Suppression of Sleeping Beauty-induced Gliomagenicity in Ts1Cje Mice, a Model of Down Syndrome.

BACKGROUND/AIM: Individuals with Down syndrome (DS), attributed to triplication of human chromosome 21 (Hsa21), exhibit a reduced incidence of solid tumors. However, the prevalence of glioblastoma among individuals with DS remains a contentious issue in epidemiological studies. Therefore, this study examined the gliomagenicity in Ts1Cje mice, a murine model of DS. MATERIALS AND METHODS: We employed the Sleeping Beauty transposon system for the integration of human oncogenes into cells of the subventricular zone of neonatal mice. RESULTS: Notably, Sleeping Beauty-mediated de novo murine gliomagenesis was significantly suppressed in Ts1Cje mice compared to wild-type mice. In glioblastomas of Ts1je mice, we observed an augmented presence of M1-polarized tumor-associated macrophages and microglia, known for their anti-tumor efficacy in the early stage of tumor development. CONCLUSION: Our findings in a mouse model of DS offer novel perspectives on the diminished gliomagenicity observed in individuals with DS.

Host-to-graft propagation of inoculated α-synuclein into transplanted human induced pluripotent stem cell-derived midbrain dopaminergic neurons.

Introduction: Cell therapeutic clinical trials using fetal mesencephalic tissue provided a proof-of-concept for regenerative therapy in patients with Parkinson's disease. Postmortem studies of patients with fetal grafts revealed that α-synuclein+ Lewy body (LB)-like inclusions emerged in long-term transplantation and might worsen clinical outcomes even if the grafts survived and innervated in the recipients. Various studies aimed at addressing whether host-derived α-synuclein could be transferred to the grafted neurons to assess α-synuclein+ inclusion appearance in the grafts. However, determining whether α-synuclein in the grafted neurons has been propagated from the host is difficult due to the intrinsic α-synuclein expression. Methods: We induced midbrain dopaminergic (mDA) neurons from human induced pluripotent stem cells (hiPSCs) and transplanted them into the striatum of immunodeficient rats. The recombinant human α-synuclein preformed fibrils (PFFs) were inoculated into the cerebral cortex after transplantation of SNCA-/- hiPSC-derived mDA neural progenitors into the striatum of immunodeficient rats to evaluate the host-to-graft propagation of human α-synuclein PFFs. Additionally, we examined the incorporation of human α-synuclein PFFs into SNCA-/- hiPSC-derived mDA neurons using in vitro culture system. Results: We detected human α-synuclein-immunoreactivity in SNCA-/- hiPSC-derived mDA neurons that lacked endogenous α-synuclein expression in vitro. Additionally, we observed host-to-graft α-synuclein propagation into the grafted SNCA-/- hiPSC-derived mDA neurons. Conclusion: We have successfully proven that intracerebral inoculated α-synuclein PFFs are propagated and incorporated from the host into grafted SNCA-/- hiPSC-derived mDA neurons. Our results contribute toward the basic understanding of the molecular mechanisms related to LB-like α-synuclein deposit formation in grafted mDA neurons.

Combination of Drugs and Cell Transplantation: More Beneficial Stem Cell-Based Regenerative Therapies Targeting Neurological Disorders.

Cell transplantation therapy using pluripotent/multipotent stem cells has gained attention as a novel therapeutic strategy for treating neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, ischemic stroke, and spinal cord injury. To fully realize the potential of cell transplantation therapy, new therapeutic options that increase cell engraftments must be developed, either through modifications to the grafted cells themselves or through changes in the microenvironment surrounding the grafted region. Together these developments could potentially restore lost neuronal function by better supporting grafted cells. In addition, drug administration can improve the outcome of cell transplantation therapy through better accessibility and delivery to the target region following cell transplantation. Here we introduce examples of drug repurposing approaches for more successful transplantation therapies based on preclinical experiments with clinically approved drugs. Drug repurposing is an advantageous drug development strategy because drugs that have already been clinically approved can be repurposed to treat other diseases faster and at lower cost. Therefore, drug repurposing is a reasonable approach to enhance the outcomes of cell transplantation therapies for neurological diseases. Ideal repurposing candidates would result in more efficient cell transplantation therapies and provide a new and beneficial therapeutic combination.

