The role of RhoA kinase inhibition in human placenta-derived multipotent cells on neural phenotype and cell survival

Biomaterials 34 (2013) 3223e3230

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The role of RhoA kinase inhibition in human placenta-derived multipotent cells on neural phenotype and cell survival Chih-Hsiang Wang a, b, Chia-Ching Wu c, Shan-Hui Hsu d, Jun-Yang Liou e, Yu-Wei Li b, Kenneth K. Wu e, Yiu-Kay Lai a, **, B. Linju Yen b, f, * a

Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan Regenerative Medicine Research Group, Institute of Cellular & System Medicine (ICSM), National Health Research Institutes (NHRI), Zhunan, Taiwan Department of Cell Biology & Anatomy, National Chung-Kung University, Tainan, Taiwan d Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan e Cardiovascular & Metabolic Medicine Research Group, ICSM, NHRI, Zhunan, Taiwan f Department of Obstetrics/Gynecology, Cathay Shiji Hospital, Shiji, Taiwan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 October 2012 Accepted 26 December 2012 Available online 12 February 2013

Current advances in stem cell biology have brought much hope for therapy of neuro-degenerative diseases. However, neural stem cells (NSCs) are rare adult stem cells, and the use of non-NSCs requires efficient and high-yielding lineage-specific differentiation prior to transplantation for efficacy. We report on the efficient differentiation of placental-derived multipotent cells (PDMCs) into a neural phenotype with use of Y-27632, a clinically compliant small molecular inhibitor of Rho kinase (ROCK) which is a major mediator of cytoskeleton dynamics. Y-27632 does not induce differentiation of PDMC toward the mesodermal lineages of adipogenesis and osteogenesis, but rather a neural-like morphology, with rapid development of cell extensions and processes within 24 h. Compared with conventional neurogenic differentiation agents, Y-27632 induces a higher percentage of neural-like cells in PDMCs without arresting proliferation or cell cycle dynamics. Y-27632-treated PDMCs express several neural lineage genes at the RNA and protein level, including nestin, MAP2, and GFAP. The effect of the ROCK inhibitor is cell-specific to PDMCs, and is mainly mediated through the ROCK2 isoform and its downstream target, myosin II. Our data suggest that ROCK inhibition and cytoskeletal rearrangement may allow for induction of a neural phenotype in PDMCs without compromising cell survival. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Multipotent mesenchymal stem cells Placenta Neural differentiation Rho kinase (ROCK) Y-27632 Cytoskeleton

1. Introduction Adult neural stem cells (NSCs) are a group of cells that have the capacity to self-renew and to differentiate into neuronal and glial lineages in the developed nervous system [1]. However, these stem cells are scarce, and are difficult to access or culture ex-vivo. In addition to NSCs, recent data has shown that a number of nonneural adult stem cellsdwhich are more abundant and include bone marrow mesenchymal stem cells (BMMSCs)dcan be differentiated into neural lineage cells [2e4]. The mechanisms involved in this process, however, remain relatively unclear. While non-NSCs capable of neural differentiation broaden the sources available for neural regeneration, the fact remains that

* Corresponding author. 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan. Tel.: þ886 037 246 166x37501; fax: þ886 037 546 166. ** Corresponding author. No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan. Tel.: þ886 3 5742751; fax: þ886 3 5717237. E-mail addresses: [email protected] (Y.-K. Lai), [email protected] (B.L. Yen). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.12.034

adult stem cellsdincluding BMMSCsd require invasive procedures to obtain cells, and cell numbers decrease with increasing age of the donor [5]. In the pursuit of alternative sources of human stem cells, we have isolated multilineage progenitors from the term human placenta, a source without ethical concerns [6]. These placentaderived multipotent cells (PDMCs) express a surface marker profile similar to BMMSCs and are capable of differentiation into all three germ layers including neural phenotypes [6,7]. Moreover, PDMCs harbor significant immunosuppressive effects towards T lymphocytes and natural killer lymphocytes [8,9]. These results suggest that PDMCs may be a potential source of easily accessible human MSCs for therapeutic use [10]. Currently, many protocols have been used to induce neural differentiation from various ASCs. One common drawback has been the toxic nature of many of the reagents used (for review, see Ref. [11]). Indeed, one of the most commonly used compound for neural differentiation, retinoic acid (RA), is cytotoxic at high concentrations in in vitro cultures, making therapeutic application difficult [12]. Thus, in this study, we explore the use of Y-27632da

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clinically compliant inhibitor of Rho kinase (ROCK), which is an important mediator of cytoskeletal dynamics [13]dfor neural differentiation of PDMCs. The specificity of the phenomenon and mechanisms involved is explored in this study.

