Roles of activated astrocytes in bone marrow stromal cell proliferation and differentiation

Neuroscience 160 (2009) 319 –329

ROLES OF ACTIVATED ASTROCYTES IN BONE MARROW STROMAL CELL PROLIFERATION AND DIFFERENTIATION F.-W. WANG,a D.-Y. JIA,a Z.-H. DU,a,b J. FU,a S.-D. ZHAO,a S.-M. LIU,a Y.-M. ZHANG,a E.-A. LINGc AND A.-J. HAOa*

Key words: astrocytes, inflammation, proliferation, neural differentiation, bone marrow stromal cells, interleukin-6.

a Key Laboratory of the Ministry of Education for Experimental Teratology, Department of Histology and Embryology, Shandong University School of Medicine, No.44, Wenhua Xi Road, Jinan, Shandong 250012, PR China

Adult neural tissue has limited regenerative capability. In view of this, stem cell– based therapy, in particular, the transplantation of bone marrow stromal cells (BMSCs) to repair various central nervous system (CNS) injuries and neurodegenerative diseases has been the focus of many studies in recent years. There is ample evidence to support that BMSCs transplantation could significantly promote the functional recovery in CNS disorders (Dezawa et al., 2004; Hofstetter et al., 2002; Kurozumi et al., 2005; Ohta et al., 2004). However, some studies have argued that little or merely modest functional recovery was achieved after BMSCs transplantation (Yoshihara et al., 2006). The discrepancy may be attributed to the different microenvironment in which the cells reside. It is generally agreed that local microenvironment (such as glial cells and cytokines) is the determinant of fate specification for transplanted BMSCs (Constantinescu, 2000; Seki, 2003; Dengke et al., 2008; Moyse et al., 2008). It is well documented that astrocytes, a major residential glial cells in the CNS, constitute the major component of local CNS microenvironment. Previous results have shown that any injury or disease to the CNS would elicit a characteristic neuroinflammatory reaction in the CNS (Wang and Shuaib, 2002; Lucas et al., 2006; Schwab and McGeer, 2008). Astrocytes, one of the key players mediating the inflammatory response, were markedly activated and secreted a plethora of pro- and anti-inflammatory cytokines, chemokines and trophic factors (Lieberman et al., 1989; Ridet et al., 1997; Lau and Yu, 2001; Lafon-Cazal et al., 2003). These factors constituted local special microenvironment and played important roles in neuronal survival, maturation, synaptogenesis and neurogenesis in the CNS (Trendelenburg and Dirnagl, 2005; Emsley et al., 2004; Mauch et al., 2001; Ullian et al., 2001; Song et al., 2002; Wagner et al., 1999). Recently, there is growing evidence that astrocytes can also induce embryonic stem cells, hematopoietic stem cells, BMSCs and other stem cells to differentiate into neural cells (Nakayama et al., 2003; Hao et al., 2003; Joannides et al., 2003, 2004). In the injured or diseased CNS, enhanced regeneration and differentiation of neural stem/progenitor cells (NSPCs), as well as marked activation of astrocytes were observed (Liu et al., 1998; Jin et al., 2004; Horky et al., 2006; Das and Basu, 2008; Eddleston and Mucke, 1993; Pekny and Nilsson, 2005) suggesting that activated astrocytes may contribute significantly to neurogenesis of NSPCs in the inflammatory microenvironment. On the other hand, it remains uncertain whether inflammation-activated astro-

b Reproductive Medicine and Infertility Center, Qingdao Women and Children’s Medical Center, Qingdao, PR China c Department of Anatomy, Yong Loo Lin School of Medicine, Block MD10, 4 Medical Drive, National University of Singapore, Singapore, 117597

