TRANSPLANTATION OF MESENCHYMAL STEM CELLS FOR THE TREATMENT OF PARKINSON’S DISEASE IN A MOUSE MODEL CHAO YIN XIA DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2008                                                                                          TRANSPLANTATION OF MESENCHYMAL STEM CELLS FOR THE TREATMENT OF PARKINSON’S DISEASE IN A MOUSE MODEL CHAO YIN XIA MD A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2008 i  ACKNOWLEDGMENTS First of all, I would like to give my deepest thanks to my mentor, Associate Professor Tay Sam Wah Samuel, Department of Anatomy, National University of Singapore, for his inspirational and righteous role model, invaluable guidance, constant encouragement, infinite patience, and friendly critics throughout my PhD study. I am also greatly indebted to Assistant Professor He Bei Ping, Department of Anatomy, National University of Singapore, for his valuable suggestions and help during this study. I am very grateful to Professor Ling Eng Ang, ex-Head of Anatomy Department, and Professor Bay Boon Huat, current Head of Anatomy Department, National University of Singapore, for their constant support and encouragement, and for their full support in using the excellent research facilities. I would like to express my appreciation to Mrs Ng Geok Lan, Ms Chan Yee Gek and Dr Wu Ya Jun for their excellent technical assistance; Mr P. Gobalakrishnan for his constant guidance in photomicrography; Mdm Ang Lye Gek Carolyne, Mdm Diljit Kaur, Mdm Teo Li Ching Violet for their secretarial assistance. I would like to thank all other staff members and my fellow graduate students at the Department of Anatomy, National University of Singapore for their help and support. I am very happy to give thanks to National University of Singapore for ii  offering me the research scholarship and my supervisor a research grant (R181-000-096-112), without which this work would not have been brought to a reality. I would like to take this opportunity to express my sincere thanks to my dear parents, brother, sister-in-law and niece for their full and endless support for my study. Finally, I am greatly indebted to my beloved husband, Dr Dong Yuan Hong for his encouragement and endless support during my study. iii  This thesis is dedicated to my beloved daughter Dong Min for celebrating 100 days after her birth iv  PUBLICATIONS Various portions of the present study have been published or accepted for publication. International Journals: 1. Chao YX, He BP, Cao Q, Tay SSW (2007) Protein aggregate-containing neuron-like cells are differentiated from bone marrow mesenchymal stem cells from mice with neurofilament light subunit gene deficiency. Neurosci Lett 417(3): 240-5. 2. Chao YX, He BP, Tay SSW. Mesenchymal stem cell transplantation attenuates blood brain barrier damage and neuroinflammation and protects dopaminergic neurons against MPTP toxicity in the substantia nigra in a model of Parkinson’s disease. Submitted, under revision. 3. Chao YX, He BP, Tay SSW. The activating immunoreceptor NKG2D are involved in MPTP induced Parkinson’s disease models. Manuscript in preparation. 4. Chao YX, He BP, Tay SSW. Immune potential of mesenchymal stem cells in vitro. Manuscript in preparation. Conference publications: 1. Chao YX, Tay SSW, Cao Q, He BP (2005) Effect of NFL gene deficiency on neuronal differentiation of bone marrow mesenchymal stem cells. Society for Neuroscience 35th Annual Meeting, Washington DC, USA. 2. Chao YX, He BP, Tay SSW (2006) Immune Potential of Mesenchymal Stem Cells. Society for Neuroscience 36th Annual Meeting, Atlanta, USA. 3. Chao YX, He BP, Tay SSW (2007) Increased blood-brain barrier permeability and infiltration of inflammatory factors in a mouse model of MPTP induced Parkinson’s disease. 7th IBRO World Congress of Neuroscience, Melbourne, Australia. 4. Chao YX, He BP, Tay SSW (2007) Mesenchymal stem cells transplantation attenuates peripheral infiltration of inflammatory factors and protects dopaminergic neurons in the substantia nigra pars compacta against MPTP toxicity. 7th IBRO World Congress of Neuroscience, Melbourne, Australia. 