Island, NY, USA) and cultured in expansion medium consisting of Dulbecco’s modified Eagle medium (DMEM; Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco-Invitrogen) [6]. At 80e90% confluence, the cells were subcultured at the dilution of 1:2 to 1:3.

2. Methods and materials

2.2. Differentiation studies

2.1. Isolation and culture of PDMCs

Osteogenic and adipogenic differentiation studies were performed as previous described [6]. For adipogenic differentiation, cells were cultured in complete medium with the addition of 0.5 mM isobutyl-methylxanthine, 1 mM dexamethasone, 10 mM insulin, and 60 mM indomethacin (all from SigmaeAldrich, St Louis, MO, USA) for induction. For osteogenic differentiation, cells were cultured in complete medium along with 0.1 mM dexamethasone, 10 mM b-glycerol phosphate, and 50 mM

PDMCs were isolated from term human placenta tissue (38- to 40-week gestation) obtained from healthy donors with informed consent approved by the institutional review board. As reported previously, placental tissue was dissected enzymatically digested with 0.25% trypsin-EDTA (Gibco-Invitrogen Corp., Grand

Fig. 1. Rho kinase (ROCK) inhibition in placenta-derived multipotent cells (PDMCs) does not induce adipogenic or osteogenic differentiation, but rather a neural-like morphology. Comparison of PDMCs cultured in complete medium (CM) to (A) adipogenic medium (AGM), (B) osteogenic medium (OGM), or (C) neurogenic (NGM) with or without the ROCK inhibitor Y-27632 (10 mM in CM), for 24 and 72 h. Adipogensis was assessed by Oil Red O staining for the presence of oil droplets, and osteogenesis was assessed by staining for alkaline phosphatase (red arrow). Bar: 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

C.-H. Wang et al. / Biomaterials 34 (2013) 3223e3230 ascorbic acid (all from SigmaeAldrich). Neurogenic differentiation was performed by culturing cells in serum-free medium with addition of 0.5 mM retinoic acid (RA) [4] or complete medium with 10 mM of Y-27632 (all from SigmaeAldrich). 2.3. Live-cell imaging ˇ

Live-cell imaging was performed on the Olympus Cell R system (Olympus, Tokyo, Japan). Cells were seeded, allowed to attached for 24 h, and then moved into the sample chamber on the stage, which was supplemented with 5% CO2 and 95% air and cultured at 37  C. For each sample, 3 different sites were traced. Images were captured every 30 min and finally merged by Cell R program. ˇ