Abstract—Local microenvironment plays an important role in determining the fate choice of stem cells in the central nervous system (CNS). Astrocytes, a major component of local microenvironment in the CNS, have been demonstrated to influence the proliferation and neural differentiation of stem cells including neural stem/progenitor cells, embryonic stem cells and bone marrow stromal cells (BMSCs). However, it has remained to be ascertained if inflammation-activated astrocytes can affect the behavior of BMSCs. To this end, astrocyte-conditioned medium (ACM) was prepared in this study for treatment of BMSCs. The ACM derived from Wistar rat astrocytes stimulated by lipopolysaccharide for 12, 36 or 72 h, respectively, served as inflammatory ACM (12 h ACM, 36 h ACM and 72 h ACM), while that from unstimulated astrocytes was used as normal control astrocyte-conditioned medium (N-ACM). The results showed that the proliferation and neural differentiation of BMSCs grown in inflammatory ACM were significantly increased compared with those grown in N-ACM. The efficiency of BMSCs exposed to 36 h ACM was significantly greater than that of those exposed to 12 or 72 h ACM. Following neutralization of interleukin-6 (IL-6) of the ACM, both the proliferation and astrocytic differentiation of BMSCs were decreased; on the other hand, the neuronal differentiation was significantly increased. The present findings suggest that inflammation-activated astrocytes can facilitate the proliferation and neural differentiation of BMSCs and activated astrocytes at different phase after CNS injuries might have distinct effects on BMSCs. Moreover, astrocytederived IL-6 participates in the proliferation and neural differentiation of BMSCs. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹86-531-88382047; fax: ⫹86-531-88382052. E-mail address: [email protected] (A.-J. Hao). Abbreviations: ACM, astrocyte-conditioned medium; BMP, bone morphogenetic protein; BMSC, bone marrow stromal cell; DAPI, 4,6diamino-2-phenylindole; DMEM, Dulbecco’s modified Eagle medium; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyannate; GFAP, glial fibrillary acidic protein; IL-6, interleukin-6; LPS, lipopolysaccharide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; N-ACM, normal astrocyte-conditioned medium; NSE, neuron specific enolase; NSPC, neural stem/progenitor cell; PLL, poly-l-lysine; RT-PCR, reverse transcription–polymerase chain reaction; STAT, signal transducers and activators of transcription.

0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.02.068

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cytes have a beneficial or detrimental effect on BMSCs. Although recent studies have demonstrated that the factors produced by astrocytes play key roles in modulating the fate specification of stem cells (Song et al., 2002; Nakayama et al., 2003; Hao et al., 2003; Joannides et al., 2003, 2004), the detailed molecules involved in the process are still unknown. In this connection, attention has been drawn to interleukin-6 (IL-6), a pleiotropic inflammatory factor, because its expression was significantly increased in various CNS disorders (Nakamura et al., 2005; Acalovschi et al., 2003; Yu and Lau, 2000; Loddick et al., 1998). Furthermore, some studies have extended that IL-6 can affect the proliferation or neural differentiation of NSPCs and other cells (Taga and Fukuda, 2005; Bernad et al., 1994; Mukaino et al., 2008). However, it has remained to be elucidated whether IL-6 participates and plays an important role in astrocyte-regulated proliferation and neural differentiation of BMSCs. This study therefore sought to determine the effects of inflammation-activated astrocytes and their secretion of IL-6 on the proliferation and neural differentiation of BMSCs in vitro.

EXPERIMENTAL PROCEDURES In the handling and care of all animals, the International Guiding Principles for Animal Research, as stipulated by the World Health Organization (1985) and as adopted by the Laboratory Animal Center, Shandong University were followed. During the study, the number of animals used and their suffering was minimized.

Primary astrocyte culture and conditioned medium collection

science, USA), respectively, and then detected by flow cytometry (FC500, Beckman Coulter, USA). Corresponding mouse IgG isotypical antibodies served as the control.

Cell viability assay by MTT Cell viability was determined by the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) assay. BMSCs were plated into 96-well culture plates (Corning Costar, USA) at density of 1⫻104 cells/ml with 200 ␮l culture medium per well. When reaching 60% confluence, the cells were treated with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, for 2 days. Then, 20 ␮L MTT solution (5 mg/ml) was added to each well and incubated at 37 °C for 4 h. The medium was aspirated and 200 ␮l dimethyl sulfoxide was added. The absorbance value was measured in a multiwell spectrophotometer (Bio-Rad, USA) at 490 nm. Four independent experiments were conducted.

