Journal of Medicinal Research Vol. 6(7), pp. 1297-1303, 23 February, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1406 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

The effects of extract of gigantea on the expression of neural markers in mesenchymal stem cells

Kunchit Kongros1, Ahnond Bunyaratvej2,3, Jarupa Viyoch1 and Monnipha Sila-asna2*

1Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand. 2Cell Engineering and Tissue Growth Center, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand. 3PYY Foundation (Foundation for Sustainable Development), PO Box 19 Kratumban, Samut Sakorn 74110, Thailand.

Accepted 3 January, 2012

The seed extract of was evaluated, its efficacy on the differentiation of mesenchymal stem cells (MSCs) into neural lineage. The collected were extracted by using 1% aqueous acetic acid. According to High-performance liquid chromatography (HPLC) analysis, the average concentration of L-3,4-dihydroxyphenylalanine (L-DOPA) was 117.57.6 µg/mg of crude extract. The MSCs were cultured in neural induction medium with or without the extract. The induction medium was then replaced with the serum free medium. The induction process was monitored by phase contrast microscopy, immunocytochemistry and real-time polymerase chain reaction. After 14 days of cultivation, the cells cultured in the presence of the extract showed higher degree of nestin and β-III tubulin messenger ribonucleic acid (mRNA) expression than those cultured without the extract. The maximum gene expression was observed in cells cultured with 50 μg/ml of the extract. The induced MSCs showed a change in morphology to that of neuron-like cells. Immunocytochemical staining showed the expression of neural markers such as nestin and microtubule associated protein-2 (MAP-2) on day 7 and 28, respectively. This study suggests that the extract of M. gigantea, which contained L- DOPA and/or the other unidentified active compounds, could induce MSCs to express neural protein and gene markers.

Key words: Mucuna gigantean, L-DOPA, mesenchymal stem cells, neuron-like cells.

INTRODUCTION

Mucuna pruriens has been described as one of the (Hirsch et al., 1988; Olanow and Tatton, 1999). Mucuna therapeutic agents for Parkinson’s disease (PD) spp. have been described to contain many bioactive (Manyam, 1990), a disease which is characterized as substances, especially L-DOPA (Adebowale et al., 2005; pathologically progressive degeneration of neurons Damodaran and Ramaswamy, 1937; Daxenbichler et al., 1972; Manyam et al., 2004; Modi et al., 2008; Prakash et al., 2001), which is a potent precursor of the brain neurotransmitter (Hornykiewicz, 2002; *Corresponding author. E-mail: [email protected]. Tel: Kostrzewa, et al, 2005; Nagatsua and Sawadab, 2009). +66 2800 3783 or +668 1915 2877. Fax: +6628003783. The M. pruriens has been shown to be more effective for PD treatment than synthetic L-DOPA (Hussain and Abbreviations: bFGF, Basic fibroblast growth factor; DMEM, dulbecco’s modified eagle medium; EGF, epidermal growth Manyam, 1997; Manyam et al., 2004). Another factor; FBS, fetal bovine serum; PD, Parkinson’s disease; MAP- found in the southern part of Thailand, Mucuna gigantea, 2, microtubule associated protein-2; MSCs, mesenchymal stem similar to the other Mucuna spp., has also been reported cells; NM, neural induction medium; NM+MG, neural induction to contain L-DOPA (Daxenbichler et al., 1972; Rajaram medium with M. gigantea; NSCs, neural stem cells. and Janardhanan, 1991; Wilmot-Dear, 2008). This plant, 1298 J. Med. Plants Res.

