Seung U. Kim

Therapeutic effect of BDNF-overexpressing human neural stem cells (HB1.F3.BDNF) in a rodent model of middle cerebral artery occlusion.

Ischemic stroke mainly caused by middle cerebral artery occlusion (MCAo) represents the major type of stroke; however, there are still very limited therapeutic options for the stroke-damaged patients. In this study, we evaluated the neurogenic and therapeutic potentials of human neural stem cells (NSCs) overexpressing brain-derived neurotrophic factor (HB1.F3.BDNF) following transplantation into a rodent model of MCAo. F3.BDNF human NSCs (F3.BDNF) were transplanted into the contralateral side of striatum at 7 days after MCAo, and the transplanted animals were monitored up to 8 weeks using animal MRI and various behavioral tests before they were sacrificed for immunohistochemical analysis. Interestingly, animal MRI results indicate that the majority of contralaterally transplanted neural stem cells were migrated to the peri-infarct area, showing a pathotropism. Transplanted animals exhibited significant behavioral improvements in stepping, rotarod, and modified neurological severity score (mNSS) tests. We also found that the transplanted human cells were colocalized with nestin, DCX, MAP2, DARPP-32, TH, GAD65/67-positive cells, of which results can be correlated with neural regeneration and behavioral recovery in the transplanted animals. More importantly, we were able to detect high levels of human BDNF protein expression, presumably derived from the transplanted F3.BDNF. Taken together, these results provide strong evidence that human neural stem cells (F3.BDNF) are effective in treating stroke animal models.

Endothelial STAT3 Activation Increases Vascular Leakage Through Downregulating Tight Junction Proteins: Implications for Diabetic Retinopathy.

Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro‐inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL‐6‐induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO‐1 and occludin. In a co‐culture model with microglia and endothelial cells under a high glucose condition, the microglia‐derived IL‐6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL‐6‐induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO‐1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL‐6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 232: 1123–1134, 2017.

In vivo bioluminescence imaging for prolonged survival of transplanted human neural stem cells using 3D biocompatible scaffold in corticectomized rat model.

Stem cell-based treatment of traumatic brain injury has been limited in its capacity to bring about complete functional recovery, because of the poor survival rate of the implanted stem cells. It is known that biocompatible biomaterials play a critical role in enhancing survival and proliferation of transplanted stem cells via provision of mechanical support. In this study, we noninvasively monitored in vivo behavior of implanted neural stem cells embedded within poly-l-lactic acid (PLLA) scaffold, and showed that they survived over prolonged periods in corticectomized rat model. Corticectomized rat models were established by motor-cortex ablation of the rat. F3 cells expressing enhanced firefly luciferase (F3-effLuc) were established through retroviral infection. The F3-effLuc within PLLA was monitored using IVIS-100 imaging system 7 days after corticectomized surgery. F3-effLuc within PLLA robustly adhered, and gradually increased luciferase signals of F3-effLuc within PLLA were detected in a day dependent manner. The implantation of F3-effLuc cells/PLLA complex into corticectomized rats showed longer-lasting luciferase activity than F3-effLuc cells alone. The bioluminescence signals from the PLLA-encapsulated cells were maintained for 14 days, compared with 8 days for the non-encapsulated cells. Immunostaining results revealed expression of the early neuronal marker, Tuj-1, in PLLA-F3-effLuc cells in the motor-cortex-ablated area. We observed noninvasively that the mechanical support by PLLA scaffold increased the survival of implanted neural stem cells in the corticectomized rat. The image-guided approach easily proved that scaffolds could provide supportive effect to implanted cells, increasing their viability in terms of enhancing therapeutic efficacy of stem-cell therapy.

Pancreatic tumor mass in a xenograft mouse model is decreased by treatment with therapeutic stem cells following introduction of therapeutic genes

Pancreatic cancer is the fourth most common cause of cancer-related mortality. In the present study, we employed 2 types of therapeutic stem cells expressing cytosine deaminase (CD) with or without human interferon-β (IFN‑β), HB1.F3.CD and HB1.F3.CD.IFN-β cells, respectively, to selectively treat pancreatic cancer. The CD gene converts the non-toxic prodrug, 5-flurorocytosine (5-FC), into the toxic agent, 5-fluorouracil (5-FU). In addition, human IFN-β is a potent cytokine that has antitumor effects. To generate a xenograft mouse model, PANC-1 cells (2x10(6)/mouse) cultured in DMEM containing 10% FBS were mixed with Matrigel and were subcutaneously injected into Balb/c nu/nu mice. In the migration assay, the stem cells expressing the CD or IFN-β gene effectively migrated toward the pancreatic cancer cells, suggesting the presence of chemoattractant factors secreted by the pancreatic tumors. In the co-culture and MTT assay, antitumor activity of the therapeutic stem cells was observed in the presence of 5-FC was shown that the growth of PANC-1 cells was inhibited. Furthermore, these effects were confirmed in the xenograft mouse model bearing tumors originating from PANC-1 cells. Analyses by histological and fluorescence microscopy showed that treatment with the stem cells resulted in the inhibition of pancreatic cancer growth in the presence of 5-FC. Taken together, these results indicate that stem cells expressing the CD and/or IFN-β gene can be used to effectively treat pancreatic cancer and reduce the side-effects associated with conventional therapies.

Anticancer Effects of the Engineered Stem Cells Transduced with Therapeutic Genes via a Selective Tumor Tropism Caused by Vascular Endothelial Growth Factor Toward HeLa Cervical Cancer Cells

The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTECs) expressing bacterial cytosine deaminase (CD) and/or human interferon-beta (IFN-β) gene against HeLa cervical cancer and the migration factors of the GESTECs toward the cancer cells. Anticancer effect of GESTECs was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed so as to assess the migration capability of the stem cells to cervical cancer cells. Next, several chemoattractant ligands and their receptors related to a selective migration of the stem cells toward HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by CD gene and it caused the cell death in a co-culture system. When IFN-β was additionally expressed with CD gene by these GESTECs, the anticancer activity was significantly increased. In the migration assay, the GESTECs selectively migrated to HeLa cervical cancer cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GESTECs. These GESTECs transduced with CD gene and IFN-β may provide a potential of a novel gene therapy for anticervical cancer treatments via their selective tumor tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTECs.

Bioimaging of microRNA124a-independent neuronal differentiation of human G2 neural stem cells.

Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell‐specific miRNAs in living organisms. Here, we examined the microarray‐based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron‐specific miR‐9 and miR‐124a during neuronal differentiation of G2 cellsin vitro andin vivo. Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline‐dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real‐time PCR showed that expression of endogenous mature miR‐9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron‐specific mature miR‐124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR‐9 and CMV/Gluc/3×PT_miR‐124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR‐9 was gradually decreased bothin vitro andin vivo in G2 cells induced to differentiate into neurons. However,in vitro andin vivo bioluminescence signals for CMV/Gluc/3×PT_miR‐124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron‐specific miR‐124a is not necessary for doxycycline‐dependent neurogenesis of G2 cells.