Proliferation analysis by BrdU labeling and immunostaining BMSCs were plated on coverslips pre-coated with poly-l-lysine (PLL, Sigma-Aldrich) in six-well plates (Corning Costar, USA) at 3⫻104 cells/ml. When reaching 60% confluence, the cells were treated with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, for 2 days in the absence or presence of 20 ng/ml anti-IL-6 antibody (Chemicon, USA). BrdU (10 ␮g/ml, Sigma-Aldrich, USA) was then added to each medium for 6 h and the cells were fixed with 4% paraformaldehyde, denatured by 2 N HCl for 30 min at 37 °C, and processed for immunocytochemistry. The cells were counterstained by 4,6-diamino-2-phenylindole (DAPI, Sigma-Aldrich, USA). The percentage of BrdU-positive cells over total DAPI cells was determined by randomly counting 10 nonoverlapping microscopic fields for each coverslip in four separate experiments.

Analysis of cell cycle distribution by flow cytometry BMSCs were plated in culture flasks at 1⫻105 cells/ml. When reaching 60% confluence, the cells were treated with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, for 2 days. Then the cells were collected and fixed by cold 70% ethanol, stained by propidium iodide solution (containing RNase) and detected by flow cytometry (FC500, Beckman Coulter, USA) to analyze the fractions of cell population in S-phase. Three independent experiments were conducted.

Primary astrocytes were prepared from 1-day-old Wistar rats as described before (McCarthy et al., 1980) with modifications. Briefly, the cortical tissues freed of meninges and blood vessels were mechanically dissociated and the cell suspension was seeded at density of 2⫻106 cells/ml in Dulbecco’s modified Eagle medium (DMEM, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA), 2 mM l-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, USA). When the culture was reaching confluence, most microglia and oligodendrocytes were removed by an orbital shaker, and then the cells were replated. Astrocytes of passage 3 were treated with fresh DMEM plus 1 ␮g/ml lipopolysaccharide (LPS, Sigma-Aldrich, USA) for 12, 36, and 72 h. The supernatant was then collected to obtain the inflammatory astrocyte-conditioned medium (ACM) at the above time points (12 h ACM, 36 h ACM, 72 h ACM). Meanwhile, the supernatant from astrocytes treated with fresh DMEM alone for 36 h was collected to be used as normal astrocyte-conditioned medium (N-ACM). The ACMs were collected, centrifuged, filtered and stored at ⫺80 °C until tested or later use.

BMSCs at passage 3 were plated either on PLL-coated coverslips in six-well plates or in 25 cm2 culture flasks (Corning, USA). When reaching 60% confluence, the cells were first treated with preinduction medium (DMEM plus 20% FBS) for 24 h. Following this, the medium were replaced by DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, for 4 days with or without 20 ng/ml anti-IL-6 antibody (Abcam, USA). After induction, the cells were processed for immunostaining or reverse transcription-polymerase chain reaction (RT-PCR) analysis.

Rat BMSCs culture

Immunocytochemistry

Primary BMSCs were prepared following the described method of Pittenger et al. (1999) with some modifications. Briefly, the tibias and femurs of 150 g Wistar rats were dissected out and the marrow was flushed out. The cell suspension was plated in DMEM plus 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin and nonadherent cells were removed by replacing the medium every 3 days. After 10 days of incubation, the cell culture was trypsinized and replated. At passage 3, BMSCs were incubated with fluorescein isothiocyannate (FITC)-labeled mouse monoclonal antirat CD29 and CD90 antibodies (eBio-

The induced BMSCs were fixed with 4% paraformaldehyde and permeabilized in 0.3% Triton X-100. After this, the cells were incubated with polyclonal anti–neuron specific enolase (NSE) antibody (1:100, Upstate, USA) or monoclonal anti– glial fibrillary acidic protein (GFAP) antibody (1:600, Chemicon, USA), respectively, overnight at 4 °C. Tetrametrylrhodarnine isothiocyante (TRITC)-conjugated antirabbit IgG and FITC-conjugated antimouse IgG (Sigma-Aldrich, USA) were used as secondary antibodies. The cells were counterstained by DAPI and the proportion of NSE and GFAP positive cells was obtained, respectively.