2.4. Measurement of cell growth PDMCs were seeded into 96-well plates at a density of 3 x 103 cell/well and cultured with complete medium (control), complete medium with 10 mM of Y-27632, or neurogenesis medium for 24, 48 and 72 h. Cell proliferation was determined by the (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigmae Aldrich) assay according to manufacturer’s instructions. The absorbance values were measure at 570 nm with SpectraMAX 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA). 2.5. Cell cycle analysis Cells were harvested by trypsinization and washed with PBS. The pellets were then fixed in 70% ethanol for 1 h at room temperature, followed by incubation in 0.5 ml staining solution (0.1% Triton X-100 in PBS, 100 mg/ml RNase, and 50 mg/ml propidium iodide) at 37  C for 20 min. The cell cycle distribution was analyzed by using FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA, USA). 2.6. Western blotting Total cell lysates (20 mg) were separated by 10% SDS-PAGE, transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA), and then incubated with the following antibodies at 4  C overnight: anti-RhoA (Upstate), anti-ROCK1 (Chemicon, Temecula, CA, USA), and anti-ROCK2 (Upstate, Lake Placid, NY, USA). Incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature was then performed. Signals were detected with Immobilon Western chemiluminescent HRP substrate (Millipore). 2.7. Real-time RT-PCR (qPCR) Total RNA was extracted form cultured cells using TRIÒ reagent (SigmaeAldrich), and quantified with NanoDrop spectrophotometer (Nyxor Biotech, Paris, France). 5 mg of RNA were converted to cDNA by using RevertAidTM H Minus reverse transcriptase (Fermentas, Burlington, Canada) according to the manufacturer’s instructions. qPCR was performed using ABI PRISM 7500 system (Applied Biosystem, Foster City, CA, USA) with SYBRÒfast qPCR kit kit (KAPA Biosystems, Woburn, MA, USA). The sequences of primers are as follows: hNestin, 50 -AACAGCGACGGAGGTCTCTA-30 and 50 -TTCTCTTGTCCCGCAG ACTT-30 ; hGFAP, 50 -AGGAAGATTGAGTCGCTGGA-30 and 50 -ATACTGCGTGCGGATCTCTT-30 ; hMAP2, 50 -CCAGGTGGCGGACGTGTGAA-30 and 50 -GCCACGCTGGATCTGCCTGG-30 ; hNG2, 5'-GCTGTGGCTGTGTCTTTTGA-3' and 5'-CTGTGTGACCTGGAAGAGGA-3'; hSOX1, 5'-AAAGTCAAAACGAGGCGAGA-3' and 5'-AAGTGCTTGGACCTGCCTTA-3' ; hSOX2, 5'-TTGCGTGAGTGTGGATGGGATGGTG3' and 5'-GGGAAATGGGAGGGGTGCAAAAGAGG-3'; and hB-actin, 50 -TGGCACCACACCTTCTACAATGAGC-30 and 50 -GCACAGCTTCTCCTTAATGTCACGC-30 . All reactions were performed in triplicates and normalized with reference gene expression.

3. Results We first assessed whether the Rho/ROCK pathway is involved in differentiation of PDMCs. PDMCs express many BMMSC markers and are capable of multilineage differentiation [6,7], and Y-27632 has been reported to induce neural differentiation under hypoxia in mouse BMMSCs and osteogenic differentiation in human BMMSCs [14,15]. To analyze the role of Rho/ROCK pathway in the differentiation of PDMCs, we performed inhibition of this pathway by using the specific small molecular inhibitor Y-27632. We found that Y27632 does not induce adipogenic or osteogenic differentiation of PDMCs, as assessed by Oil red O staining or alkaline phosphatase staining respectively (Fig. 1A and B). However, a clear morphological change with cell-process formationdhighly similar to neural cellsdwas induced, and compared to conventional neurogenic differentiation medium using RA in which only a minority of cells were affected, Y-27632 induces a neural-like phenotype in nearly all the cells within 24 h (Fig. 1C). To characterize the induction of apparent neural differentiation, we performed qPCR to verify whether neural-lineage genes were expressed in the differentiated PDMCs. After 24 h of induction with Y-27632, we found significant up-regulation of nestin, an NSC marker; MAP2, a mature neuronal marker, and GFAP, an astrocyte and NSC marker. The expression of oligodendrocyte marker, NG2, on the other hand, was not significantly increased (Fig. 2). We then checked for protein expression of neural-lineage markers in the differentiated PDMCs. Immunofluorescent staining for protein expression of GFAP, nestin, and MAP2 shows enhancement after Y27632 treatment, supporting the qPCR data (Fig. 3). Since Y-27632 is an inhibitor of ROCK, which is an important signaling pathway in cytoskeletal kinetics [16], we investigated the effects of ROCK inhibition on PDMC cytoskeletal elements. Using live-cell imaging, we assessed for changes to the cytoskeleton during neural differentiation of PDMCs. Unlike PDMCs grown in expansion medium, which spread normally on the plate bottom and showed low mobility (Supplemental Movie 1), Y-27632- and RA-treated PDMCs exhibit a higher motility and form neuron-like processes (Supplemental Movies 2 and 3). In response to Y-27632 treatment, PDMCs rapidly form cytoplasmic protrusions, followed by elongation of these protrusions into long extensions. This phenomenon can be observed as early as 3 h after treatment. Moreover, Y-27632-treated cells showed a longer cytoplasmic extension than

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2.9. RNA interference experiments Cells were transfected with small interfering RNA (siRNA) specific to RhoA, ROCK1, ROCK2 (siGENOME SMARTpool; Dharmacon, Inc. Lafayette, MO, USA) or non-target siRNA (siCONTROL Nontargeted siRNA 1; Dharmacon, Inc.) according to manufacturer’s instructions. After 24 h, the transfection medium was removed and replaced with complete medium. The inhibitory efficiency was confirmed by western blotting.