Generation of striatal neurons from human induced pluripotent stem cells by controlling extrinsic signals with small molecules.

Human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling human brain development and treating neurodegenerative diseases. Here we established a robust protocol with high scalability for generating striatal medium spiny neurons (MSNs) from hiPSCs using small molecules under two- and three-dimensional culture conditions. Using this protocol, GSH2+ lateral ganglionic eminence (LGE) progenitors were generated in two-dimensional culture by Sonic hedgehog signaling activation using purmorphamine, WNT signaling inhibition using XAV939, and dual-SMAD inhibition using LDN193189 and A83-01. Quantitative PCR analysis revealed sequential expression of LGE and striatal genes during differentiation. These LGE progenitors subsequently gave rise to DARPP32+ MSNs exhibiting spontaneous and evoked monophasic spiking activity. Applying this protocol in three-dimensional culture, we generated striatal neurospheres with gene expression profiles and cell layer organization resembling that of the developing striatum, including distinct ventricular and subventricular zones and DARPP32+ neurons at the surface. This protocol provides a useful experimental model for studying striatal development and yields cells potentially applicable for regenerative medicine to treat striatum-related disorders such as Huntington's disease.

A novel dipeptide type inhibitor of the Wnt/ß-catenin pathway suppresses proliferation of acute myelogenous leukemia cells.

The Wnt/ß-catenin pathway is an attractive target for the treatment of acute myelogenous leukemia (AML), since aberrant activation of the Wnt/ß-catenin pathway contributes to carcinogenesis in various types of cancers including AML. Screening of an in-house compound library, constructed at Kyoto Pharmaceutical University, identified a novel compound designated "31" that was found to be an inhibitor of the Wnt/ß-catenin pathway. The compound inhibited T-cell factor (TCF) activity in a TCF firefly luciferase-reporter assay and suppressed the proliferation of several human AML cell lines in a dose-dependent manner. Compound 31 arrested the cell cycle of AML cells at the G1 stage and induced apoptosis. Decrease in protein and mRNA expression level of Wnt pathway-related molecules was confirmed by the analyses of western blotting and quantitative reverse transcription-polymerase chain reaction. In addition, compound 31 combined with idarubicin synergistically inhibited the proliferation of AML cells. In conclusion, these results strongly suggest that compound 31 has potential as a novel anti-AML agent targeting the Wnt/ß-catenin signaling pathway.

Neuroprotective effects of 5-aminolevulinic acid against neurodegeneration in rat models of Parkinson's disease and stroke.

Oxidative stress is associated with the progression of the neurodegenerative diseases Parkinson's disease (PD) and cerebral ischemia. Recently, 5-aminolevulinic acid (5-ALA), an intermediate in the porphyrin synthesis pathway, was reported to exert antioxidative effects on macrophages and cardiomyocytes. Here, we demonstrated the neuroprotective effects of 5-ALA using rat models of PD and ischemia as well as in vitro in SH-SY5Y cells. 5-ALA partially prevented neurodegeneration in each condition. These results suggest that 5-ALA has a potential for promising therapeutic agent to protect against neurodegeneration exacerbated by oxidative stress.

Mouse Bone Marrow-derived Microglia-like Cells Secrete Transforming Growth Factor-ß1 and Promote Microglial Aß Phagocytosis and Reduction of Brain Aß.