Neural differentiation of BMSCs

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Reverse transcription–polymerase chain reaction (RT-PCR) Total RNA was extracted from induced cell cultures using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA concentration was determined by a spectrophotometer at 260 nm. Identical amounts of RNA were reverse transcribed into cDNA, which was subsequently amplified by PCR with specific primers. NSE primer: forward, 5=-GGTCCAAGTTCACAGCCAAT-3=; reverse, 5=-GTCGGGACAGCAAGAAAGAG-3=, 94 °C, 1 min, 55 °C, 55 s, 72 °C, 90 s, 30 cycles. GFAP primer: forward,5=-TCTGCCCAGTGAGTAAAGGTGA-3=; reverse, 5=-GGTGTGGAGTGCCTTCGTATT-3=, 94 °C, 45 s, 58 °C, 30 s, 72 °C, 45 s, 30 cycles. IL-6 primer: forward, 5=-GAGAAAAGAGTTGTGCAATGGC-3=; reverse, 5=-ACTAGGTTTGCCGAGTAGACC-3=, 94 °C, 30 s, 62 °C, 40 s, 68 °C, 40 s, 40 cycles. ␤-Actin primer: forward, 5=-GTGGGGCGCCCCAGGCACCA-3=; reverse, 5=-CTTCCTTAATGTCACGCACGATTTC-3=, 94 °C, 45 s, 58 °C, 30 s, 72 °C, 45 s, 30 cycles. PCR products separated on a 1.5% agarose/TAE gel were visualized by staining with ethidium bromide. The densitometric analysis of the data was normalized to ␤-actin.

Western blotting analysis Cells were rinsed with cold phosphate buffered saline and lysed in cold lysis buffer containing 10 mM Tris–HCl, pH 8.0, 240 mM

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NaCl, 5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1 mM sodium vanadate, and 1 g/ml of leupeptin, pepstatin, aprotinin. Cell lysates were incubated at 4 °C for 20 min. The sample was centrifuged at 12,000 rpm for 10 min at 4 °C, then the supernatant was collected and protein content was assayed colorimetrically. Ten micrograms of total proteins was loaded onto a 4%–20% gradient polyacrylamide gel, electrophoretically transferred to polyvinylidene difluoride membrane and probed with rabbit anti-NSE antibodies (Upstate, USA) and mouse anti-GFAP antibodies (Chemicon, USA), respectively. Monoclonal anti-␤-actin (Sigma-Aldrich, USA) was used as an internal control. Secondary antibodies were horseradish peroxidase conjugated to either goat antirabbit IgG or antimouse IgG (Sigma-Aldrich, USA). The membranes were developed using an ECL detection system (Pierce, Rockford, IL, USA). The intensity of bands was determined using the Image-Pro Plus 6.0 software.

Analysis of IL-6 expression by ELISA The procedures were carried out according to the description of an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, # R6000B). Briefly, serial dilutions of protein standards and ACM samples were added to 96-well ELISA plates, then biotinylated anti-IL-6 antibody was added. After rinsing several times with wash buffer, the prepared solution of avidin, horseradish peroxi-

Fig. 1. Characterization of BMSC cultures. (A) Primary BMSCs at about 7th day formed obvious cell colonies after initial plating (arrow indicates the cell colonies). (B) Nearly all the cells at passage 3 were elongated and bipolar and have a fibroblastic appearance. Scale bar⫽100 ␮m. (C, D) The fluorescence-activated cell sorting (FACS) demonstrate that the majority of the BMSCs at passage 3 are positive for CD29 (C) and CD90 (D).

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dase (HRP)-conjugated complex was added followed by addition of substrate solution. Finally, the reaction was stopped with the stopping solution and the optical density was detected at 450 nm. Three independent experiments were conducted.

Statistical analysis All the results were expressed as mean⫾SD. Statistical analysis of data was done by Student’s t-test or one-way analysis of variance (ANOVA). P⬍0.05 was considered to be statistically significant.

RESULTS Characterization of rat BMSC cultures BMSCs could be isolated by their adherence to the culture flasks. The primary cultures developed to form many cell

colonies at about 7th day after initial plating (Fig. 1A). Beginning at passage 3, BMSCs became relatively homogeneous in appearance. The majority of cells appeared to be more spindle-shaped or fibroblastic when reaching confluence (Fig. 1B). Fluorescent cell sorting at passage 3 demonstrated that nearly all the cells were positive for CD29 (Fig. 1C) or CD90 (Fig. 1D). Effects of ACM at different inflammatory states on the proliferation of BMSCs To examine the effects of different ACM on the proliferation of BMSCs, the analysis of MTT, BrdU incorporation and cell cycle detection was performed. First, MTT results (Fig. 2A) showed that ACM obviously improved the cell viability