* *

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2.8. Immunofluorescent staining

Y-27632

12 Folds

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX 100 (SigmaeAldrich). Cells were then blocked by 5% BSA in PBS for 1 h, and incubated with following primary antibodies at 4  C overnight at a 1:200 dilution: anti-GFAP, anti-Nestin, and anti-MAP2 (all from Chemicon). After that, cells were rinsed three times with PBS and incubated with FITC- or PE-conjugated secondary antibodies at a dilution of 1:200 at 37  C for 1 h, and stained with DAPI (40 , 6diamino-2-phenylindole dihyrochloride, 1:10,000; Molecular Probes, Eugene, OR, USA) to identify cell nuclei. Staining was visualized using a fluorescence microscope (Olympus, Tokyo, Japan).

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Fig. 2. Increased gene expression of nestin and microtubule-associated protein 2 (MAP2) in PDMCs after ROCK inhibition. PDMCs were treated with complete medium (control), NGM, or Y-27632 (incomplete medium) for 24 h and assayed by real-time PCR for expression of neurogenic genes. *, p < 0.05.

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Fig. 3. Increased protein expression of nestin and MAP2 in PDMCs after ROCK inhibition. (A) Immunofluorescence staining for glial fibrillary acidic protein (GFAP), MAP2 and nestin in PDMCs after culture complete medium (control), complete medium þ Y-27632 and NGM for 24 h. (B) Quantification of intensity of immunofluorescent signal; *, p < 0.05.

those in RA culture, which can be evident with conventional phase-contrast light microscopy. We further used phalloidin to visualize actin filament organization in PDMCs. After 24 h of incubation and acquiring the neural-like morphology, Y-27632treated cells lose the normal distribution of microtubules, and displayed microtubule-rich on the extension region (Fig. 4A). These data indicate that cytoskeletal changes are involved in the neural differentiation induced by Y-27632 in PDMCs. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2012.12.034. Since the cytoskeleton is ubiquitous in all cells, we sought to ascertain the specificity of the neural-like morphology induced by Y-27632 in PDMCs. To assess this, we treated a human hepatocellular liver carcinoma cell line and human choriocarcinoma cell line (a placental cancer cell line, sharing organ-specificity with PDMCs), HepG2 and JEG-3, respectively, with Y-27632, and after 24 h of

incubation, both cell types remained unchanged morphologically (Fig. 4B). Phalloidin staining also showed that the actin distribution of the cytoskeleton remained unchanged after Y-27632 treatment. These results revealed that the neural differentiation seen in PDMCs after ROCK inhibition is specific for this cell type. One drawback with current methods of neural differentiation is the toxicity induced by the reagents used. We therefore investigated whether inhibition of Rho/ROCK pathway by Y-27632 affected cell proliferation and the cell cycle. We found that Y27632-treated PDMCs displayed similar growth curve as untreated cells even up to 72 h after treatment, whereas the RA-treated cell stop proliferation by 24 h of treatment (Fig. 5A). Analysis of cell cycle kinetics by flow cytometry shows that Y-27632-treated PDMCs showed slight changes to the S and G1 phases, whereas the RA-treated cells were mostly arrested at the G0/G1 phase (Fig. 5B).

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Fig. 4. ROCK inhibition induces neural-like morphological changes in PDMCs via cytoskeletal rearrangement but not in epithelial cells. (A) PDMCs were cultured in control medium only, Y-27632 (10 mM in control medium) or NGM. Upper column: phase contrast; Lower column: immunofluorescent staining for F-actin (rhodamine phalliodin) with nuclear staining by DAPI. (B) HepG2 and JEG cells were cultured in complete medium with or without the presence of 10 mM of Y-27632 for 24 h. Cytoskeleton changes were confirmed by immunofluorescent staining for F-actin by rhodamine phalliodin and DAPI for nuclear staining.

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Time (hr) Fig. 5. Y-27632 minimally affects cell proliferation and cell cycle dynamics of PDMCs. (A) Cell growth as assessed by MTT and (B) cell cycle changes as assessed by flow cytometry after propidium iodide staining for DNA content in PDMCs after culturing in complete medium (control), NGM or Y-27632 (10 mM in complete medium) for 24, 48 and 72 h.