Figure 1. Parts of M. gigantea (Willd) DC: A) seeds; B) leaves; C) flowers and; D) pods.

therefore, may be an alternative source of L-DOPA for the potential activity of the extract for neurodegenerative the treatment of neurodegenerative disorders including disorder treatment mediated through stem cell PD. differentiation. Neural stem cells (NSCs) are immature self-renewing cells that have the potential to differentiate into neurons, astrocytes and oligodendrocytes (Chu et al., 2003; MATERIALS AND METHODS

McKay, 1997). However, their clinical utilities have the Plant material limitations. The human MSCs has been known about the capability to differentiate into the cells with neural M. gigantea (Wild.) DC., a twiner with trifoliate leaves, green flowers phenotype at under appropriate conditions including the and pods covered with hairs (Figure 1), were collected in July 2006 from Samut Songkhram province, the southern part of Thailand. A presence of cytokines and chemical inducers in vitro voucher specimen (BKF 12345) was deposited at the Forest (Deng et al., 2001; Jin et al., 2003; Kabos et al., 2002; Herbarium, Department of National Parks, Wildlife and Plant Kim et al., 2002; Lei et al., 2007; Lu et al., 2006; Tao et Conservation, Ministry of Natural Resources and Environment, al., 2005; Sanchez-Ramos et al., 2000; Woodbury et al., Bangkok, Thailand. 2000, 2002). Medicinal plants, such as Salvia miltiorrhiza, also show ability to induce the differentiation of MSCs Preparation of M. gigantea seed extract into neuron-like cells (Ma et al., 2005). In addition, MSCs can be easily collected from several tissues. Therefore, it The whole seeds were ground into powder and macerated with 1% has been suggested as the alternative source of NSCs aqueous acetic acid (LabScan Asia Company Limited, Bangkok, for studying the treatment of injury or diseases of nervous Thailand) containing 0.1% sodium sulfite (Riedel-de Haën, Hannover, Germany) for 6 h at 60°C. Then, the filtered extract was system. lyophilized and stored in the light-protected container at -20°C Although there have been reported about the effect of (Behera et al., 2010). L-DOPA on neural cells and neural cell lines in vitro (Liu et al., 2004; Maeda et al., 1997; Pardo et al., 1995; Determination of L-DOPA content in the extract Pedrosa and Soares-da-Silva, 2002; Walkinshaw and Waters, 1995), the effect of the plant extract containing L- To control the quality of the extract, its L-DOPA content was DOPA on differentiation of MSCs into neuron-like cells analyzed by using HPLC. Standard L-DOPA was purchased from has never been reported. Therefore, in this study, we Sigma-Aldrich (St. Louise, Missouri, USA). Standard solutions of L- examined the effects of crude extract from M. gigantea DOPA were freshly prepared by dissolving dried standard in 0.1 N HCl and were diluted to the concentration of 12.5, 25, 50, 100 and seeds on directional differentiation of MSCs into neural 250 µg/ml. Samples and standard were analyzed by HPLC LC- lineage. 20AT, SPD-M10AVP diode array detector and DGU-20A5 degasser The expression of protein markers and genes (Shimadzu Company Limited, Kyoto, Japan). The column was a associated to differentiated cells such as nestin (marker Gemini-NX 5u C18 110A, 25 cm x 4.6 mm with 5 µm particle size of neural progenitor), β-III tubulin (marker of immature (Phenomenex; Torrance, California, USA). Chromatographic neuron), or microtubule associated protein-2 (MAP-2; conditions were used as previously described (Siddhuraju and Becker, 2001) with some modifications. The running process was a marker of mature neurons) (Cáceres et al., 1986; gradient with two types of solvents: the eluting solution was made Dráberová et al., 1998; Kelly et al., 2009; Svendsen et up of water, methanol (LabScan Asia Company Limited) and al., 1998). The finding from this study would first suggest orthophosphoric acid (85%; Carlo Erba Reagents, Rodano, Milano, Kongros et al. 1299

humidified incubator with 5% CO2 at 37°C. Cultures were split by - ethylene diamine tetraacetic acid (EDTA) treatment (0.05% trypsin and 0.02% EDTA; Gibco, BRL) at 75% confluence.