Fig. 2. ACM promotes proliferation of BMSCs. Cells were treated with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, for 2 days and processed for detection of cell proliferation. (A) MTT assay reveals the cell viability. Each bar represents the mean⫾SD (n⫽4). (B) BrdU incorporation analysis shows the representative immunofluorescence images and quantification of BrdU-positive BMSCs (expressed as a percentage of DAPIpositive cells). Data were presented as mean⫾SD (n⫽4). BrdU, red; DAPI, blue. Scale bar⫽100 ␮m. (C) Quantification of the proportion of BMSCs populations at S-phase in different groups detected by FACS. Data are presented as mean⫾SD (n⫽3). * P⬍0.01, vs. DMEM; ** P⬍0.05, vs. N-ACM; # P⬍0.05, vs. 12 h and 72 h ACM; ⽧ P⬍0.05, vs. 12 h ACM. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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Fig. 3. ACM promotes neuronal differentiation of BMSCs. Cells were cultured with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, for 4 days and were stained with anti-NSE (red) and counterstained with DAPI (blue). (A) showing the representative immunofluorescence images of NSE-positive cells, Scale bar⫽100 ␮m. Quantification of NSE-positive cells over the total BMSCs was also shown in (A). Data were presented as mean⫾SD (n⫽4). (B) Western blotting analysis shows the protein expression of NSE in different induced BMSCs. ␤-Actin serves as an internal control. * P⬍0.01, vs. DMEM; ** P⬍0.01, vs. N-ACM; # P⬍0.05, vs. 12 and 72 h ACM. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

of BMSCs compared with DMEM alone (P⬍0.01). Furthermore, inflammatory ACM significantly increased cell viability of BMSCs (12 h, 36 h, 72 h ACM was 0.30⫾0.04, 0.40⫾0.05, 0.35⫾0.03, respectively) in comparison with N-ACM (0.26⫾0.02, P⬍0.05). Besides, the viability of cells treated with 36 h ACM was evidently higher than that treated with 12 or 72 h ACM, respectively (P⬍0.05). ACM also significantly increased the percentage of BrdUpositive cells in BMSCs compared with DMEM alone (P⬍ 0.01). The ratio of BrdU-positive cells in BMSCs treated with inflammatory ACM (12 h, 36 h, 72 h ACM was 24.84⫾ 1.59%, 31.92⫾2.21%, 27.68⫾2.07%, respectively) was markedly enhanced in comparison with N-ACM (21.24%⫾1.67%, P⬍0.05) (Fig. 2B). The proportion of BrdU-positive cells in BMSCs exposed to 36 h ACM was significantly higher than that exposed to 12 or 72 h ACM (P⬍0.05). Finally, the results of fluorescence activated cell sorting (FACS) (Fig. 2C) showed that inflammatory ACM (12 h, 36 h, 72 h ACM was 14.32⫾2.63%, 20.31⫾2.87%, 18.45⫾2.17%, respectively) also prominently increased the percentage of cells in S-phase of BMSCs compared with N-ACM (11.47%⫾1.58%, P⬍0.05). Furthermore, the proportion of cell populations in S-phase of BMSCs treated with 36 h ACM or 72 h ACM was markedly higher than that treated with 12 h ACM (P⬍0.05).

Effects of ACM at different inflammatory states on the neuronal differentiation of BMSCs To investigate the effects of different ACM on neuronal differentiation of BMSCs, the expression of NSE was tested using immunocytochemistry and Western blotting methods, respectively, (Fig. 3). The results of immunostaining showed that the percentage of NSE-positive cells in BMSCs treated with ACM was significantly higher than that of DMEM treated cells (P⬍0.01, Fig. 3A). Furthermore, compared with N-ACM (8.7%⫾1.6%), the proportion of NSEpositive cells in BMSCs treated with inflammatory ACM (12 h, 36 h, 72 h ACM was 15.1⫾1.8%, 21.3⫾2.4%, 12.6⫾1.5%, respectively) was markedly elevated (P⬍0.01). Besides, the effects of 36 h ACM on the neuronal differentiation of BMSCs were evidently greater than that of 12 or 72 h ACM (P⬍0.05). The results of Western blotting showed that the expression change of NSE protein was consistent with the immunostaining results (Fig. 3B). Effects of ACM at different inflammatory states on the astrocytic differentiation of BMSCs To explore the influences of different ACM on astrocytic differentiation of BMSCs, the expression of specific astrocytic protein, namely, GFAP, was tested using immunocy-

