Chung Heon Ryu

Migration of human umbilical cord blood mesenchymal stem cells mediated by stromal cell-derived factor-1/CXCR4 axis via Akt, ERK, and p38 signal transduction pathways

Human mesenchymal stem cells (hMSCs) have been used for cell-based therapies in degenerative disease and as vehicles for delivering therapeutic genes to sites of injury and tumors. Recently, umbilical cord blood (UCB) was identified as a source for MSCs, and human UCB-derived MSCs (hUCB-MSCs) can serve as an alternative source of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, migration signaling pathways required for homing and recruitment of hUCB-MSCs are not fully understood. Stromal cell-derived factor-1 (SDF-1), a ligand for the CXCR4 chemokine receptor, plays a pivotal role in mobilization and homing of stem cells and modulates different biological responses in various stem cells. In this study, expression of CXCR4 in hUCB-MSCs was studied by western blot analysis and the functional role of SDF-1 was assessed. SDF-1 induced the migration of hUCB-MSCs in a dose-dependent manner. The induced migration was inhibited by the CXCR4-specific peptide antagonist (AMD3100) and by inhibitors of phosphoinositide 3-kinase (LY294002), mitogen-activated protein kinase/extracellular signal related kinase (PD98059) and p38MAPK inhibitor (SB203580). hUCB-MSCs treated with SDF-1 displayed increased phosphorylation of Akt, ERK and p38, which were inhibited by AMD3100. Small-interfering RNA-mediated knock-down of Akt, ERK and p38 blocked SDF-1 induced hUCB-MSC migration. In addition, SDF-1-induced actin polymerization was also blocked by these inhibitors. Taken together, these results demonstrate that Akt, ERK and p38 signal transduction pathways may be involved in SDF-1-mediated migration of hUCB-MSCs.

Gene Therapy of Intracranial Glioma Using Interleukin 12–Secreting Human Umbilical Cord Blood–Derived Mesenchymal Stem Cells

Clinical trials of gene therapy using a viral delivery system for glioma have been limited. Recently, gene therapy using stem cells as the vehicles for delivery of therapeutic agents has emerged as a new treatment strategy for malignant brain tumors. In this study, we used human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) as delivery vehicles with glioma-targeting capabilities, and modified interleukin-12 (IL-12p40N220Q; IL-12M) as a novel therapeutic gene. We also engineered UCB-MSCs to secret IL-12M (UCB-MSC-IL12M) via tetrameric cell-permeable peptide (4HP4)-mediated adenoviral transduction. We confirmed the migratory capacity of UCB-MSC-IL12M toward GL26 mouse glioma cells by an in vitro migration assay and in vivo injection of UCB-MSC-IL12M into the ipsilateral hemisphere of implanted gliomas in C57BL/6 mice. In vivo efficacy experiments showed that intratumoral injection of UCB-MSC-IL12M significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with control mice. Antitumor effects were associated with increased local IL-12M levels, followed by interferon-γ secretion and T-cell infiltration in intracranial gliomas, as well as antiangiogenesis. Interestingly, tumor-free mice after UCB-MSC-IL12M treatment were resistant to ipsilateral and contralateral tumor rechallenge, which was closely associated with tumor-specific long-term T-cell immunity. Thus, our results provide the rationale for designing novel experimental protocols to induce long-term antitumor immunity against intracranial gliomas using UCB-MSCs as an effective delivery vehicle for therapeutic cytokines including IL-12M.

Irradiation Enhances the Tumor Tropism and Therapeutic Potential of Tumor Necrosis Factor‐Related Apoptosis‐Inducing Ligand‐Secreting Human Umbilical Cord Blood‐Derived Mesenchymal Stem Cells in Glioma Therapy