Accumulation of amyloid-ß (Aß) in brain tissue contributes to the pathophysiology of Alzheimer's disease (AD). We recently reported that intrahippocampal transplantation of mouse bone marrow-derived microglia-like (BMDML) cells suppresses brain amyloid pathology and cognitive impairment in a mouse model of AD. How these transplanted cells interact with resident microglia remains unknown. In the present study, we evaluated the effects of cytokines secreted from mouse BMDML cells on cultured mouse microglia. Conditioned medium from BMDML cells increased microglial Aß phagocytosis. High levels of transforming growth factor-ß1 (TGF-ß1) were present in the conditioned medium, and BMDML cells and microglia expressed Tgf-ß1 mRNA and TGF-ß receptor type 1 (TGF-ßR1) protein, respectively. BMDML conditioned medium also induced microglial Smad2/3 phosphorylation. A TGF-ßR1 inhibitor suppressed Smad2/3 phosphorylation and promotion of microglial Aß phagocytosis induced by conditioned medium. Recombinant mouse TGF-ß1 similarly increased microglial Aß phagocytosis and induced Smad2/3 phosphorylation, which were suppressed by the TGF-ßR1 inhibitor. Brain TGF-ß1 levels and resident microglial TGF-ß1R expression were increased by intrahippocampal injection of BMDML cells in a mouse model of AD. Cotreatment with the TGF-ßR1 inhibitor suppressed the ability of transplanted BMDML cells to increase microglial TGF-ß1R expression and decrease hippocampal Aß levels. Taken together, these findings suggested that transplanted BMDML cells secreted TGF-ß1 to stimulate Aß phagocytosis by resident microglia and decrease brain Aß pathology.

Accelerative effects of carbazole-type alkaloids from Murraya koenigii on neurite outgrowth and their derivative's in vivo study for spatial memory.

Murraya koenigii is a medicinal plant that contains several carbazole-type alkaloids as its characteristic constituents. Blood-brain barrier permeable constituents of M. koenigii accelerated neurite outgrowth in PC-12 cells. Nine compounds were isolated from M. koenigii and their effects on neurite outgrowth were examined. Murrayamine-E (8) at 10 µM showed significant effect. Focusing on the carbazole skeleton, we synthesized derivatives to attenuate cytotoxicity. 9-Benzyl-9H-carbazol-4-ol (15) exhibited strong neurite outgrowth accelerative effect. In addition, the novel object recognition test and the Morris water maze test were performed to evaluate memory improvement of 15 in APdE9 mice. Compound 15 tended to improve spatial memory in the Morris water maze test. These results suggest that carbazole derivative 15 would be a seed compound for Alzheimer's disease drug.

Perturbation of the immune cells and prenatal neurogenesis by the triplication of the Erg gene in mouse models of Down syndrome.

Some mouse models of Down syndrome (DS), including Ts1Cje mice, exhibit impaired prenatal neurogenesis with yet unknown molecular mechanism. To gain insights into the impaired neurogenesis, a transcriptomic and flow cytometry analysis of E14.5 Ts1Cje embryo brain was performed. Our analysis revealed that the neutrophil and monocyte ratios in the CD45-positive hematopoietic cells were relatively increased, in agreement with the altered expression of inflammation/immune-related genes, in Ts1Cje embryonic brain, whereas the relative number of brain macrophages was decreased in comparison to wild-type mice. Similar upregulation of inflammation-associated mRNAs was observed in other DS mouse models, with variable trisomic region lengths. We used genetic manipulation to assess the contribution of Erg, a trisomic gene in these DS models, known to regulation hemato-immune cells. The perturbed proportions of immune cells in Ts1Cje mouse brain were restored in Ts1Cje-Erg+/+/Mld2 mice, which are disomic for functional Erg but otherwise trisomic on a Ts1Cje background. Moreover, the embryonic neurogenesis defects observed in Ts1Cje cortex were reduced in Ts1Cje-Erg+/+/Mld2 embryos. Our findings suggest that Erg gene triplication contributes to the dysregulation of the homeostatic proportion of the populations of immune cells in the embryonic brain and decreased prenatal cortical neurogenesis in the prenatal brain with DS.

Role of transient receptor potential vanilloid subtype 4 in the regulation of azoymethane/dextran sulphate sodium-induced colitis-associated cancer in mice.

