent many of the ethical concerns surrounding the use of fetal tissues to repair damaged tissues (Lo and Parham 2009), but technical problems in isolation, expansion, differentiation, and maintenance of genotype/phenotype stability have limited routine use of adult-derived stem cells in clinical medicine (Dolgin 2011; Hussein et al. 2011; Kim et al. 2010; Mayshar et al. 2010; Kocamaz et al. 2012; Wu et al. 2013; Sun et al. 2014). Derivation of neurons and glial cells from adult stem cells is a central challenge in modern medicine because most neurodegenerative diseases are incurable. In general, adult brain and other tissues contain relatively few stem cells, so obtaining sufficient numbers for transplantation requires efficient isolation (harvesting) and in vitro expansion. Furthermore, stem cells must differentiate into the appropriate neural phenotype, so the signaling mechanisms controlling differentiation must be identified. Bone marrow stem cells (BMSCs) can differentiate into a variety of distinct cell types, including neurons, under appropriate culture conditions (Kumar et al. 2012; Woodbury et al. 2000). Bone marrow also contains a population of stem cells derived from the neural crest, and both BMSCs and these neural crest-derived cells can differentiate into neurons (Wislet-Gendebien et al. 2012; Yamachika et al. 2012), underscoring the potential of bone marrow as a source of therapeutic cells to treat neurodegenerative diseases. Beneficial effects may be mediated by two distinct mechanisms. First, BMSC-derived neurons may replace those destroyed by disease. Second, BMSCs appear to suppress inflammation (Maggini et al. 2010; Ramasamy et al. 2008; Shi et al. 2010), which has led to speculation that these cells may benefit neurological disease such as multiple sclerosis characteristic by aberrant immune responses (Uccelli et al. 2011). Despite this potential, the efficiency of neuronal differentiation must be substantially improved. The Rho GTPases and downstream effectors such as Rho kinases regulate proliferation, cell adhesion, migration, and apoptosis in response to growth factor and integrin signaling by affecting cytoskeletal dynamics and protein expression or stability (David et al. 2012; Heasman and Ridley 2008; Ozdemir et al.2012). Moreover, The Rho kinase inhibitor appears to enhance the survival of stem cells from a variety of sources, thereby improving the yield of differentiated cells (Rizzino 2010), and inhibits the apoptosis of stem cells-derived neuronal progenitors

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following animal transplantation (Koyanagi et al. 2008). In this study, we examined if the Rho kinase inhibitor can also enhance the yield of neurons and glial cells from BMSCs in the presence of epidermal growth factor (EGF) and basic fibroblastic growth factors (bFGF).

Materials and methods Animals Sprague–Dawley male rats (4 weeks old, 100–120 g) were provided by the Animal Center of Xuzhou Medical College [license No. SYXK (Su) 2007–0037]. All procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of China, and approved by the Animal Ethical Committee of Xuzhou Medical College. Experiments were carried out in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. Isolation and expansion of BMSCs Rat BMSCs were isolated from whole bone marrow and purified by preferential adherence as described (Grassel et al. 2012; Li et al. 2013). The rat was anesthetized using 10 % chloral hydrate and sacrificed by cervical dislocation under sterile conditions. The femurs and tibias were exposed, excised, and the marrow cavities flushed into a Petri dish with PBS containing 0.25 % trypsin to yield a single cell suspension. After two centrifugations with PBS, whole bone marrow cells were resuspended in DMEM-F12 medium (GIBCO, USA) at 1 9 106 cells/ml, and then maintained at 37 °C under humidified 5 % CO2 in DMEMF12 medium supplemented with 10 % fetal bovine serum (ExCell Biology, South America), and 100 U/ml penicillin and streptomycin (Beyotime Institute of Biotechnology, Jiangsu, China) for 24 h. Hereafter, the medium was changed every 72 h. BMSCs was passaged at 80–90 % confluency. Flow cytometric analysis Identification of BMSCs at the third passage was determined by flow cytometry. Flow cytometric analysis was according to a previously reported method (Asumda and Chase 2011; Delorme et al. 2008; Kazemnejad et al.2009), with some modifications which will be mentioned. The human bone marrow BMSCs were detached from the tissue culture flasks at the third passage and counted. About 1 9 106 cells were incubated on ice for 30 min with goat