5. Chao YX, He BP, Tay SSW (2007) Activation of JAK/STAT signalling in dopaminergic neurons following 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine treatment and mesenchymal stem cells transplantation in mice. Society for Neuroscience 37th Annual Meeting, San Diego, USA. v  6. Chao YX, He BP, Tay SSW (2007) Involvement of peripheral inflammatory factors in the pathogenesis of MPTP induced Parkinson’s disease. 6th ASEAN Microscopy Conference, Pahang, Malaysia. vi  TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………………………….i DEDICATIONS……………………………………………………… ………… iii PUBLICATIONS….………………………………………………………………iv TABLE OF CONTENTS………………………………………………………vi ABBREVIATIONS…………………………………………………………… .xiv SUMMARY………………………………………………………………………xix CHAPTER 1: INTRODUCTION………………………………………… .1 1. Etiology of PD………………………………………………………………… 1.1. Genetic mutations in PD…………………………………………………… 1.1.1. PARK1 (α-Synuclein, SNCA, PARK4) ……………………………… 1.1.2. PARK2 (Parkin, an E3-ubiquitin ligase) …………………………… .4 1.1.3. PARK6: PTEN-induced kinase 1(PINK1)…………………………… .5 1.1.4. PARK7: DJ-1……………… …………………… . ………………… 1.1.5. PARK8: leucine-rich repeat kinase (LRRK2)… .6 1.2. Environmental factors related to PD ……………………………………… 2. Animal models…………………………………………………………………… .7 2.1. The 6-OHDA model………………………………………………………… 2.2. The MPTP model……………………………………………… 2.3. The rotenone model……………………………………………… .10 vii  2.4. The Paraquat and Maneb………………………………………………… .11 3. Review on the pathogenesis of PD……………………………………………… 12 3.1. Neuronal alteration in the SNc of PD…………………………………… .…12 3.1.1. Loss of dopaminergic neurons and Lewy body formation………… .12 3.1.2. Mitochondrial dysfunction and oxidative stress…………………… .13 3.2. Inflammation as a causative factor in the pathogenesis of PD…………… 14 3.2.1. Immune reaction in the CNS of PD……………………………… .14 3.2.2. Involvement of systemic immunity……………………………… …17 3.3. Impairment of the blood-brain barrier…………………………………… .19 4. Review on the management of PD…………………………………………… ….22 4.1. Anti-inflammation………………………………………………………… .22 4.2. Stem cell transplantation………………………………………………… .23 4.2.1. Historical discovery of MSCs……………………………………… 23 4.2.2. Biological characteristics of MSCs………………………………… 24 4.2.3. Proliferation of MSCs……………………………………………… 24 4.2.4. Multipotent differentiation of MSCs……………………………… 25 4.2.5. Immune modulatory effect of MSCs……………………………… 26 4.2.6. Mechanisms of immune modulation of MSCs…………………… .26 4.2.7. Systemic administration of MSCs………………………………… 28 4.2.8. Therapeutic potential of MSCs to treat CNS diseases and injuries… 29 5. Hypothesis……………………………………………………………………… 31 6. Aims of the present study……………………………………………………… .32 viii  6.1. Analysis of the dopaminergic neuron degeneration in the SNc after MPTP-treatment……………………………………………………… .32 6.1.1. Study of pathological changes of the dopaminergic neurons in the SNc after MPTP-treatment……………………………………………………… ….32 6.1.2. Study of the activation of microglial cells in the SNc after MPTP-treatment.………………………………………………………… 33 6.1.3. Study of the changes in integrity of blood-brain barrier in the SNc after MPTP-treatment………………………………………………………… .33 6.1.4. 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Neuroscience Letters 417 (2007) 240–245 Protein aggregate-containing neuron-like cells are differentiated from bone marrow mesenchymal stem cells from mice with neurofilament light subunit gene deficiency Yin Xia Chao, Bei Ping He ∗ , Qiong Cao, Samuel Sam Wah Tay ∗ Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, MD10, Medical Drive, Singapore 117597, Singapore Received 21 July 2006; received in revised form February 2007; accepted 19 February 2007 Abstract Autologous bone marrow mesenchymal stem cell (MSC) transplantation has great potential in cell therapy used for the treatment of neurodegenerative disorders. Since many genetic deficiencies have been reported in pathogenesis of the diseases, genetic backgrounds of donor stem cells should be concerned. In this study, effects of neurofilament light subunit (NFL) gene deficiency on proliferation and neuronal differentiation of MSCs were studied in vitro. Lower proliferation rate was observed in NFL−/− MSCs. When exposed to retinoic acid (RA), both NFL−/− and normal MSCs could express several markers of neuronal lineage, such as Nestin, MAP-2, NeuN, O4 and GFAP. However, the NFL expression at mRNA and protein