Ca2+-permeable ion channels, such as transient receptor channels, are one of the potential therapeutic targets in cancer. Transient receptor potential vanilloid subtype 4 (TRPV4) is a nonselective cation channel associated with cancer progression. This study investigates the roles of TRPV4 in the pathogenesis of colitis-associated cancer (CAC) in mice. The role of TRPV4 was examined in azoxymethane (AOM)/dextran sulphate sodium (DSS)-induced murine CAC model. The formation of colon tumours induced by AOM/DSS treatment was significantly attenuated in TRPV4-deficient mice (TRPV4KO). TRPV4 was co-localised with markers of angiogenesis and macrophages. AOM/DSS treatment upregulated the expression of CD105, vascular endothelial growth factor receptor 2, and TRPV4 in wildtype, but the upregulation of CD105 was significantly attenuated in TRPV4KO. Bone marrow chimera experiments indicated that TRPV4, expressed in both vascular endothelial cells and bone marrow-derived macrophages, played a significant role in colitis-associated tumorigenesis. There was no significant difference in the population of hematopoietic cells, neutrophils, and monocytes between untreated and AOM/DSS-treated WT and TRPV4KO on flow cytometric analysis. TRPV4 activation by a selective agonist induced TNF-α and CXCL2 release in macrophages. Furthermore, TRPV4 activation enhanced the proliferation of human umbilical vein endothelial cells. These results suggest that TRPV4 expressed in neovascular endothelial cells and bone marrow-derived macrophages contributes to the progression of CAC in mice.

Peripheral Blood-Derived Microglia-Like Cells Decrease Amyloid-ß Burden and Ameliorate Cognitive Impairment in a Mouse Model of Alzheimer's Disease.

Amyloid-ß (Aß) accumulation in the brain triggers the onset of Alzheimer's disease (AD), and its prevention and elimination are high priorities for anti-AD therapeutic strategies. Microglia, the resident immune cells in the brain, promote Aß clearance by phagocytosis. Previously, we demonstrated that injection of primary cultured rat microglia and mouse bone marrow-derived microglia-like cells into the brain decreases the level of Aß and that intrahippocampal injection of these cells ameliorates cognitive impairment in a mouse model of AD. To advance this cell therapeutic strategy to the clinical stage, less invasive ways of preparing autologous microglia-like cells from elderly patients are required. In this study, we demonstrated that hematopoietic stem cells mobilized from the bone marrow to peripheral blood by administering granulocyte colony-stimulating factor and a CXCR4 antagonist to mice differentiated into microglia-like cells upon stimulation with colony-stimulating factor 1 and interleukin-34. The peripheral blood-derived microglia-like (PBDML) cells expressed microglial markers and engaged in Aß phagocytosis. Although PBDML cells were in an anti-inflammatory state under nonstimulated conditions, they expressed mRNAs encoding proinflammatory cytokines following lipopolysaccharide treatment. PBDML cells injected into the hippocampi of a mouse AD model survived for at least 36 days while phagocytosing Aß, contributed to a reduction in brain Aß burden, and ameliorated cognitive impairment in the mice. These results strongly suggest that PBDML cells are a promising source for the development of a novel cell therapy against AD.

DJ-1 Contributes to Self-renewal of Stem Cells in the U87-MG Glioblastoma Cell Line.

BACKGROUND/AIM: DJ-1, an oncogenic molecule, helps to maintain somatic stem cells by reducing the intracellular level of reactive oxygen species (ROS). This study investigated the role of DJ-1 in glioma stem cells (GSCs). MATERIALS AND METHODS: U87-MG (U87) and U251-MG (U251) glioblastoma cell lines that express wild-type and mutant p53, respectively, were used. These were cultured with DJ-1-targeting siRNA and subjected to a variety of in vitro experiments or intracranial transplantation into nude mice. RESULTS: Knockdown of DJ-1 reduced clonogenicity only in U87 cells, which was rescued by p53 depletion. ROS accumulated in DJ-1-depleted cells, although treatment with N-acetyl cysteine, which quenches ROS, did not affect exhaustion of CSCs among U87 cells by DJ-1 knockdown. In a serial transplantation study, DJ-1 knockdown prolonged the survival of mice in secondary transplantation. CONCLUSION: DJ-1 plays a pivotal role in maintenance of stem cell self-renewal in the U87 cell line.

Bone-Marrow-Derived Microglia-Like Cells Ameliorate Brain Amyloid Pathology and Cognitive Impairment in a Mouse Model of Alzheimer's Disease.

