Sin-Soo Jeun

Gene Therapy Using TRAIL-Secreting Human Umbilical Cord Blood–Derived Mesenchymal Stem Cells against Intracranial Glioma

Adenovirus-mediated gene therapies against brain tumors have been limited by the difficulty in tracking glioma cells infiltrating the brain parenchyma. Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSC) are particularly attractive cells for clinical use in cell-based therapies. In the present study, we evaluated the tumor targeting properties and antitumor effects of UCB-MSCs as gene delivery vehicles for glioma therapy. We efficiently engineered UCB-MSCs to deliver a secretable trimeric form of tumor necrosis factor-related apoptosis-inducing ligand (stTRAIL) via adenoviral transduction mediated by cell-permeable peptides. We then confirmed the migratory capacity of engineered UCB-MSCs toward tumor cells by an in vitro migration assay and by in vivo injection of UCB-MSCs into the tumor mass or the opposite hemisphere of established human glioma in nude mice. Moreover, in vitro coculture, experiments on Transwell plates, and in vivo survival experiments showed that MSC-based stTRAIL gene delivery has more therapeutic efficacy compared with direct injection of adenovirus encoding the stTRAIL gene into a tumor mass. In vivo efficacy experiments showed that intratumoral injection of engineered UCB-MSCs (MSCs-stTRAIL) significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with controls. These results suggest that human UCB-MSCs have potential use as effective delivery vehicles for therapeutic genes in the treatment of intracranial glioma.

Brain-derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways

Brain‐derived neurotrophic factor (BDNF) plays an important role in the differentiation, development, and survival of neural stem cells. In this study, we analyzed its effects on the stimulation of human umbilical cord blood‐derived mesenchymal stem cells in terms of their potential to differentiate into neuron‐like cells, their survival characteristics, and the molecular mechanisms involved. The treatment of cells with neural induction medium (NIM) and BDNF generated more cells that were neuron‐like and produced stronger expression of neural‐lineage markers than cells treated with NIM and without BDNF. Raf‐1 and ERK phosphorylation and p35 expression levels increased significantly in cells treated with both NIM and BDNF. This treatment also effectively blocked cell death following neural induction and increased Akt phosphorylation and Bcl2 expression compared with cells treated with NIM without BDNF. Inhibition of ERKs inhibited the BDNF‐stimulated up‐regulation of p35 and Bcl2. In addition, the inhibition of PI3K abrogated Akt phosphorylation and Bcl2 expression, but not p35 expression. Thus, MAPK/ERK‐dependent p35 up‐regulation and MAPK/ERK‐dependent and PI3K/Akt‐dependent Bcl2 up‐regulation contribute to BDNF‐stimulated neural differentiation and to the survival of differentiated cells.

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.

Overexpression of CXC chemokine receptors is required for the superior glioma-tracking property of umbilical cord blood-derived mesenchymal stem cells.

Our observations indicate that umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) have a strong migration capacity toward the human glioma cell line, U-87 MG, LN18, U138, and U251, when compared to several other cancer cell lines. In order to identify soluble factors that function to attract UCB-MSCs, we used cytokine antibody arrays to screen changed cytokines in conditioned media from U-87 MG cells. Among these, interleukin-8 (IL-8) and growth-related oncogene (GRO-alpha) enhanced UCB-MSC migration. Furthermore, antibodies treatment against the IL-8 receptors reduced these migration events and overexpression of IL-8 in cells with lower level of IL-8 such as A549 could induce UCB-MSC migration. Since we found that the capacity of UCB-MSC migration is much higher than that of bone marrow-derived MSCs (BM-MSCs) toward either U-87 MG cells or recombinant IL-8, we compared the levels of the IL-8 receptor, CXC chemokine receptor 1 (CXCR1) and CXCR2 between two kinds of MSCs by RT-PCR and immunostaining. Expression levels of two receptors were much higher in UCB-MSCs than in BM-MSCs. These data suggest that higher levels of two IL-8 receptors could influence downstream signaling events affecting superior UCB-MSC migration toward the glioma cells.

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.

Acupuncture Stimulation for Motor Cortex Activities: A 3T fMRI Study

The acupoint, GB34, located in the back of the knee, is known to be effective in recovering motor function after a stroke. This study uses a functional magnetic resonance imaging (fMRI) study with 3T scanner to investigate whether or not acupuncture of GB34 produces a significant response of the modulation of somatomotor areas. A fMRI of the whole brain was performed in ten normal healthy subjects during two task stimulations of acupuncture manipulation on GB34 and sham points, inserting and twisting the needle for 25 seconds at a rate of approximately 120 times per minute; the needle manipulation was paused for a duration of 25 seconds as a control state. The process was repeated four times to have four epochs of stimulation. Bilateral sensorimotor areas (BA 3, 4, 6 and 7) showed approximately 6% of stimulation-related BOLD signal contrast, whereas very few areas were activated when sham stimulation was given. Acupuncture stimulation in GB34 modulates the cortical activities of the somatomotor area in humans. The present findings may shed light on the CNS mechanism of motor function by acupuncture, and form a basis for future investigations of motor modulation circuits in stroke patients.

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.