levels was observed only in normal MSCs but absent in NFL−/− MSCs. Significant reductions in amount of neurofilament heavy subunit (NFH) protein and number of neuron-like cells were detected in differentiated NFL−/− MSCs. Interestingly, NFH positive protein accumulations were observed in the neuron-like cells derived from NFL−/− MSCs. These accumulations were perinuclear and morphologically similar to protein aggregations in motoneurons of the spinal cord in NFL−/− mice. The results suggest that NFL gene deficiency could retard MSCs proliferation and neuronal generation, even though the capability of neuronal lineage differentiation of MSCs may not be deterred. Moreover, the NFL−/− MSCs differentiated neuron-like cells carried on the genetic and pathologic deficiency, suggesting that the genetic quality of donor cells must not only be tested, but also modified before transplantation. This also points towards the possibility of creating a stem cell-derived cell model for pathogenesis study. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Mesenchymal stem cells; Neurofilament light subunit; Neuronal differentiation; Proliferation Stem cell transplantation offers great hope in treatment of neurodegenerative disorders. Although embryonic stem cells and stem cells derived from fetal brain have been considered as candidates for transplantation therapy [5,7,20], limitations in histocompatibility and ethical concerns would hinder their clinical application. Autologous stem cells, free from histocompatibility and ethical concerns, may therefore be advantageous in cell therapy. MSCs are one of the most hopeful cell sources in treatment of neurodegenerative diseases [3,4,8]. Experiments have shown that efficient induction of neurons, without glial differentiation, could be achieved in human or rat MSCs by transfer of Notch1 intracellular domain gene plus adminis- ∗ Corresponding authors. Tel.: +65 65167809; fax: +65 67787643. E-mail addresses: anthebp@nus.edu.sg (B.P. He), anttaysw@nus.edu.sg (S.S.W. Tay). 0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.02.082 tration of certain trophic factors [7]. In addition, glial cell line-derived neurotrophic factor could also effectively induce cell differentiation from MSCs to dopamine-producing cells and result in functional recovery after such differentiated cells were grafted into the brain of Parkinson’s disease (PD) model [4]. For the cell replacement strategy, it is understandable that the quality of donor cells must be considered because a large number of gene deficiencies or mutations are currently known to be responsible for various neurodegenerative diseases. For example, ␣-synuclein gene mutation has been confirmed to be associated with familial PD [13] and SOD-1 mutation with familial amyotrophic lateral sclerosis (ALS) [14]. An intriguing possibility to consider is whether similar abnormalities in gene expression occur in differentiated stem cells derived from the same patients or from fetal stem cells with certain gene deficiencies. Y.X. Chao et al. / Neuroscience Letters 417 (2007) 240–245 In this study, we have adopted the transgenic mice with targeted disruption of NFL gene [21] to address our concern on the issue of genetic background of stem cells in clinical application. There are five types of intermediate filament proteins (IFs) expressed in mature neurons, including ␣-internexin, peripherin and three neurofilaments, i.e., NFL, NFM and NFH. These subunits co-assemble as heteropolymers through their central helical domain in normal mature neurons. The NFL deficiency has been reported in some neurological disorders, including Alzheimer’s disease (AD), PD and ALS [17,18] and the significant down-regulation in the expression of NFL mRNA has been observed in degenerating neurons of ALS, PD and AD [2,10,11,19]. Furthermore, protein aggregation has been clearly detected in vivo in the cytoplasm as well as processes of neurons after targeted disruption of NFL because of mal-assembly and intracellular redistribution of IFs [21]. It is important to note that the abnormal accumulation of IFs in neurons is a pathological hallmark for many human neurodegenerative diseases. Both clinical manifestations of NFL in neurodegenerative