Induction of cell differentiation in culture

MSCs were plated at a density of 1x104 cells/cm2 on poly-L- ornithine (Sigma-Aldrich) and laminin (R & D systems, Minneapolis, MN, USA)-coated tissue culture dishes and cultured in the maintenance medium (DMEM with 10% FBS and 1% Penicillin/Streptomycin). After that, the cells were cultured in neural induction medium (NM); DMEM/F12 (1:1) (Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 2% B27 (Gibco, Invitrogen), 100 U/ml Penicillin/ 100 µg/ml Streptomycin, 20 µg/ml human recombinant basic fibroblast growth factor (bFGF; Sigma-Aldrich) and 20 ng/ml human recombinant epidermal growth factor (EGF; Sigma-Aldrich). The M. gigantea seed extract was added into the medium to study its effect at the concentration of 25, 50 or 100 µg/ml. After 7 days (for gene expression study) or 21 days (for protein expression study), the NM was replaced by DMEM/F12 (1:1) supplemented with 0.2 mM ascorbic acid, 1% N2 (Gibco, Invitrogen) and 100 U/ml Penicillin/100 µg/ml Streptomycin. Media were changed every 3 to 4 days. The concentration that gave the maximum expression of mRNA was used in the further studies. The morphological changes and protein expressions of cells in cultures were characterized by using phase contrast microscope (Te-2000u Eclipse; Nikon, Japan), and immunocytochemistry, respectively.

Real-time polymerase chain reaction (RT-PCR)

Total ribonucleic acid (RNA) was extracted from samples using Trizol (Invitrogen, New York, USA), and complementary deoxyribonucleic acid (cDNA) was generated with Revertaid reverse transcription (RT) (Fermentas, Burlington. Ontario, Canada) as directed by manufacturers. The cDNA was then stored at -20°C until use. The RT- PCR reaction mixtures were prepared in a 20-µl solution containing cDNA, TaqMan forward and reverse primers, probes in TaqMan Gene Expression Assay Mix and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, California, USA). RT- PCR was performed on ABI Prism 7300 RT- PCR System (Applied Biosystems), using ABI TaqMan gene expression assays as follows: nestin Hs00707120, β-III tubulin Figure 2. The HPLC Chromatograms of: A) standard L- Hs00964962, hydroxylase Hs01002188 and DOPA and; B) the seed extract of M. gigantea at the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) concentration of 100 µg/ml and 1 mg/ml, respectively. Hs99999905 (Applied Biosystems). The RT- PCR reactions were performed using thermal cycling with conditions at 50°C for 2 min and at 95°C for 10 min as initial steps followed by 45 cycles of 15 s at 95°C and 1 min at 60°C for amplification. All assays were Italy) in the ratio of 975.5:19.5:1 (v/v/v), pH 2.0, and washing performed in triplicate. The relative mRNA levels were calculated solution was 70% methanol. The injection volume was 20 µl and the using the comparative Ct method, using GAPDH as the flow rate was 1.2 ml/min. The quantification of L-DOPA was based endogenous control. on the peak area at 280 nm. The analyzing procedure was carried out in triplicate. Immunocytochemistry

Culture of MSCs Cells plated on poly-L-ornithine/laminin-coated culture dishes were washed in phosphate buffer saline (PBS) and fixed with cold MSCs were taken from bone marrow cells from subjects aged 31 to methanol for 15 to 20 min. Fixed cells were washed once with PBS 39 years with informed consent, and were provided by the Cell before staining. For staining, cells were permeabilized in 0.1% Engineering and Tissue Growth Center, Institute of Molecular Triton X-100 in PBS for 10 min. The cells were incubated for 30 min Biosciences, Mahidol University, Nakhon Pathom, Thailand. These with 3% bovine serum albumin. The primary antibodies (1:200), cells were isolated as the adherent bone marrow cells as described which were specific to neuron, were then added to the cells and anywhere. Cells were maintained in Dulbecco’s modified eagle incubated for 2 h in the dark at room temperature. After that, cells medium, DMEM (Gibco, BRL, Gaithersburg, MD, USA), containing were washed in PBS and incubated with secondary antibody 10% fetal bovine serum (FBS; Gibco, BRL) and 100 U/ml Penicillin/ conjugated with fluorescent dye (1:200) for 1 h. After washing with 100 µg/ml Streptomycin (Gibco, BRL, Gaithersburg, MD, USA), in a PBS, the nuclei were counterstained with propidium iodide (Sigma- 1300 J. Med. Plants Res.