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serum, resuspended in phosophate-buffered saline (PBS) and pelleted by centrifugation for 4 min at 2,000 r/min. Subsequently, the cells were stained for 30 min at 4 °C with fluorescent isothiocyanate (FITC)-conjugated CD29(Biolegend, USA), CD34, CD44, CD45 (Boisynthesis biotechnology Co., LTD., Beijing, China), and CD90 (eBioscience, USA). An isotype control with FITC-labeled was included in each experiment and specific staining was measured from the cross point of the isotype with the specific antibody graph. The labeled cells were thoroughly washed with PBS and analyzed on a flow cytometer (FACS Calibur Becton, Dickinson, United States) using WinMidi software (Scripps Research Institute; San Diego, United States). Immunofluorescence staining At the third passage, BMSCs were subcultured in 6-well plates at 1 9 105 cells/well with Neurobasal medium contained 1 % B27 (Gibco, USA) for 3 weeks. Rho kinase inhibitor, Y-27632 (Sigma, USA) was dissolved and diluted with the same volume of physiological saline. The medium also contained 10 lg/l EGF and 10 lg/l bFGF (Peprotech, USA) with or without the Rho kinase inhibitor Y-27632 (10 lM). Treated BMSCs were washed in PBS and fixed with PBS plus 4 % formaldehyde, followed by freezing in 100 % methanol at -20 °C for 10 min. Fixed cells were blocked and permeabilized in 0.01 MPBS containing 5 % bovine serum albumin and 0.3 % Triton X-100 at 37 °C for 60 min. Fixed and blocked cells were then incubated in chicken polyclonal anti-NSE antibody (1:2,000, Thermo Scientific, USA) and rabbit polyclonal anti-GFAP antibody (1:5,000, Thermo Scientific) overnight at 4 °C. Immunolabeling was visualized using DyLightTM 488 donkey anti-chicken IgY (1:400, Jackson ImmunoResarch Laboratories, USA) and DyLightTM 549 goat anti-rabbit IgG (1:500, MultiSciences Biotech, China) for 2 h at room temperature in 0.01 M PBS. And the nuclear was stained with DAPI (5 mg/ml) at room temperature for 15 min. Cells were examined and a representative field of each experiment photographed at 409 magnification under the IX71 confocal fluorescent microscope (OLYMPUS, Japan). Quantification was done by counting numbers of the total number of cells and specific immunopositive cells in five randomly chosen optical fields, and the ratio of immunopositive cells to the total number of cells was recorded. Western blotting BMSCs at an initial density of 1 9 105 cells/well in 6-well plates were cultured for 3 weeks as described above, and then harvested in RIPA buffer (1 % NP-40, 50 mM, 150 mM NaCl; Beyotime). Proteins were quantified using a

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BCA Protein Assay Kit (Beyotime) and equal amounts per gel lane (67.21 g) separated by 10 % SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes (Millipore Corporation, Billerica, MA, USA). The non-specific sites on each blot were blocked with 5 % milk powder diluted in TBS with 0.05 % Tween 20 (TBST) for 3 h, and then incubated with chicken polyclonal anti-NSE antibody, rabbit polyclonal anti-GFAP antibody and rabbit polyclonal anti-GAPDH antibody (Cell Signaling Technology, USA) overnight at 4 °C, washed with TBST buffer, and then incubated in alkaline phosphatase (AKP)-labeled goat anti-chicken secondary (1:400, Jiahe Biotechnology, Shanghai, China) and AKP-labeled goat anti-rabbit secondary antibody (1:500, MultiSciences Biotech Co. China) for 1 h at room temperature. Antibody binding was detected using a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate assay kit (Sigma, St. Louis, MO). Immunoblot images were captured and digitized using a Millipore Digital Bioimaging System (Bedford, MA) and net band intensities quantified using Adobe Photoshop software (Adobe, San Jose, CA).The protein expressions were calculated as optical density (OD)target protein/ODGAPDH.

Statistical analysis Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Data were expressed as mean ± standard deviation (SD) of three independent experiments. Statistical significance was evaluated by Student’s t-tests. A P value of \0.05 was considered statistically significant (P \ 0.05).

Results The Rho kinase inhibitor Y-27632 promoted differentiated cell proliferation or survival Rat BMSCs were isolated from whole bone marrow and purified by preferential adherence. The BMSC nature of these cells was confirmed based on positivity for CD90, CD29, and CD44, and negativity for CD34 and CD45 by flow cytometry (Fig. 1). Final purity of BMSCs was about 85 % as indicated by CD90/CD29/CD34/CD44/CD45-positive status. After two passages, BMSCs were seeded into 6-well plates and incubated in differentiation medium (Neurobasal medium contained 1 % B27 supplemented with 10 % fetal bovine serum, 10 lg/l EGF, and 10 lg/l bFGF) with or without Y-27632 (10 lM). After 4 days, cell numbers were markedly higher in cultures containing Y-27632 (2.6 9 105 ± 0.2 9 105 vs. 1.2 9 105 ± 0.4 9 105) (Fig. 2), suggesting that Rho kinase inhibitor enhances differentiated cell proliferation or survival.

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Fig. 1 CD antigen expressions of bone marrow mesenchymal stem cells (BMSCs) at passage 3 by flow cytometry analysis. The BMSC nature of these cells was confirmed based on positivity for CD29 (a), CD90 (b) and CD44 (c), and negativity for CD34 (d) and CD45 (e). The isotype control is showed in green, and blue histogram indicates reactivity with the antibody indicated. (Color figure online)

Y-27632 promoted BMSC differentiation into neurons and glial cells Double immunofluorescence staining showed that some BMSCs expressed the neuronal marker NSE or the astroglial marker GFAP after 2 weeks culture (Fig. 3) and 3 weeks

