Sang In Park

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.

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Therapy Promotes Functional Recovery of Contused Rat Spinal Cord through Enhancement of Endogenous Cell Proliferation and Oligogenesis

Numerous studies have shown the benefits of mesenchymal stem cells (MSCs) on the repair of spinal cord injury (SCI) model and on behavioral improvement, but the underlying mechanisms remain unclear. In this study, to investigate possible mechanisms by which MSCs contribute to the alleviation of neurologic deficits, we examined the potential effect of human umbilical cord blood-derived MSCs (hUCB-MSCs) on the endogenous cell proliferation and oligogenesis after SCI. SCI was injured by contusion using a weight-drop impactor and hUCB-MSCs were transplanted into the boundary zone of the injured site. Animals received a daily injection of bromodeoxyuridine (BrdU) for 7 days after treatment to identity newly synthesized cells of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells was evident. Behavior analysis revealed that locomotor functions of hUCB-MSCs group were restored significantly and the cavity volume was smaller in the MSCs-transplanted rats compared to the control group. In MSCs-transplanted group, TUNEL-positive cells were decreased and BrdU-positive cells were significantly increased rats compared with control group. In addition, more of BrdU-positive cells expressed neural stem/progenitor cell nestin and oligo-lineage cell such as NG2, CNPase, MBP and glial fibrillary acidic protein typical of astrocytes in the MSC-transplanted rats. Thus, endogenous cell proliferation and oligogenesis contribute to MSC-promoted functional recovery following SCI.

Upregulation of Vascular Endothelial Growth Factor Receptors Flt-1 and Flk-1 Following Acute Spinal Cord Contusion in Rats:

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors. (J Histochem Cytochem 55: 821–830, 2007)

Multiple stem cell traits of expanded rat bone marrow stromal cells.

Bone marrow stromal cells (BMSC) exhibit many traits of a stem cell population. Knowing that BMSC have the ability to self-renew, proliferate and differentiate into a variety of cell types, questions may arise as to whether these traits differ between the cells that have different expansion times. In this study, we examined the stem cell potentiality of BMSC through their characterization, proliferative capacity and the ability to differentiate into multiple lineages in the cultured 2nd passage cells and 10th passage cells. The results were as follows: (1) the 10th passage cells had a larger and more flatted morphology than the 2nd passage cells and also exhibited a decreased labeling for BMSC-related antigens such as CD90, CD73. (2) The cell proliferative capacity was approximately 2 times greater in the 2nd passage cells, and the apoptosis phenomenon was detected in the 10th passage cells. (3) The ability to differentiate into mesodermal tissue (osteocytes, adipocytes), as well as into ectodermal tissue (neurons) was more effective in the 2nd passage cells. Taken together, early stage BMSC would be a valuable cell source for various in vitro applications, as well as cell therapy.

Neural differentiation of brain-derived neurotrophic factor-expressing human umbilical cord blood-derived mesenchymal stem cells in culture via TrkB-mediated ERK and β-catenin phosphorylation and following transplantation into the developing brain.

The ability of mesenchymal stem cells (MSCs) to differentiate into neural cells makes them potential replacement therapeutic candidates in neurological diseases. Presently, overexpression of brain-derived neurotrophic factor (BDNF), which is crucial in the regulation of neural progenitor cell differentiation and maturation during development, was sufficient to convert the mesodermal cell fate of human umbilical cord blood-derived MSCs (hUCB-MSCs) into a neuronal fate in culture, in the absence of specialized induction chemicals. BDNF overexpressing hUCB-MSCs (MSCs-BDNF) yielded an increased number of neuron-like cells and, surprisingly, increased the expression of neuronal phenotype markers in a time-dependent manner compared with control hUCB-MSCs. In addition, MSCs-BDNF exhibited a decreased labeling for MSCs-related antigens such as CD44, CD73, and CD90, and decreased potential to differentiate into mesodermal lineages. Phosphorylation of the receptor tyrosine kinase B (TrkB), which is a receptor of BDNF, was increased significantly in MSC-BDNF. BDNF overexpression also increased the phosphorylation of β-catenin and extracellular signal-regulated kinases (ERKs). Inhibition of TrkB availability by treatment with the TrkB-specific inhibitor K252a blocked the BDNF-stimulated phosphorylation of β-catenin and ERKs, indicating the involvement of both the β-catenin and ERKs signals in the BDNF-stimulated and TrkB-mediated neural differentiation of hUCB-MSCs. Reduction of β-catenin availability using small interfering RNA-mediated gene silencing inhibited ERKs phosphorylation. However, β-catenin activation was maintained. In addition, inhibition of β-catenin and ERKs expression levels abrogated the BDNF-stimulated upregulation of neuronal phenotype markers. Furthermore, MSC-BDNF survived and migrated more extensively when grafted into the lateral ventricles of neonatal mouse brain, and differentiated significantly into neurons in the olfactory bulb and periventricular astrocytes. These results indicate that BDNF induces the neural differentiation of hUCB-MSCs in culture via the TrkB-mediated phosphorylation of ERKs and β-catenin and following transplantation into the developing brain.