diseases and several lines of converging evidence have shown that disruption of NFL may result in not only the formation of protein aggregations in neurons but also retardation in neuronal proliferation in the spinal cord of NFL−/− mice during development [21]. This study has therefore examined the consequence of targeted disruption of NFL on MSCs proliferation and differentiation in vitro. All procedures were in accordance with guidelines of Institutional Animal Care and Use Committee. Ten NFL−/− mice (5 males and females, a generous gift of Dr. M.J. Strong and Dr. J.P. Julien) and 10 non-transgenic controls (5 males and females) of the C57BL/6J strain at 8–10 weeks of age were employed to obtain MSCs. A 22-gauge needle filled with Dulbecco’s Modified Eagle’s Medium (DMEM) was used to flush out whole bone marrow from the tibia and femur. The recovered cells were filtered through a 70 ␮m mesh and plated in 75 mm tissue culture plates containing DMEM supplemented with 10% fetal bovine serum, mM l-glutamate, 100 U/ml penicillin and 100 ␮g/ml streptomycin. Twenty-four hours later, non-adherent cells were removed with total replacement of culture medium. At confluency, the remaining adherent cells were further subcultured at the 1:3 dilutions for weeks. Finally, MSCs were ready for subsequent experiments. To determine whether targeted disruption of NFL gene could impose any inhibitory effect on MSCs growth, the proliferation was assessed using Cell Titer96TM AQueous One Solution Cell Proliferation Assay kit (Promega, USA). MSCs at a density of × 103 cells/well with 200 ␮l medium were cultured in 96-well plates in triplicate at 37 ◦ C and 5% CO2 environment. The cell viability was measured at different time points for consecutive days according to the protocol provided by the company. Briefly, 20 ␮l of MTS (5 mg/ml) were added into each well of 96-well plates for h and then cell viability spectrophotometrically quantified using an ELISA plate reader at 490 nm (BioRad, USA). As no additional interruption in cell metabolism and no cell death were induced in the cultures, the disparity in cell viability would therefore mainly represent the difference in the number of viable cells [12]. A standard curve was 241 generated by serial dilution of normal MSCs, wherein the maximum was 5.1 × 104 cells/well and the minimum 2.55 × 103 . The number of MSCs was extrapolated from the standard curve. To study if differentiated neuron-like cells from NFL−/− MSCs would express NFL or NFH and form protein aggregates, MSCs at 80% confluence were trypsinized and then sub-cultured in 6-well plates at a density of × 105 cells/well with or without poly-l-lysine coated coverslips. Epidermal growth factor (20 ng/ml, Sigma, USA) was added to culture medium for 24 h before it was replaced with trans-retinoic acid (RA) (30 ␮M, Sigma) containing media. The cultures were re-fed with fresh medium containing 30 ␮M RA every or days. At 10 days after RA treatment, cells were studied by immunocytochemistry, RT-PCR and Western blot. Neural differentiation of MSCs was demonstrated using immunohistological staining. RA-treated MSCs on coverslips were fixed in 4% paraformaldehyde for 10 at room temperature. After fixation, the coverslips were incubated with 5% normal serum for h. Following blocking, rabbit anti-MAP2 (1:500), rabbit anti-NeuN (1:1000), rabbit anti-glial fibrillary acidic protein (GFAP) (1:1000), rabbit anti-NFH (1:5000), rabbit anti-O4 (1:1000) and mouse anti-Nestin (1:500, all antibodies from Chemicon, USA) were applied, respectively, at ◦ C overnight. Specific labeling for various proteins was either revealed by DAB colorization after the cells were processed with goat anti-rabbit or anti-rat secondary antibody for h and Elite ABC complex for 30 min, or detected after incubation with secondary antibodies conjugated with FITC or Cy3. For ABC method, the coverslips with cells were dehydrated in alcohol, cleared in xylene and then mounted using permount. For immunofluorescent method, the coverslips were mounted on slides using Vectashield mounting medium. The staining was viewed using conventional and confocal microscopes. NFH Fig. 1. Lower proliferation rate of NFL−/− MSCs. Since NFL−/− and normal MSCs were cultured in the same condition and no obvious cell death observed in various time intervals, significant decreases in OD value of formazan product in MTS assay in NFL−/− MSCs in comparison with that in normal MSCs at different time point have indicated less number of viable NFL−/− MSCs in the culture (* p < 0.01). 