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culture (Fig. 4). The numbers of NSE-positive and GFAPpositive cells (Fig. 4) were significantly higher in cultures treated with Y-27832 (NSE?: 109.20 ± 8.430 vs. 83.20 ± 8.677 per field of view, positive ratio: 0.3193 ± 0.0161 vs. 0.2140 ± 0.0119; GFAP?: 107.50 ± 8.683 vs. 96.30 ± 8.486 per field of view, positive ratio 0.2710

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Fig. 2 Effect of Rho kinase inhibitor Y-27632 on differentiated cell proliferation or survival. BMSCs at passage 3 were cultured for 4 days in the differentiation medium alone [Y-27632(-)] (a) or the differentiation medium supplemented with 10 lM Y-27632 [Y-27632(?)] (b) (Bar, 100 lm). c the differentiated cell number (9105). The data was shown as mean ± standard deviation (SD). *P \ 0.05 versus Y-27632 (-) group

Fig. 3 Effects of Y-27632 on expressions of NSE and GFAP protein in the BMSCs at the third passage cultured with Neurobasal medium contained 1 % B27, 10 lg/l EGF and 10 lg/l bFGF with or without Y-27632 (10 lM) for 2 weeks. The expressions of NSE (a, b) and GFAP (c, d) protein were revealed by fluorescence confocal microscopy. Panels b and d are magnification views from Panels a and c, respectively. Bar, 200 lm

± 0.0115 vs. 0.1822 ± 0.0152; P \ 0.05 for both). These results were confirmed by Western blot (Fig. 5), which revealed significantly higher total NSE and GFAP protein expression in cultures treated with Y-27632 (NSE: 0.74 ± 0.05 vs. 1.03 ± 0.06; GFAP:0.64 ± 0.08 vs. 0.97 ± 0.05, both P \ 0.01).

Discussion Woodbury and Sanchez first demonstrated the capacity of BMSCs to differentiate into neuron-like cells in vitro more than a decade ago (Woodbury et al. 2000). However, the efficiency of induction is still very low despite advances in

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Fig. 4 Effects of Y-27632 on numbler of NSE and GFAP-positive cells in the BMSCs at the third passage cultured with Neurobasal medium contained 1 % B27, 10 lg/l EGF and 10 lg/l bFGF with or without Y-27632 (10 lM) for 3 weeks. The numbler and ratio of NSE (a, b, e) and GFAP-positive (c, d, f) cells was revealed by

fluorescence confocal microscopy (Bar, 200 lm). The number and ratio of positive cells was expressed as mean ± SD per field of view from three independent BMSCs preparations. *P \ 0.05 versus Y-27632 (-) group. Green: DyLightTM 488; Orange: DyLightTM 549. (Color figure online)

culture protocols, greatly limiting any possibility of using these cells to treat neurological diseases. In this study, we demonstrate that the efficiency of differentiation can be increased by inhibition of Rho kinases. Indeed, these results add to a growing body of work suggesting that Rho kinase inhibitors hold significant potential in regenerative medicine (Rizzino 2010). Rho GTPases are monomeric G-proteins that act as key transducers of extracellular signals controlling actin cytoskeleton dynamics (David et al. 2012; Heasman and Ridley 2008; Rizzino 2010). Many signaling pathways activated by ligand-bound integrins, growth factor receptors, and heterotrimeric G-protein-coupled receptors (GPCRs) alter the activities of nucleotide exchange factors that in turn regulate the conversion of Rho GTPase from the inactive (GDP-bound) state to an active (GTPbound) state. Through effects on the neuronal cytoskeleton, Rho and downstream kinases are essential regulators of axonal migration, neuronal growth cone motility, and

dendritic morphogenesis (Linseman and Loucks 2008; Murakoshi et al. 2011). In addition, a number of recent studies have implicated Rho family GTPases in the regulation of neuronal survival and apoptosis (Loucks et al. 2006; Takakura et al. 2000). This current study demonstrated that the efficiency of BMSC differentiation into neural cells in the presence of EGF and bFGF was significantly higher in cultures treated with the Rho kinase inhibitor Y-27632. In principle, these effects could be mediated by enhanced differentiated cell proliferation or survival resulting from inhibition of ROCK-dependent apoptosis, and (or) changes in the probability of neural differentiation. Further experiments are required to distinguish among these possibilities. Moreover, it is not known if Rho kinase inhibitor can also enhance neural differentiation in vivo, although enhanced survival of BMSC-derived neural progenitors in vivo has been demonstrated (Koyanagi et al. 2008).

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Fig. 5 Effects of Y-27632 on NSE and GFAP protein expression levels in the BMSCs at the third passage cultured with Neurobasal medium contained 1 % B27, 10 lg/l EGF and 10 lg/l bFGF with or without Y-27632 (10 lM) for 3 weeks. NSE (a) and GFAP (b) Protein expressions were determined by Western blots. GAPDH expression was used as the protein loading control. The data was shown as mean ± SD. **P \ 0.01 versus Y-27632 (-) group Acknowledgments The authors thank the authorities of the Central Laboratory of the Affiliated Hospital of Xuzhou Medical College for providing research facilities for this work. Furthermore, this study was supported by a grant of the National Natural Science Foundation of China (No. 81371243). Conflicts of interest of interest.

The authors declare that they have no conflict

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The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells.

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical ...
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