Effect of combination therapy with sodium ozagrel and panax ginseng on transient cerebral ischemia model in rats.

Sodium ozagrel (SO) prevents platelet aggregation and vasoconstriction in the cerebral ischemia. It plays an important role in the prevention of brain damage induced by cerebral ischemia/reperfusion. Recently, many animal studies have suggested that the Panax ginseng (PG) has neuroprotective effects in the ischemic brain. In this study, we assessed the neuroprotective effects that come from a combination therapy of SO and PG in rat models with middle cerebral artery occlusion (MCAO). Animals with MCAO were assigned randomly to one of the following four groups: (1) control (Con) group, (2) SO group (3 mg/kg, intravenously), (3) PG group (200 mg/kg, oral feeding), and (4) SO + PG group. The rats were subjected to a neurobehavior test including adhesive removal test and rotarod test at 1, 3, 7, 10, and 15 days after MCAO. The cerebral ischemic volume was quantified by Metamorph imaging software after 2-3-5-triphenyltetrazolium (TTC) staining. The neuronal cell survival and astrocytes expansion were assessed by immunohistofluorescence staining. In the adhesive removal test, the rats of PG or SO + PG group showed significantly better performance than those of the control group (Con: 88.1 ± 24.8, PG: 43.6 ± 11, SO + PG: 11.8 ± 7, P < .05). Notably, the combination therapy group (SO + PG) showed better performance than the SO group alone (SO: 56 ± 12, SO + PG: 11.8 ± 7, P < .05). In TTC staining for infarct volume, cerebral ischemic areas were also significantly reduced in the PG group and SO + PG group (Con: 219 ± 32, PG: 117 ± 8, SO + PG: 99 ± 11, P < .05). Immunohistofluorescence staining results showed that the group which received SO + PG group therapy had neuron cells in the normal range. They also had a low number of astrocytes and apoptotic cells compared with the control or SO group in the peri-infarction area. During astrocytes staining, compared to the SO + PG group, the PG group showed only minor differences in the number of NeuN-positive cells and quantitative analysis of infarct volume. In conclusion, these studies showed that in MCAO rat models, the combination therapy with SO and PG may provide better neuroprotective effects such as higher neuronal cell survival and inhibition of astrocytes expansion than monotherapy with SO alone.

Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.

A Pilot Study for the Neuroprotective Effect of Gongjin-dan on Transient Middle Cerebral Artery Occlusion-Induced Ischemic Rat Brain

In this study, we investigated whether gongjin-dan improves functional recovery and has neuroprotective effects on reducing the infarct volume after transient middle cerebral artery occlusion (MCAo). Infarct volume was measured using TTC staining and glucose utilization by F-18 FDG PET. Functional improvement was evaluated with the Rota-rod, treadmill, Garcia score test, and adhesive removal test. At 14 days after MCAo, neuronal cell survival, astrocytes expansion, and apoptosis were assessed by immunohistofluorescence staining in the peri-infarct region. Also, the expression of neurotrophic factors and inflammatory cytokines such as VEGF, BDNF, Cox-2, TNF-α, IL-1β, and IL-1α was measured in ischemic hemisphere regions. The gongjin-dan-treated group showed both reduced infarct volume and increased glucose utilization. Behavior tests demonstrated a significant improvement compared to the control. Also in the gongjin-dan treated group, NeuN-positive cells were increased and number of astrocytes, microglia, and apoptotic cells was significantly decreased compared with the control group in the ischemic peri-infarct area. Furthermore, the expression of VEGF and BDNF was increased and level of Cox-2, TNF-α, IL-1β, and IL-1α was decreased. These results suggest that gongjin-dan may improve functional outcome through the rapid restoration of metabolism and can be considered as a potential neuroprotective agent.

Therapeutic Effects of Acupuncture through Enhancement of Functional Angiogenesis and Granulogenesis in Rat Wound Healing

Acupuncture regulates inflammation process and growth factors by increasing blood circulation in affected areas. In this study, we examined whether acupuncture has an effect on wound healing in injured rat. Rats were assigned randomly into two groups: control group and acupuncture group. Acupuncture treatment was carried out at 8 sites around the wounded area. We analyzed the wound area, inflammatory cytokines, proliferation of resident cells, and angiogenesis and induction of extracelluar matrix remodeling. At 7 days after-wounding the wound size in acupuncture-treat group was decreased more significantly compared to control group. In addition, the protein levels of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly decreased compared to the control at 2 and 7 days post-wounding. Also, we analyzed newly generated cells by performing immunostaining for PCNA and using several phenotype markers such as CD-31, α-SMA, and collagen type I. In acupuncture-treated group, PCNA-positive cell was increased and PCNA labeled CD-31-positive vessels, α-SMA- and collagen type I-positive fibroblastic cells, were increased compared to the control group at 7 days post-wounding. These results suggest that acupuncture may improve wound healing through decreasing pro-inflammatory response, increasing cell proliferation and angiogenesis, and inducing extracellular matrix remodeling.