242 Y.X. Chao et al. / Neuroscience Letters 417 (2007) 240–245 positive neuron-like cells were blindly counted from three coverslips of each group. The expressions of NFL mRNA in RA-treated MSCs were analyzed with RT-PCR. Total RNA was extracted using RNeasy Mini Kit (Qiagen, USA). NFL sense primer was -atcagcaacgacctcaagtctat-3 and antisense primer tactatacctgcatggcgctaat-3 [21]. As the primer covers the knockout domain of the peptide, it is possible to identify two different-sized mRNA, i.e., the normal NFL mRNA and disrupted NFL mRNA whereby a 720 bp fragment was replaced by a 1000 bp neo cassette coding for neomycin. One microgram of total RNA per sample was denaturated at 70 ◦ C for 10 min. The RNA template was added to RT-PCR reaction mixture containing ␮l of reverse transcription 10× buffer, mM MgCl2 , 10 mM DTT, mM dNTPs, 0.5 ␮g Oligo(dT)15, unit/␮l RNAse inhibitor and 15 units/␮g AMV Reverse Transcriptase (Promega, USA). The final mixture was incubated at 42 ◦ C for 15 and then 95 ◦ C for min. After reverse transcription reactions, cDNA amplification was performed in a final volume of 25 ␮l containing 1× PCR buffer, 0.2 mM dNTPs, 0.4 ␮M primers, unit Taq polymerase (Qiagen, USA), and and ␮l cDNA in the first and second rounds of PCR, respectively. The reaction mixture was subjected to 40 PCR cycles at 94 ◦ C for and at 60 ◦ C for min. The last of each PCR cycle were run at 72 ◦ C. Final extensions were performed at 68 ◦ C for before soaking of the amplified PCR products in −20 ◦ C. The amplified PCR products were electrophoresed for 45 at 95 V through 5% agarose gel containing 0.5 ␮g/ml ethidium bromide. One hundred base pairs DNA ladder (Promega, USA) was used as a molecular weight marker and gel was visualized under UV light. Western blotting was employed to analyze expressions of NFL and NFH proteins. The cell homogenates (25 ␮g) were separated using 10% SDS-polyacrylamide gels and the proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes. After incubation with 5% non-fat milk in 0.1% Tween-20 TBS (TTBS) for h, the PVDF membrane was then incubated overnight with monoclonal antibody to NFL (1:500) or polyclonal antibody to NFH (1:5000) in 1% bovine serum albumin in TTBS. The membrane was then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000) or goat anti-rabbit IgG (1:5000, Pierce, USA) for Fig. 2. MSCs differentiation. After RA treatment, NFH−/− MSCs can be induced to differentiate into cells expressing Nestin (A, marker for neural precursor cells, red), MAP-2 (B, for neurons, green), NeuN (C, for neurons, red), O4 (D, for oligodendrocytes, green) or GFAP (E, for astrocytes, red). In MAP-2, NeuN, GFAP and O4 staining, the nuclei are counterstained with DAPI (blue). A NeuN-positive cell with its nucleus stained in red (C) is observed to locate closely with a NeuN-negative cell. Both NFL−/− and normal MSCs could be induced to transdifferentiate into NFH positive neuron-like cells. However, in NFL−/− MSCs derived neuron-like cells, perikaryal NFH positive accumulation is observed in immunohistological staining (F, arrow), compared with the normal control (G). The protein aggregations can also be observed in the cytoplasm of motoneurons in the spinal cord of NFL−/− mice (H, arrow), which serve as positive control (bar = 50 ␮m for all micrographs). Y.X. Chao et al. / Neuroscience Letters 417 (2007) 240–245 h at room temperature. Immunoreactivity was visualized using a chemiluminescent substrate (Supersignal West Pico, Pierce). Loading controls were carried out by incubating the blots at 50 ◦ C for 30 with stripping buffer (100 mM 2mercaptoethanol, 2% SDS and 62.5 mM Tris–hydrochloride, pH 6.8), followed by reprobing with a mouse monoclonal antibody to ␤-actin (1:5000 in TTBS, Sigma) and horseradish peroxidaseconjugated anti-mouse IgG (1:5000 in TTBS, Pierce). Exposed films containing blots were scanned and the densities of the bands measured