Figure 3. The relationship between M. gigantea (MG) concentration and neuronal mRNA expressions in MSCs. The MSCs were cultured in neural induction medium with various concentrations (25, 50 and 100 µg/ml) of seed extract from M. gigantea for 7 days. The medium was subsequently replaced with the serum-free medium containing ascorbic acid. The expressions of: (A) nestin; and (B) β-III tubulin mRNA relative to GAPDH in MSCs at day 14 of culture were detected by using real time PCR. The maximum gene expressions of: (A) nestin; and (B) β-III tubulin were observed when the concentration of extract was 50 μg/ml. These data are expressed as mean  SEM from 4 separate experiments.

Aldrich) at room temperature in the dark for 10 min and washed mRNA expressions were marked higher in cells cultured again with PBS. The stained cells were observed under fluorescent with the addition of 50 g/ml extract neural induction microscope (Te-2000u Eclipse; Nikon, Tokyo, Japan). For the negative control, the primary antibody was replaced with normal medium with M. gigantea (NM + MG), and the expression mouse, goat, or rabbit sera (all by Chemicon, Temecula, California, of nestin correlated well with that of β-III tubulin. At higher USA). The following primary antibodies were used: nestin, for concentration (100 g/ml), M. gigantea tended to provide neural progenitors, MAP-2 and tyrosine hydroxylase, for neurons inhibitory effect on the differentiation of MSCs isolated (all by Chemicon, Temecula, California, USA). from patients, resulting in the decreased expressions of gene markers.

RESULTS Morphology of MSCs HPLC analysis of M. gigantea seed extract After MSCs were seeded onto poly-L-ornithine/laminin- According to HPLC analysis, the retention time of coated dish, they spread on the culture dish and standard L-DOPA was about 5.4 min (Figure 2). The developed their morphology as elongated or large flat calibration curve for L-DOPA (at five concentrations; cells in maintenance medium. The induced MSCs could 12.5, 25, 50, 100 and 250 µg/ml) plotted against the peak develop long, thin cellular processes and refractive cell area was shown a linearity with the correlation coefficient bodies, like neuron, when they were cultured in NM of 1. The percentage yield of lyophilized crude extract (Figure 4). MSCs could form the cluster from day 7. was about 37% (w/w). The average concentration of L- However, in the presence of NM + MG at concentration of DOPA found in 1 mg of lyophilized crude extract was 50 g/ml, the clumping of cells did not appear and 117.5  7.6 µg. neuron-like cell morphology was markedly observed.

Neural mRNA expressions in MSCs Neural protein marker expressions in MSCs

The neural gene expression, in induce MSCs, was To confirm neural differentiation of MSCs, neural markers evaluated by using nestin, β-III tubulin and tyrosine of neural progenitor (nestin) and mature neural cells hydroxylase gene marker. Induced MSCs could express (MAP-2) were used to assess cell type-specific protein nestin and β-III tubulin mRNA (Figure 3) at day 14 of expressions by immunocytochemical staining. Induced culture, but not for tyrosine hydroxylase mRNA. The MSCs with 50 g/ml of the extract NM + MG and without Kongros et al. 1301

Figure 4. The MSCs observed by phase contrast microscope. The MSCs were cultured in the induction medium (NM) without or with the seed extract of M. gigantea (NM + MG) at the concentration of 50 µg/ml for 21 days. The cell culture media were subsequently changed at 3-day interval using serum-free medium containing ascorbic acid. Cells displayed long, thin cellular process and refractive cell bodies, like neurons. Clumping of cells was found in condition without the extract (NM).