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Fig. 4. ACM promotes astrocytic differentiation of BMSCs. Cells were cultured with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, for 4 days and were stained with anti-GFAP (green) and counterstained with DAPI (blue). (A) The representative immunofluorescence images of GFAP-positive cells. Scale bar⫽50 ␮m. Quantification of GFAP-positive cells over the total BMSCs was also shown in (A). Data were presented as mean⫾SD (n⫽4). (B) Western blotting analysis shows the protein expression of GFAP in different induced BMSCs. ␤-Actin serves as an internal control. * P⬍0.01, vs. DMEM; ** P⬍0.01, vs. N-ACM. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

tochemistry and Western blotting methods, respectively (Fig. 4). As shown in Fig. 4A, the proportion of GFAPpositive cells in BMSCs treated with ACM was significantly increased compared with DMEM (P⬍0.01). Moreover, the ratio of GFAP-positive cells in BMSCs grown in inflammatory ACM (12 h, 36 h, 72 h ACM was 12.4⫾2.1%, 14.7⫾1.6%, 11.3⫾1.4%, respectively) was significantly higher than that grown in N-ACM (7.9%⫾1.7%, P⬍0.05). However, there was no obvious expression change in GFAP immunostaining among 12 h, 36 h, and 72 h ACM (P⬎0.05). In addition, the results of Western blotting showed that the expression change in GFAP protein in induced BMSCs was in line with the results of the immunocytochemistry (Fig. 4B).

normal astrocytes. Besides, the expression of IL-6 mRNA in 24 h 36 h and 48 h ACM was also significantly greater than that of 12 or 72 h ACM, respectively. IL-6 affects ACM-induced proliferation of BMSCs To determine whether IL-6 participates in the proliferation of BMSCs, anti-IL-6 antibody was added to DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively. The results showed that after blocking IL-6, the percentage of BrdU-positive cells was significantly decreased compared with the control group (Fig. 6). In particular, in the 36 and 72 h ACM groups, respectively, the reduction was significant (P⬍0.05).

Analysis of IL-6 expression The results of ELISA (Fig. 5A) demonstrated that the secretion of IL-6 from LPS-stimulated astrocytes was significantly increased compared with that from normal astrocytes (P⬍0.05). In addition, the level of IL-6 in 24 h ACM (1704⫾232 pg/ml), 36 h ACM (1859⫾338 pg/ml) and 48 h ACM (1584⫾286 pg/ml) was much greater than that of 12 h ACM (1180⫾216 pg/ml) or 72 h ACM (784⫾315 pg/ml), respectively. Furthermore, RT-PCR analysis (Fig. 5B) also showed astrocytes stimulated by LPS obviously increased the expression of IL-6 mRNA compared with

IL-6 affects ACM-induced neuronal differentiation of BMSCs Anti-IL-6 antibody was added to DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, to investigate the effects of IL-6 on the neuronal differentiation of BMSCs. The results showed that after blocking IL-6, the percentage of NSE-positive cells significantly increased compared with the corresponding control group (Fig. 7A). Meanwhile, the increase in NSE mRNA expression was also observed after neutralization of IL-6 (Fig. 7B). Moreover, the differ-

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Fig. 5. (A) Results of ELISA showing the expression of IL-6 released from unstimulated astrocytes or from astrocytes stimulated by LPS (1 ␮g/ml) for 12 h, 24 h, 36 h, 48 h, or 72 h, respectively. Data were presented as mean⫾SD (n⫽3). (B) RT-PCR analysis showing the expression change of IL-6 mRNA in astrocytes. ␤-Actin serves as the internal control. * P⬍0.05, vs. N-ACM; ** P⬍0.01, vs. N-ACM; # P⬍0.05, vs. 12 and 72 h ACM.

ence in inflammatory ACM groups (12 h, 36 h, 72 h ACM) was significant (P⬍0.05).

IL-6 affects ACM-induced astrocytic differentiation of BMSCs To further investigate the effects of IL-6 on the astrocytic differentiation of BMSCs, anti-IL-6 antibody was also added to DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively. It was shown that after blocking IL-6, the proportion of GFAP-positive cells significantly decreased in comparison with the corresponding control group (Fig. 8A). Concomitantly, the GFAP mRNA expression was reduced (Fig. 8B). Furthermore, the difference in inflammatory ACM groups (12 h, 36 h, 72 h ACM) was significant (P⬍0.05).