using Quantity One Version software (BioRad). Densities of investigated bands were normalized against those of ␤-actin and the mean ratios calculated. Cell proliferation, differentiation and Western blot were analyzed using ANOVA. Significance was set at p < 0.05 or more stringent. Although plating cell numbers were the same, NFL−/− MSCs grew in culture dishes at a lower density in comparison with normal MSCs when viewed at daily check-up. Trypan blue staining showed that no obvious cell death occurred in the culture medium of both types of MSCs till at least days of culture. However, the MTS assay has shown that the proliferation of NFL−/− MSCs measured by viable cells was significantly lower than that of normal MSCs (Fig. 1). In vitro multiple-lineage differentiation of adult MSCs from both mice was confirmed based on our established protocols [6,9]. Especially, after the RA treatment, MSCs of both genotypes have differentiated into Nestin(+) neural precursor cells, MAP-2(+) or NeuN(+) neuron-like cells, GFAP(+) astrocytelike and O4(+) oligodendrocyte-like cells (Fig. 2A–E). RT-PCR results have confirmed that the RA treatment could induce expressions of NFH mRNA in both MSCs and NFL mRNA in normal but not NFL−/− MSCs. Interestingly, an abnormal band about 400 bp larger than normal NFL gene was detected in NFL−/− MSCs after the RA treatment (Fig. 3). In concert with mRNA expressions, the Western blot showed an absence in NFL protein expression in differentiated NFL−/− MSCs. Although NFH expression can be detected in both MSCs, a significant reduction in amount of NFH protein was observed in differentiated NFL−/− MSCs (p < 0.05) (Fig. 4). The number of NFH positive neuron-like cells increased from 9.92 ± 1.41 (without induction) to 33.39 ± 3.30 per 1000 cells (after induction) in C57BL/6J MSCs but the increase was from 6.79 ± 0.59 to 12.73 ± 1.94 per 1000 cells in NFL−/− MSCs. Less neuron-like cells were differentiated from NFL−/− MSCs after induction 243 Fig. 3. NFL gene expression. The primers were designed to cover neomycin gene (1100 bp), the selective marker for NFL knockout by replacement of the depleted NFL sequence (700 bp). The introduction of the neomycin gene into the NFL depletion sequence has resulted in an increase in size of gene expressed from 840 bp in normal MSCs to 1240 bp in NFL−/− MSCs after RA treatment, suggesting that the quality of differentiated neuron-like cells might have been altered even though NFL−/− MSCs still possess the ability to differentiate into neuron-like cells. in comparison to C57BL/6J MSCs (p < 0.01). Significantly, dense protein accumulation indicated by NFH immunostaining was found in the cytoplasm of differentiated NFL−/− MSCs (Fig. 2F) and the number of neuron-like cells with protein aggregation increased to 8.04 ± 2.32 per 1000 cells at 10 days after induction, in comparison to 1.61 ± 1.62 per 1000 cells without induction (p < 0.01). No such dense NFH accumulation was found in the cytoplasm of normal MSCs with or without the RA treatment (Fig. 2G). Morphologically, the protein accumulation located at perinuclear position greatly resembled protein aggregation in the cytoplasm of motoneurons in the spinal cord of NFL−/− mice (Fig. 2H). It would be ideal to replace diseased or injured neurons with newly generated neurons from self-originated stem cells in cell therapy. Although MSCs from the bone marrow have been confirmed to possess stem cell properties of self-renewal and multi-transdifferentiation, doubts are still cast on the ability of MSCs to differentiate into neurons. In this study, we have employed the neurodegenerative mouse lack of NFL to isolate adult MSCs and demonstrated their ability in neural transdifferentiation from another point of view. Neurofilaments are important and specific molecular components of neurons which are normally involved in the maintenance of neuronal caliber [1,16]. In concert with other reports [4,6,9], our results showed that MSCs from both mice could be induced by the RA treatment to express GFAP, O4, Nestin, MAP-2 or NeuN, suggesting that MSCs possess neuronal lineage differentiation potential. Our results further demonstrated that MSCs from normal mice could differentiate into cells