the extract NM were positive staining with nestin since neural maker expression (nestin and MAP-2) between day 7 of induction, and much stronger expression at day induced MSCs with and without the extract was difficult to 14 (Figure 5A and C). MSCs also showed positive observe as both conditions provided the expression of staining with antibodies specific to MAP-2 on day 28 of protein markers. However, it is interested to see that the culture (Figure 5B and D). mRNA expression of nestin, a neural precursor marker, MSCs was markedly higher in cells cultured with the extract than those without the extract. DISCUSSION In addition, the expression of β-III tubulin, an immature neuron marker, was also exhibited in the similar manner. M. pruriens has been used as herbal medicines for the It is likely that, the extract enhanced the expression of treatment the PD because of L-DOPA content (Manyam neural genes. To explain this effect, the further et al., 1990, 2004; Hussain and Manyam, 1997). L-DOPA investigation about its mechanism on cells is needed. is a potent precursor of the brain neurotransmitter It should be taken into consideration as we found that dopamine in catecholaminergic neurons and is the most the M. gigantea extract at high concentration (100 µg/mL) effective therapeutic agent for Parkinson’s disease tended to decrease neural gene expressions. The (Hornykiewicz, 2002; Kostrzewa et al, 2005; Nagatsu and previous reports revealed that L-DOPA is toxic for Sawadab, 2009). Like M. pruriens, we also found L- dopaminergic neurons in culture, neural stem cell lines DOPA in M. gigantea seed extract. and neuroblastoma cell lines (Liu et al., 2004; Maeda et In the present study, we found that neural precursors al., 1997; Pardo et al., 1995; Pedrosa and Soares-da- and mature neurons could be cultured from human Silva, 2002; Walkinshaw and Waters, 1995). The MSCs, and the marked morphological changes into exposure of Neuro-2A cells, a cell line with origin in a neuron-like cells were found in case of the treatment with mouse neuroblastoma, or C17.2 neural stem cells to L- the extract as determination by phase contrast DOPA or dopamine reduced cell viability in a microscopic examination. It has been reported that L- concentration- and time- dependent manner (Liu et al., DOPA increases the adherence of MSCs to the culture 2004; Pedrosa and Soares-da-Silva, 2002). The surface and also increases the proliferation and mechanism of L-DOPA induce apoptosis may contribute mitochondrial activity of MSCs (Kim et al., 2011). Such by the activation of the mitochondria-dependent pathway report supports our experimental finding that the cells and caspase-3 protease (Liu et al., 2004; Pedrosa and cultured in neural induction medium in the presence of Soares-da-Silva, 2002). the extract tended to spread on poly-L-ornithine and In the previous study, the cytotoxicity of the L-DOPA laminin-coated dish rather than clump as sphere. This might be prevented by the antioxidants such as ascorbic could lead to increase in MSC proliferation and acid, glutathione, N-acetyl-L-cysteine, sodium differentiation. By immunocytochemistry, the difference in metabisulfite (Mytilineou et al., 1993; Pardo et al., 1995; 1302 J. Med. Plants Res.

Figure 5. The immunocytochemical characterization of neuron-like cells developed from MSCs in culture. The commercial MSCs cultured in the induction medium without (NM) or with the seed extract of Mucuna gigantea (NM + MG) at the concentration of 50 µg/ml for 21 days. The medium was subsequently replaced with the serum-free medium containing ascorbic acid. Cells with neural precursor marker (nestin; A, C) were positively stained as green color on day 14 and mature neural marker (MAP-2; B,D) on day 28. Nucleus (red) was also counterstained with propidium iodide (PI).

Pedrosa and Soares-da-Silva, 2002) or glia-conditioned Growth Center, Institute of Molecular Biosciences, medium (Mena et al., 1997) at the appropriate Mahidol University, Nakhon Pathom, Thailand. We also concentration. would like to thank the Faculty of Pharmaceutical From this study, M. gigantea seed extract was found to Sciences and Center of Excellence for Innovation in contain L-DOPA. Induced MSCs could differentiate into Chemistry, Naresuan University, Thailand for the other neuron-like cells. The addition of seed extract of M. facility supports. gigantea resulted in increased neural mRNA expression in such neuron-like cells. The seed extract of M. gigantea, therefore, may be an alternative source of L- REFERENCES

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