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Y-27632 inhibits RhoA activity via ROCK, which is the downstream kinase of RhoA and exists as two isoforms: ROCK1 and ROCK2. To investigate the specific pathway involved in Y-27632mediated neural differentiation, we performed specific siRNA knockdown of RhoA, ROCK1, and ROCK2 (Fig. 6A). We found that PDMCs with specific knockdown of ROCK2 show a neuron-like morphology with long cytoplasmic extension very similar to Y-

27632-treatment (Fig. 6B). Knockdown of RhoA or ROCK1 also resulted in minor morphological changes, whereas PDMCs transfected with non-target siRNA showed no morphological difference compared with untransfected cells. These results indicate that ROCK2 is the major regulator through which Y-27632 act on in the neural differentiation of PDMCs. We further investigated for the involvement of downstream targets of ROCK2, which include

Fig. 6. Y-27632 induction of a neural-like phenotype in PDMCs is mediated in large part through inhibition of ROCK2 and myosin II. PDMCs were transfected with small interfering RNA (siRNA) which specifically inhibit RhoA, ROCK1, ROCK2, or non-target sequence. (A) Protein expression of RhoA, ROCK1, and ROCK2 in PDMCs after specific siRNA inhibition as assessed by western-blotting, and (B) morphological changes (phase contrast). (C) Cell morphology of PDMCs after treatment with blebbistatin, an inhibitor of myosin II, in complete medium. (D) Diagram of Y-27632 effects on PDMCs as mediated through the RhoA/ROCK2/myosin II axis. Cytoskeleton rearrangement by Y-27632, ROCK2 inhibition (with minor contributions from ROCK1), or myosin II inhibition by blebbistatin induces a neural-like phenotype in PDMCs.

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myosin II, microtubule and actin [16]. Treatment with blebbistatin, an inhibitor of myosin II, triggers a similar morphological change as ROCK inhibition in PDMCs (Fig. 6C). On the other hand, inhibition of microtubule with nocodozole, or inhibition of actin with cytochalasin D, did not induce similar changes (data not shown). Therefore, Y-27632-mediated neural differentiation of PDMCs is mediated in large part through ROCK2 and its downstream target, myosin II (Fig. 6D). 4. Discussion Continued advances in the field of ASC research have brought much excitement in terms of finding therapeutic cures for neurodegenerative diseases. Many of these diseases are progressively fatal, and such patients are now among the first targets for clinical trials of stem cell therapy [17]. As promising as the basic science data has been, one important concern is the ability for efficient in vitro differentiation prior to transplantation. The in vivo environment is complex and known to influence transplanted stem cells, which by nature are highly malleable and may differentiate into lineages not therapeutic for the problem at hand [18]. Thus, in this study we have evaluated the neural differentiation potential of the small molecular compound Y-27632 on PDMCs and the mechanistic pathway involved. Our findings show that Y-27632, which is an inhibitor of ROCK and currently approved for clinical use, can induce a neural-like morphology in PDMCs with high efficiency and yield, both important considerations for therapeutic application. It is known that MSCs can be differentiated into many lineages depending the extrinsic factors in their microenvironment. In our previous study, we found that PDMCs are capable of differentiating into neural-lineage cells by using RA or isobutymethylxanthine (IBMX); however, undifferentiated cells still exist even after 6 days of induction [7]. Moreover, significant toxicity exists with many of these agents [12,21]. While cell toxicity is minimized with the use of growth factors such as brain-derived growth factor, neural growth factor, or N2B27 medium for neural differentiation, these methods generally require even longer periods of time for differentiation and are costly [4,11,19,20]. Indeed, we found that PDMCs cultured in the commonly used neurogenic differentiation RA-serum-free medium undergo cell cycle arrest at the G0/G1 phase, halting further proliferation (Fig. 5B); Y-27632treated PDMCs, on the other hand, had minimal alterations to the cell cycle. Collectively, our data shows that inhibition of Rho-ROCK pathway by the clinical approved compound Y-27632 in PDMCs can provide an efficient and high-yielding method for neural differentiation. Small GTPases of the Rho family and its downstream kinases, ROCK1 and ROCK2, are known as key regulators of cytoskeletal dynamics, affecting many cellular processes such as proliferation and differentiation [22,23]. While differentiation affects cell morphology, changes in cell shape can in it of itself affect cell fate and differentiation into specific lineages [24,25], including neural cell types [26]. There is growing evidence showing that the RhoA-ROCK pathway plays an essential role in neuronal morphogenesis [23], neuritogenesis [27] and in neuron regeneration [28]. Very recently, there have been reports of Rho/ROCK modulation of lineagespecific differentiation in BMMSCs. However, the data is still scarce and results are mixed; McBeath et al found that Rho/ROCK modulate human BMMSCs in terms of osteogenic versus adipocytic differentiation [14], but Pacary et al found neurogenic differentiation of mouse BMMSCs to be affected by Y-27632 only under hypoxia [15]. We found that Y-27632 does not induce differentiation of PDMCs into mesodermal lineages, but in fact help mediate differentiation into a neural-phenotype under conditions of normoxia. Our data contrasts with the previous two reports, and this is