expressing both Fig. 4. Western blot analysis of NFL and NFH expressions. After RA treatment, NFL expression (A) is totally absent in NFL−/− MSCs. NFH expression (A) can be observed in both NFL−/− and normal MSCs, however, the amount of NFH (B) is significantly reduced (p < 0.05) in the NFL−/− MSCs. 244 Y.X. Chao et al. / Neuroscience Letters 417 (2007) 240–245 NFH and NFL, pointing out another evidence of neuronal properties of differentiated MSCs. Interestingly, no NFL expression at both mRNA and protein levels was detected in differentiated NFL−/− cells after the RA treatment. However, the dense perinuclear protein accumulation indicated by NFH staining was observed in some differentiated NFL−/− cells and morphologically the NFH-positive protein aggregation was similar to that in the cytoplasm of the motoneuron in the spinal cord of NFL−/− mice. The neurofilament aggregations in the perikarya of neurons are thought to be caused by the failure in normal assembly of NFL, NFM and NFH triplets after the targeted disruption of NFL in mice [21]. Protein accumulation is one of hallmarks in neurodegenerative diseases, such as ALS [21], PD [18] and AD [17]. Although NFL gene may not be a real causative factor for PD [15], down-regulation of NFL mRNA expression has been reported in some cases of these diseases [2,10,11,19]. The ability of MSCs to differentiate into protein aggregate-containing neuron-like cells in this study has definitely not only added further token to the MSC potential in generation of neurons, but also provided a possible approach as a cell model to study pathogenesis of neurodegenerative diseases. There were about 20% less neurons in the spinal cord of NFL−/− mice [21], suggesting a deficiency in neuronal differentiation in neuronal progenitors. In agreement with differentiation deficiency of neuronal progenitors in vivo, our cell proliferation assessment has shown a significant decrease in proliferation of MSCs in vitro in comparison with that in normal MSCs. Significant reductions in amount of NFH protein and in the number of neuron-like cells in differentiated NFL−/− MSCs compared with that in differentiated normal MSCs may also indicate a deficiency in neuronal differentiation. Although we not understand the exact mechanism involved in this reduced proliferation and neuronal differentiation in both neuronal progenitors and MSCs, NFL might be somehow important for stem cell renewal and differentiation both in vivo and in vitro. In conclusion, the generation of protein-accumulating neurons in vitro has not only confirmed the neuron generating ability of MSCs but also points out a serious concern for application of stem cells in cell therapy, where the differentiation could inherit the feature of the original gene deficiency. The newly generated genetically deficient cells might not be able to fulfill the purpose of replacement of original diseased cells because the adult stem cells may possess the same genetic deficiency and the embryonic stem cells may have other genetic deficiencies. It is important in cell replacement strategy to monitor the stem cell genetic background and to modify the genetic deficiency if any is present. 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Strong, Characterization of neuronal intermediate filament protein expression in cervical spinal motor neurons in sporadic amyotrophic lateral sclerosis (ALS), J. Neuropathol. Exp. Neurol. 59 (2000) 972–982. 245 [20] S.C. Zang, M. Wernig, I.D. Duncan, O. Br¨ustle, J.A. Thomson, In vitro differentiation of transplantable neural precursors from human embryonic stem cells, Nat. Biotechnol. 19 (2001) 1129–1133. [21] Q. Zhu, S. Couillard-Despres, J.P. Julien, Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments, Exp. Neurol. 148 (1997) 299–316. [...]