Microglia, the primary immune cells in the brain, sense pathogens and tissue damage, stimulate cytokine production, and phagocytosis to maintain homeostasis. Accumulation of amyloid-ß peptides (Aß) in the brain triggers the onset of Alzheimer's disease (AD). Accordingly, promotion of Aß clearance represents a promising strategy for AD therapy. We previously demonstrated that primary-cultured rat microglia phagocytose Aß, and that transplantation of these cells ameliorates the Aß burden in brains of Aß-injected rats. In this study, we demonstrate that stimulation with colony-stimulating factor-1 efficiently differentiates mouse bone marrow cells into bone marrow-derived microglia-like (BMDML) cells that express markers for microglia, including the recently identified transmembrane protein 119. BMDML cells effectively phagocytose Aß in vitro, with effects comparable to primary-cultured mouse microglia and greater than peritoneal macrophages. RT-qPCR analysis for cytokine mRNA levels revealed that BMDML cells polarize to a relatively anti-inflammatory state under non-stimulated and inflammatory conditions but exert a pro-inflammatory reaction after lipopolysaccharide treatment. Moreover, BMDML cells hippocampally injected into a mouse model of AD are morphologically similar to the ramified and amoeboid types of residential microglia. Comparisons with simulations assuming a uniform distribution of cells suggest that BMDML cells migrate directionally toward Aß plaques. We also detected Aß phagocytosis by BMDML cells, concomitant with a reduction in the number and area of Aß plaques. Finally, we observed amelioration of cognitive impairment in a mouse model of AD after hippocampal injection of BMDML cells. Our results suggest that BMDML cells have potential as a cell-based disease-modifying therapy against AD.

Alpha7 nicotinic acetylcholine receptor-specific agonist DMXBA (GTS-21) attenuates Aß accumulation through suppression of neuronal γ-secretase activity and promotion of microglial amyloid-ß phagocytosis and ameliorates cognitive impairment in a mouse model of Alzheimer's disease.

We previously demonstrated that stimulation of nicotinic acetylcholine receptors (nAChRs) increases amyloid-ß (Aß) phagocytosis in rat microglia and is closely associated with the decrease of brain Aß and amelioration of memory dysfunction in a transgenic mouse model of Alzheimer's disease (AD). Here, we examined the subtypes of nAChRs involved in these beneficial effects. In primary cultures of rat microglia, the α7 nAChR selective agonist 3-[(2,4-dimethoxy)benzylidene]-anabaseine dihydrochloride (DMXBA) promoted Aß and fluorescent latex bead phagocytosis, whereas selective α7 nAChR antagonists suppressed the enhanced Aß phagocytosis. In a transgenic mouse model of AD, administration of DMXBA attenuated brain Aß burden and memory dysfunction. Moreover, DMXBA suppressed γ-secretase activity in solubilized fractions of human neuroblastoma cells and transgenic mouse brain. These results suggested that selective activation of α7 nAChRs promoted microglial Aß phagocytosis and suppressed neuronal γ-secretase activity to contribute to the attenuation of the brain Aß burden and cognitive impairment. Thus, we propose neuronal and microglial α7 nAChRs as new therapeutic targets in the treatment of AD.

Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function.

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies.

CG13250, a novel bromodomain inhibitor, suppresses proliferation of multiple myeloma cells in an orthotopic mouse model.

Multiple myeloma (MM) is characterized by the clonal proliferation of neoplastic plasma cells. Despite a stream of new molecular targets based on better understanding of the disease, MM remains incurable. Epigenomic abnormalities contribute to the pathogenesis of MM. bromodomain 4 (BRD4), a member of the bromodomain and extraterminal (BET) family, binds to acetylated histones during M/G1 transition in the cell cycle promoting progression to S phase. In this study, we investigated the effects of a novel BET inhibitor CG13250 on MM cells. CG13250 inhibited ligand binding to BRD4 in a dose-dependent manner and with an IC50 value of 1.1 µM. It inhibited MM proliferation in a dose-dependent manner and arrested cells in G1, resulting in the induction of apoptosis through caspase activation. CG13250 inhibited the binding of BRD4 to c-MYC promoter regions suppressing the transcription of the c-MYC gene. Administered in vivo, CG13250 significantly prolonged survival of an orthotopic MM-bearing mice. In conclusion, CG13250 is a novel bromodomain inhibitor that is a promising molecular targeting agent against MM.

Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression.

Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer.

Inhibition of monocarboxylate transporter 1 suppresses the proliferation of glioblastoma stem cells.

Recent evidence suggests that a minor subset of cancer cells, termed cancer stem cells (CSCs), have self-renewal and tumorigenic potential. Therefore, the characterization of CSCs is important for developing therapeutic strategies against cancer. Cancer cells rely on anaerobic glycolysis to produce ATP even under normoxic conditions, resulting in the generation of excess acidic substances. Cancer cells maintain a weakly alkaline intracellular pH to support functions. Glioblastoma is an aggressive malignancy with a poor 5-year survival rate. Based on the hypothesis that ion transport-related molecules regulate the viability and function of CSCs, we investigated the expression of ion transport-related molecules in glioblastoma CSCs (GSCs). Quantitative RT-PCR analysis showed that monocarboxylate transporter1 (MCT1) were upregulated in GSCs, and inhibition of MCT1 decreased the viability of GSCs compared with that of non-GSCs. Our findings indicate that MCT1 is involved in the maintenance of GSCs and is a promising therapeutic target for glioblastoma.

Effective internalization of U251-MG-secreted exosomes into cancer cells and characterization of their lipid components.

Exosomes, the natural vehicles of various biological molecules, have been examined in several research fields including drug delivery. Although understanding of the biological functions of exosomes has increased, how exosomes are transported between cells remains unclear. We hypothesized that cell tropism is important for effective exosomal intercellular communication and that parental cells regulate exosome movement by modulating constituent exosomal molecules. Herein, we demonstrated the strong translocation of glioblastoma-derived exosomes (U251exo) into their parental (U251) cells, breast cancer (MDA-MB-231) cells, and fibrosarcoma (HT-1080). Furthermore, disruption of proteins of U251exo by enzymatic treatment did not affect their uptake. Therefore, we focused on lipid molecules of U251exo with the expectation that they are crucial for effective incorporation of U251exo by cancer cells. Phosphatidylethanolamine was identified as a unique lipid component of U251-MG cell-derived extracellular vesicles. From these results, valuable insight is provided into the targeting of U251exo to cancer cells, which will help to develop a cancer-targeted drug delivery system.

Therapeutic effects of human mesenchymal and hematopoietic stem cells on rotenone-treated parkinsonian mice.

To appreciate the potential applications of stem cell technology in neurodegenerative diseases, including Parkinson's disease (PD), it is important to understand the characteristics of the various types of stem cells. In this study, we designed a set of experiments to compare the ability of three types of human stem cells--mesenchymal stem cells (MSCs), bone marrow CD34(+) cells (BM), and cord blood CD34(+) cells (CB)--using rotenone-treated NOD/SCID mice. Rotenone was orally administered once daily at a dose of 30 mg/kg for 56 days to induce a parkinsonian phenotype. Intravenous delivery of CB into rotenone-treated mice was slightly more beneficial than that of MSCs or BM according to both histological and behavioral analyses. Human nucleus (hNu)(+) cells, which are a specific marker of human cells, were observed in the striatum of rotenone-treated mice transplanted with stem cells. These hNu(+) cells expressed tyrosine hydroxylase (TH). Additionally, α-synuclein(+)/TH(+) cells in the substantia nigra pars compacta decreased significantly following stem cell transplantation. Immunohistochemical analysis also revealed that chronic exposure to rotenone decreased glial cell line-derived neurotrophic factor immunoreactivity and that the reduction was improved by each stem cell transplantation. Gene expression analyses revealed that MSCs, BM, and CB expressed several neurotrophic factors. These results suggest that the beneficial effects of intravenous delivery of stem cells into rotenone-treated mice may result not only from a neurotrophic effect but also from endogenous brain repair mechanisms and the potential of intravenous delivery of stem cells derived from an autologous source for clinical applications in PD.