Irradiation is a standard therapy for gliomas and many other cancers. Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is one of the most promising candidates for cancer gene therapy. Here, we show that tumor irradiation enhances the tumor tropism of human umbilical cord blood‐derived mesenchymal stem cells (UCB‐MSCs) and the therapeutic effect of TRAIL delivered by UCB‐MSCs. The sequential treatment with irradiation followed by TRAIL‐secreting UCB‐MSCs (MSC‐TRAIL) synergistically enhanced apoptosis in either TRAIL‐sensitive or TRAIL‐resistant glioma cells by upregulating the death receptor 5 and by inducing caspase activation. Migration assays showed greater MSC migration toward irradiated glioma cells and the tumor site in glioma‐bearing mice compared with unirradiated tumors. Irradiated glioma cells had increased expression of interleukin‐8 (IL‐8), which leads to the upregulation of the IL‐8 receptor on MSCs. This upregulation, which is involved in the migratory capacity of UCB‐MSCs, was confirmed by siRNA inhibition and an antibody‐neutralizing assay. In vivo survival experiments in orthotopic xenografted mice showed that MSC‐based TRAIL gene delivery to irradiated tumors had greater therapeutic efficacy than a single treatment. These results suggest that clinically relevant tumor irradiation increases the therapeutic efficacy of MSC‐TRAIL by increasing tropism of MSCs and TRAIL‐induced apoptosis, which may be a more useful strategy for cancer gene therapy. STEM CELLS 2010;28:2217–2228

Therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells after intrathecal administration by lumbar puncture in a rat model of cerebral ischemia.

IntroductionStem cell transplantation is a promising therapeutic strategy for the treatment of stroke. Mesenchymal stem cells (MSCs) are a potential cell source for clinical application because they can be easily obtained and cultivated with a high proliferative capacity. The safety and efficacy of cell therapy depends on the mode of cell administration. To determine the therapeutic potential of intrathecal administration of MSCs by lumbar puncture (LP), we administrated human umbilical cord blood-derived MSCs (hUCB-MSCs) intrathecally into the lumbar spinal cord or intravenously into the tail vein in a rat model of stroke, and then investigated whether hUCB-MSCs could enter the brain, survive, and improve post-stroke neurological functional recovery.MethodshUCB-MSCs (1.0 × 106) were administrated three days after stroke induced by occlusion of the middle cerebral artery. The presence of hUCB-MSCs and their survival and differentiation in the brain tissue of the rats was examined by immunohistochemistry. Recovery of coordination of movement after administration of hUCB-MSCs was examined using a Rotarod test and adhesive-removal test on the 7th, 14th, 21st, and 28th days after ischemia. The volume of ischemic lesions seven days after the experimental procedure was evaluated using 2-3-5-triphenyltetrazolium (TTC) staining.ResultsRats receiving hUCB-MSCs intrathecally by LP had a significantly higher number of migrated cells within the ischemic area when compared with animals receiving cells intravenously. In addition, many of the cells administered intrathecally survived and a subset of them expressed mature neural-lineage markers, including the mature neuron marker NeuN and glial fibrillary acidic protein, typical of astrocytes. Animals that received hUCB-MSCs had significantly improved motor function and reduced ischemic damage when compared with untreated control animals. Regardless of the administration route, the group treated with 1 × 106 hUCB-MSCs showed better neurological recovery, without significant differences between the two treatment groups. Importantly, intrathecal administration of 5 × 105 hUCB-MSCs significantly reduced ischemic damage, but not in the intravenously treated group. Furthermore, the cells administered intrathecally survived and migrated into the ischemic area more extensively, and differentiated significantly into neurons and astrocytes.ConclusionsTogether, these results indicate that intrathecal administration of MSCs by LP may be useful and feasible for MSCs treatment of brain injuries, such as stroke, or neurodegenerative disorders.

Valproic acid enhances anti-tumor effect of mesenchymal stem cell mediated HSV-TK gene therapy in intracranial glioma.

Suicide gene therapy of glioma based on herpes simplex virus type I thymidine kinase (HSV-TK) and prodrug ganciclovir (GCV) suffers from the lack of efficacy in clinical trials, which is mostly due to low transduction efficacy and absence of bystander effect in tumor cells. Recently, stem cells as cellular delivery vehicles of prodrug converting gene has emerged as a new treatment strategy for malignant glioma. In this study, we evaluated the anti-glioma effect of suicide gene therapy using human bone marrow mesenchymal stem cells expressing HSV-TK (MSCs-TK) combined with valproic acid (VPA), which can upregulate the gap junction proteins and may enhance the bystander effect of suicide gene therapy. Expression of HSV-TK in MSCs was confirmed by RT-PCR analysis and the sensitivity of MSCs-TK to GCV was assessed. A bystander effect was observed in co-cultures of MSCs-TK and U87 glioma cells by GCV in a dose-dependent manner. VPA induced the expression of the gap junction proteins connexin (Cx) 43 and 26 in glioma cell and thereby enhanced the bystander effect in co-culture experiment. The enhanced bystander effect was inhibited by the gap junction inhibitor 18-β-glycyrrhetinic acid (18-GA). Moreover, the combined treatment with VPA and MSCs-TK synergistically enhanced apoptosis in glioma cells by caspase activation. In vivo efficacy experiments showed that combination treatment of MSCs-TK and VPA significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with single-treatment groups. In addition, TUNEL staining also demonstrated a significant increase in the number of apoptotic cells in the combination treated group compared with single-treatment groups. Taken together, these results provide the rational for designing novel experimental protocols to increase bystander killing effect against intracranial gliomas using MSCs-TK and VPA.