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very likely due to cell-specific differences, since it has been reported that mouse cellsdthe species of cells used in the Pacary study [29]dmay be more sensitive to oxygen than human cells [30]. In addition, differences in cell density in in vitro culture can greatly affect lineage commitment of stem cells and activation of cytoskeletal pathways [31e33]; in the McBeath et al report, human BMMSCs were cultured in cell densities ranging from isolated, single-cell culture to extremely high densities of 25,000 cells/cm2 [14]. Thus, these major experimental differences may very well account for the contrasting data between these two studies and ours. Y-27632 is known to inhibit both isoforms of ROCK [34], and it now appears that ROCK1 and ROCK2 have distinct role in various aspects of cell function [16], including differentiation [35]. In our study, we found that ROCK2 appears to be the main mediator of inducing neuron-like morphology in PDMCs, whereas RhoA and ROCK1 play smaller contributing roles. The effects of Y-27632 are specific for PDMCs, since treatment of the inhibitor in human epithelial cancer cell linesdone from liver and one from placenta, which is of similar origin to PDMCsddo not replicate such findings (Fig. 4B). These results indicate that the induction of the neural phenotype by Y-27632 is not only dependent on inhibition of RhoA/ ROCK pathway, but also critically influenced by the differentiation potential of the cell. This shows the importance of the context of the specific cell in elucidating the mechanisms of stem cell lineage commitment mechanisms, in terms of translating basic research to clinical applications. 5. Conclusion Our study demonstrates that PDMCs can differentiate into a neural phenotype via inhibition of RhoA/ROCK pathway by Y27632. This is mediated to a large part by ROCK2 and its downstream target, myosin II. Further investigation of the detail mechanisms of neural differentiation is needed to improve the efficiency of differentiation for clinical therapeutic use. The capacity of PDMCs to differentiate into a neural phenotype, coupled with the many advantages inherent to these multipotent cellsdthe lack of ethical concerns, ease of accessibility, and abundant cell numbersdrender these fetal progenitor cells as an attractive cell source for basic research or clinical application of cell therapy. Acknowledgment This study was supported in part by grants from the NHRI (CS102-PP-06 to B.L.Y.) and the National Science Council of Taiwan (NSC101-2321-B-400-013 to B.L.Y.). References [1] Gage FH. Mammalian neural stem cells. Science 2000;287:1433e8. [2] Ostenfeld T, Svendsen CN. Recent advances in stem cell neurobiology. Adv Tech Stand Neurosurg 2003;28:3e89. [3] Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61: 364e70. [4] Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247e56. [5] Rao MS, Mattson MP. Stem cells and aging: expanding the possibilities. Mech Ageing Dev 2001;122:713e34. [6] Yen BL, Huang HI, Chien CC, Jui HY, Ko BS, Yao M, et al. Isolation of multipotent cells from human term placenta. Stem Cells 2005;23:3e9. [7] Yen BL, Chien CC, Chen YC, Chen JT, Huang JS, Lee FK, et al. Placenta-derived multipotent cells differentiate into neuronal and glial cells in vitro. Tissue Eng Part A 2008;14:9e17. [8] Chang CJ, Yen ML, Chen YC, Chien CC, Huang HI, Bai CH, et al. Placenta-derived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-gamma. Stem Cells 2006;24:2466e77.

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