Fig. 6. IL-6 affects ACM-induced proliferation of BMSCs. Cells were treated with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, in the absence or presence of anti-IL-6 antibody for 2 days, and then incubated with BrdU for additional 6 h. Quantification of immunostaining analysis showed the reduction of proliferation of BMSCs after neutralization of IL-6. Data were presented as mean⫾ SD (n⫽4). * P⬍0.05.

DISCUSSION This study was aimed to investigate the effects of astrocytes at different inflammatory states on the proliferation and neural differentiation of BMSCs as well as the putative molecules involved in the process. A major finding was that

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Fig. 7. IL-6 affects ACM-induced neuronal differentiation of BMSCs. Cells were treated with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, in the absence or presence of anti-IL-6 antibody for 4 days and then processed for serial of experiments. (A) Quantification of immunostaining analysis showed the increase in neuronal differentiation of BMSCs after neutralization of IL-6. (B) RT-PCR analysis showing the expression change of NSE mRNA in different induced BMSCs. ␤-Actin serves as the internal control. Data were presented as mean⫾SD (n⫽4). * P⬍0.05.

inflammatory ACM significantly promoted the proliferation and neural differentiation of BMSCs; furthermore, 36 h ACM exerted greater effects on BMSCs than 12 or 72 h ACM. Moreover, IL-6 released from activated astrocytes was involved and played important roles in the proliferation and neural differentiation of BMSCs. Many studies have shown that BMSCs transplantation can significantly improve functional deficit after CNS injuries or neurodegenerative diseases (Dezawa et al., 2004; Hofstetter et al., 2002; Kurozumi et al., 2005; Ohta et al., 2004). However, the exact mechanisms had remained unclear. Since astrocytic reaction is a hallmark in CNS disease or injury, it was postulated that activated astrocytes would be involved in determining the special microenvironment to instruct the proliferation and neural differentiation of transplanted BMSCs. To investigate the effects of inflammation-activated astrocytes on BMSCs, we have established an in vitro model mimicking at least partially the microenvironment in vivo. The results showed that compared with N-ACM, inflammatory ACM significantly promoted BMSCs proliferation (Fig. 2) and neural differentiation (Figs. 3– 4) suggesting that astrocytes at inflammatory state had greater effects in regulating the behavior of BMSCs than unstimulated as-

trocytes. Previous studies have demonstrated that after subjected to stimuli, astrocytes can secrete larger amounts of a variety of cytokines (Ridet et al., 1997; Dong and Benveniste, 2001; Lafon-Cazal et al., 2003). In addition, they have been shown to induce BMSCs to differentiate into neural cells through secreting various factors under physiological condition (Joannides et al., 2003). Thus, the increased factors released by activated astrocytes would play a pivotal role in promoting proliferation and neural differentiation of BMSCs. It is relevant to note that astrocytes activated by ischemia, inflammation or mechanicallesion can significantly accelerate the proliferation and differentiation of NSPCs (Faijerson et al., 2006; Ma et al., 2005; Horner and Palmer, 2003). Taken together, it is suggested that in CNS injuries, properly activated astrocytes may be beneficial in promoting the proliferation and differentiation of stem cells and improve the final functional outcome. Furthermore, it has been reported that the factors and some cytokines released from activated astrocytes appear to be changed temporally in CNS injuries (Tokumine et al., 2003; Zhu et al., 2006). These factors would constitute different local microenvironments and have distinct effects on the behavior of stem cells. The present results have

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Fig. 8. IL-6 affects ACM-induced astrocytic differentiation of BMSCs. Cells were treated with DMEM, N-ACM, 12 h ACM, 36 h ACM, 72 h ACM, respectively, in the absence or presence of anti-IL-6 antibody for 4 days and then processed for serial of experiments. (A) The results of immunocytochemistry analysis showed that the astrocytic differentiation of BMSCs was obviously reduced after blocking IL-6. (B) RT-PCR analysis showing the expression change of GFAP mRNA in different induced BMSCs. ␤-Actin serves as the internal control. Data were presented as mean⫾SD (n⫽4). * P⬍0.05.