... illustrates that an acute insult to the SN can result in a sustained inflammatory response (Barcia et al., 2005; Sawada et al., 2007) It is therefore conceivable that in PD, as in humans, primates and rodents exposed to MPTP, an acute insult initiates an inflammatory reaction that becomes self sustaining after the initiating agent has disappeared The MPTP model of PD has been invaluable in the studying of. .. Alzheimer´s disease It affects approximately 1% of the population by the age of 65 years, increasing to 4% to 5% of the population by the age of 85 years (Dawson and Dawson, 2003) The prevalence rate in those aged 50 years old and above in Singapore is 3.0‰ and increases with age (Tan et al., 2004) .The neuropathological hallmarks are characterized by progressive loss of dopaminergic neurons in the substantia... SNc of MPTP-treated mice, real-time RT-PCR did not find mRNA expression of MBL in the brain parenchyma, suggesting the infiltration of MBL from the circulation into the brain MBL may play an important role in the activation of microglia Immunohistochemistry also revealed migration of NK cells into brain parenchyma as SUMMARY xxi a consequence of increase in permeability of the BBB The NK cell activation... to date, it is of great importance to develop animal models for the treatment of this disease A variety of experimental PD models has been established using pharmacological agents or environmental toxins in different species The following sections of this review will discuss the advantages and disadvantages of the most commonly used animal models of PD and their potential roles in revealing the pathogenesis... (Saint-Pierre, 2006) and drug abuse have been found to be related to PD in people who have been exposed to them These environmental factors will be reviewed in detail in the following part regarding the animal models of PD INTRODUCTION 7 2 Animal models Animal models are an important aid to study pathogenic mechanisms and therapeutic strategies in human diseases Since the pathogenesis and therapy of PD are still... 99% loss of dopamine in the striatum The schematic drawing of mechanism(s) of MPTP-induced cell death (Fig 1) indicates a great amount of cross-talk between the neurons and the non-neuronal milieu For those drug abusers, autopsies done years later after they developed Parkinsonism demonstrated activated microglia in the SN similar to that observed in PD cases (Langston et al, 1999) Animal model also illustrates... of the mechanisms of PD pathogenesis, for example, the mechanisms of microglial activation and their potential damage to the adjacent dopaminergic neurons Less is known about the role of astrocytes than microglia, but they are known to secrete both inflammatory and anti-inflammatory molecules and may play a role in modulating microglial activity Oligodendrocytes do not seem to play a role in promoting... significant depletion of striatal dopamine (Braak, 2003) and Lewy bodies (LBs) in survived dopaminergic neurons LBs are intracytoplasmic inclusion bodies composed mainly of neurofilament-like structures that also stain positively for ubiquitin and α-synuclein.Ubiquitin is a protein involved in the degradation of cytoplasmic proteins The function of α-synuclein is still not clear In the healthy brain, α-synuclein... of rotenone results in uniform inhibition of complex I throughout the rat brain but selective degeneration of the nigrostriatal dopaminergic neurons, selective striatal oxidative damage, and formation of ubiquitin/α-synuclein-positive inclusions in nigral cells, which are similar to the Lewy bodies of PD patients (Betarbet et al., 2000) The rotenone model appears to be an accurate model in that systemic... of PD together with the possible therapeutics 2.1 The 6-OHDA model 6-hydroxydopamine (6-OHDA) was the first chemical agent that was discovered to have specific neurotoxic effects on catecholaminergic pathways (Butcher，1975) Since systemically administered 6-OHDA is unable to cross the blood-brain barrier, it has to be injected stereotactically into the substantia nigra, the nigrostriatal tract or the .  TRANSPLANTATION OF MESENCHYMAL STEM CELLS FOR THE TREATMENT OF PARKINSON’S DISEASE IN A MOUSE MODEL CHAO YIN XIA DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF. Chao YX, He BP, Tay SSW (2007) Mesenchymal stem cells transplantation attenuates peripheral infiltration of inflammatory factors and protects dopaminergic neurons in the substantia nigra pars. 2. Chao YX, He BP, Tay SSW. Mesenchymal stem cell transplantation attenuates blood brain barrier damage and neuroinflammation and protects dopaminergic neurons against MPTP toxicity in the substantia