Valproic Acid Downregulates the Expression of MGMT and Sensitizes Temozolomide-Resistant Glioma Cells

Temozolomide (TMZ) has become a key therapeutic agent in patients with malignant gliomas; however, its survival benefit remains unsatisfactory. Valproic acid (VPA) has emerged as an anticancer drug via inhibition of histone deacetylases (HDACs), but the therapeutic advantages of a combination with VPA and TMZ remain poorly understood. The main aim of the present study was to determine whether an antitumor effect could be potentiated by a combination of VPA and TMZ, especially in TMZ-resistant cell lines. A combination of VPA and TMZ had a significantly enhanced antitumor effect in TMZ-resistant malignant glioma cells (T98 and U138). This enhanced antitumor effect correlated with VPA-mediated reduced O6-methylguanine-DNA methyltransferase (MGMT) expression, which plays an important role in cellular resistance to alkylating agents. In vitro, the combination of these drugs enhanced the apoptotic and autophagic cell death, as well as suppressed the migratory activities in TMZ-resistant cell lines. Furthermore, in vivo efficacy experiment showed that treatment of combination of VPA and TMZ significantly inhibited tumor growth compared with the monotherapy groups of mice. These results suggest that the clinical efficacy of TMZ chemotherapy in TMZ-resistant malignant glioma may be improved by combination with VPA.

Mesenchymal Stem Cells Expressing Brain-Derived Neurotrophic Factor Enhance Endogenous Neurogenesis in an Ischemic Stroke Model

Numerous studies have reported that mesenchymal stem cells (MSCs) can ameliorate neurological deficits in ischemic stroke models. Among the various hypotheses that have been suggested to explain the therapeutic mechanism underlying these observations, neurogenesis is thought to be critical. To enhance the therapeutic benefits of human bone marrow-derived MSCs (hBM-MSCs), we efficiently modified hBM-MSCs by introduction of the brain-derived neurotrophic factor (BDNF) gene via adenoviral transduction mediated by cell-permeable peptides and investigated whether BDNF-modified hBM-MSCs (MSCs-BDNF) contributed to functional recovery and endogenous neurogenesis in a rat model of middle cerebral artery occlusion (MCAO). Transplantation of MSCs induced the proliferation of 5-bromo-2′-deoxyuridine (BrdU-) positive cells in the subventricular zone. Transplantation of MSCs-BDNF enhanced the proliferation of endogenous neural stem cells more significantly, while suppressing cell death. Newborn cells differentiated into doublecortin (DCX-) positive neuroblasts and Neuronal Nuclei (NeuN-) positive mature neurons in the subventricular zone and ischemic boundary at higher rates in animals with MSCs-BDNF compared with treatment using solely phosphate buffered saline (PBS) or MSCs. Triphenyltetrazolium chloride staining and behavioral analysis revealed greater functional recovery in animals with MSCs-BDNF compared with the other groups. MSCs-BDNF exhibited effective therapeutic potential by protecting cell from apoptotic death and enhancing endogenous neurogenesis.