shown that different inflammatory ACM (12 h ACM, 36 h ACM and 72 h ACM) had distinct effects on BMSCs. In this connection, 36 h ACM appeared to exert the optimal effects in promoting the proliferation and neural differentiation of BMSCs than 12 and 72 h ACM (Figs. 2– 4). This suggests that the astrocytes at different phases following injury or stimuli had different effects on stem cells. It is well documented that astrocytes express a variety of cytokines that play an important role in controlling the behavior of stem cells (Ridet et al., 1997; Song et al., 2002). Among these, IL-6, one of the pleiotropic and multifunctional cytokines, participated in the proliferation and differentiation of NSPCs and other cells (Taga and Fukuda, 2005; Bernad et al., 1994). In the present study, the results of both ELISA and RT-PCR demonstrated that IL-6 expressed by inflammation-stimulated astrocytes was significantly increased compared with that from the unstimulated astrocytes peaking at 36 h ACM (Fig. 5). In view of this, it is speculated that IL-6 may serve as a factor regulating the proliferation and neural differentiation of BMSCs. Indeed, following the neutralization of IL-6 with its antibody, the proportion of BrdU-positive cells in BMSCs was markedly

reduced (Fig. 6) supporting the notion that IL-6 facilitates the proliferation of BMSCs. This is in agreement with recent results that IL-6 can promote the proliferation of placental-derived mesenchymal stem cells and adult spinal cord-derived neural progenitors (Li et al., 2007; Kang et al., 2007). In IL-6 deficient mice, BMSCs proliferation was significantly reduced both in vivo and in vitro studies (Rodríguez et al., 2004). It is also evident from the neutralization study that IL-6 is linked to neural differentiation. The proportion of NSE-positive cells in BMSCs was significantly increased after IL-6 neutralization (Fig. 7), while that of GFAP-positive cells was decreased (Fig. 8). This suggests that IL-6 can promote the astrocytic differentiation of BMSCs and inhibit their neuronal differentiation. Very interestingly, recent studies have reported that IL-6 can inhibit the neuronal differentiation and promote the astrocytic differentiation of NSPCs by suppressing or overexpressing IL-6 in transgenic animals in vivo and by adding or removing exogenous IL-6 in vitro (Monje et al., 2003; Okada et al., 2004; Vallières et al., 2002). The function of IL-6 on NSPCs may also be applicable for the cell differentiation of BMSCs. It is well known that IL-6 acts on target cells by

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binding to its cell surface receptors (IL-6 receptor), which form complexes with gp130, a non-kinase membrane protein. The complex then induces activation of Janus-activated kinases (JAK) and signal transducers and activators of transcription (STAT) (Heinrich et al., 1998). Phosphorylated STAT3 molecules form dimers and are subsequently translocated to the nucleus, where they lead to transactivation of their target genes (Schindler et al., 1995), and regulate the proliferation and cell differentiation of stem cells (Ihle, 2001). Arising from the above, we conclude that IL-6 plays a key and complex role (both beneficial and detrimental) in regulating the proliferation and neural differentiation of BMSCs. In addition to IL-6, other factors such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and bone morphogenetic proteins (BMPs), secreted by astrocytes may also be involved in the neural differentiation of BMSCs (Bonni et al., 1997; Yoneyama et al., 2007; Chang et al., 2003; Nakashima et al., 1999). It is reported that BMPs, one of the members of the transforming growth factor-beta superfamily, can signal through heterotetrameric serine/threonine kinase receptors. Activated BMP receptors phosphorylate downstream transcription factors Smad proteins, regulate the expression of related specific genes and inhibit the neuronal differentiation of stem cells (Derynck et al., 1998; Heldin et al., 1997; Gross et al., 1996). In conclusion, It is obvious that the roles played by the multiple factors in regulating stem cells proliferation and differentiation either alone or in combination are highly complex and this awaits further investigation. Acknowledgments—This research was supported by National Basic Research Program of China (973 Program, No. 2007CB512001); Program for New Century Excellent Talents in University of China (No. NCET 04 – 0624); National Natural Science Foundation of China (No. 30671105, 30771142); Natural Science Foundation of Shandong Province (No. Z2007C11); Key Research Program of Ministry of Education (No. 107069).

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(Accepted 24 February 2009) (Available online 9 March 2009)