Effective Combination Therapy for Malignant Glioma with TRAIL-Secreting Mesenchymal Stem Cells and Lipoxygenase Inhibitor MK886

The apoptotic ligand TRAIL is believed to have promise as a cancer gene therapy, yet many types of cancer, including gliomas, have exhibited resistance to TRAIL-induced apoptosis. Here, we show that therapeutic combination of the lipoxygenase inhibitor MK886 and TRAIL-secreting human mesenchymal stem cells (MSC-TRAIL) provide targeted and prolonged delivery of TRAIL both in vitro and in orthotopic mouse models of glioma. Treatment of either TRAIL-sensitive or TRAIL-resistant human glioma cells with MK886 and MSC-TRAIL resulted in significantly enhanced apoptosis compared with each agent alone. MK886 effectively increased the sensitivity to TRAIL-induced apoptosis via upregulation of the death receptor 5 and downregulation of the antiapoptotic protein survivin in human glioma cell lines and in primary glioma cells. This regulation was accompanied by a substantial increase in caspase activation after combined treatment. Furthermore, in vivo survival experiments and imaging analysis in orthotopic xenografted mice showed that MSC-based TRAIL gene delivery combined with MK886 into the tumors had greater therapeutic efficacy than single-agent treatment. Together, our findings indicate that MK886 combined with MSC-based TRAIL gene delivery may represent a novel strategy for improving the treatment of malignant gliomas.

IL-8 enhances the angiogenic potential of human bone marrow mesenchymal stem cells by increasing vascular endothelial growth factor

The beneficial effects of mesenchymal stem cells (MSCs) are mediated partly by the paracrine production of cytoprotective and trophic factors. Vascular endothelial growth factor (VEGF) is released from MSCs as a paracrine trophic factor and contributes to the therapeutic effects of the stem cell by regulating angiogenesis and promoting revascularization in injured tissues. Interleukin‐8 (IL‐8), an inflammatory chemokine with potent proangiogenic properties, is upregulated in the ischemic brain and has been shown to promote homing of bone marrow‐derived cells to injured sites. However, the effect of IL‐8 on MSCs paracrine function remains unknown. We found that IL‐8 induced VEGF production and phosphorylation of Akt and ERK. Both effects could be blocked by inhibitors (LY294002, PD098059) or siRNA‐mediated silencing of Akt and ERK in human bone marrow MSCs (hBM‐MSCs). IL‐8‐induced VEGF production in hBM‐MSCs significantly increased tube formation on Matrigel compared with basal secreted VEGF. In a rat stroke model, administration of IL‐8‐treated hBM‐MSCs decreased the infarction volume and increased angiogenesis in the ischemic boundary zone compared with hBM‐MSC treatment alone. In conclusion, IL‐8 stimulates VEGF production in hBM‐MSCs in part via the PI3K/Akt and MAPK/ERK signal transduction pathways and that administration of IL‐8‐treated hBM‐MSCs increases angiogenesis after stroke. This approach may be used to optimize MSC‐based therapies for numerous diseases including stroke, myocardial ischemia, and spinal cord injury.

Gene Therapy of Multiple Sclerosis Using Interferon β-Secreting Human Bone Marrow Mesenchymal Stem Cells

Interferon-beta (IFN-β), a well-established standard treatment for multiple sclerosis (MS), has proved to exhibit clinical efficacy. In this study, we first evaluated the therapeutic effects for MS using human bone marrow-derived mesenchymal stem cells (hBM-MSCs) as delivery vehicles with lesion-targeting capability and IFN-β as therapeutic gene. We also engineered hBM-MSCs to secret IFN-β (MSCs-IFNβ) via adenoviral transduction and confirmed the secretory capacity of MSCs-IFNβ by an ELISA assay. MSCs-IFNβ-treated mice showed inhibition of experimental autoimmune encephalomyelitis (EAE) onset, and the maximum and average score for all animals in each group was significantly lower in the MSCs-IFNβ-treated EAE mice when compared with the MSCs-GFP-treated EAE mice. Inflammatory infiltration and demyelination in the lumbar spinal cord also significantly decreased in the MSCs-IFNβ-treated EAE mice compared to PBS- or MSCs-GFP-treated EAE mice. Moreover, MSCs-IFNβ treatment enhanced the immunomodulatory effects, which suppressed proinflammatory cytokines (IFN-γ and TNF-α) and conversely increased anti-inflammatory cytokines (IL-4 and IL-10). Importantly, injected MSCs-IFNβ migrated into inflamed CNS and significantly reduced further injury of blood-brain barrier (BBB) permeability in EAE mice. Thus, our results provide the rationale for designing novel experimental protocols to enhance the therapeutic effects for MS using hBM-MSCs as an effective gene vehicle to deliver the therapeutic cytokines.