Hong Bae Jeon

Comparative Analysis of Human Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue, and Umbilical Cord Blood as Sources of Cell Therapy

Various source-derived mesenchymal stem cells (MSCs) have been considered for cell therapeutics in incurable diseases. To characterize MSCs from different sources, we compared human bone marrow (BM), adipose tissue (AT), and umbilical cord blood-derived MSCs (UCB-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, and paracrine activity. Although MSCs from different tissues have similar levels of surface antigen expression, immunosuppressive activity, and differentiation ability, UCB-MSCs had the highest rate of cell proliferation and clonality, and significantly lower expression of p53, p21, and p16, well known markers of senescence. Since paracrine action is the main action of MSCs, we examined the anti-inflammatory activity of each MSC under lipopolysaccharide (LPS)-induced inflammation. Co-culture of UCB-MSCs with LPS-treated rat alveolar macrophage, reduced expression of inflammatory cytokines including interleukin-1α (IL-1α), IL-6, and IL-8 via angiopoietin-1 (Ang-1). Using recombinant Ang-1 as potential soluble paracrine factor or its small interference RNA (siRNA), we found that Ang-1 secretion was responsible for this beneficial effect in part by preventing inflammation. Our results demonstrate that primitive UCB-MSCs have biological advantages in comparison to adult sources, making UCB-MSCs a useful model for clinical applications of cell therapy.

CXC chemokine receptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomas

In this study, we showed that knocking-down interleukin-8 (IL-8) in glioma cells, or its receptor, CXC chemokine receptor 1 (CXCR1) in hUCB-MSCs reduced hUCB-MSC migration toward glioma cells in a Transwell chamber. In contrast, CXCR1-transfected hUCB-MSCs (CXCR1-MSCs) showed a superior capacity to migrate toward glioma cells in a Transwell chamber compared to primary hUCB-MSCs. Furthermore, these transfected cells also demonstrated the same ability to migrate toward tumors in mice bearing intracranial human gliomas as shown by histological and in vivo imaging analysis. Our findings indicate that overexpression of CXCR1 could be a useful tool for MSC-based gene therapy to achieve a sufficient quantity of therapeutic MSCs that are localized within tumors.

Critical Role of Vascular Endothelial Growth Factor Secreted by Mesenchymal Stem Cells in Hyperoxic Lung Injury

Intratracheal transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) protects against neonatal hyperoxic lung injury by a paracrine rather than a regenerative mechanism. However, the role of paracrine factors produced by the MSCs, such as vascular endothelial growth factor (VEGF), has not been delineated. This study examined whether VEGF secreted by MSCs plays a pivotal role in protecting against neonatal hyperoxic lung injury. VEGF was knocked down in human UCB-derived MSCs by transfection with small interfering RNA specific for human VEGF. The in vitro effects of MSCs with or without VEGF knockdown or neutralizing antibody were evaluated in a rat lung epithelial (L2) cell line challenged with H2O2. To confirm these results in vivo, newborn Sprague-Dawley rats were exposed to hyperoxia (90% O2) for 14 days. MSCs (1 × 10(5) cells) with or without VEGF knockdown were administered intratracheally at postnatal Day 5. Lungs were serially harvested for biochemical and histologic analyses. VEGF knockdown and antibody abolished the in vitro benefits of MSCs on H2O2-induced cell death and the up-regulation of inflammatory cytokines in L2 cells. VEGF knockdown also abolished the in vivo protective effects of MSCs in hyperoxic lung injury, such as the attenuation of impaired alveolarization and angiogenesis, reduction in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive and ED-1-positive cells, and down-regulation of proinflammatory cytokine levels. Our data indicate that VEGF secreted by transplanted MSCs is one of the critical paracrine factors that play seminal roles in attenuating hyperoxic lung injuries in neonatal rats.

Thrombospondin-2 secreted by human umbilical cord blood-derived mesenchymal stem cells promotes chondrogenic differentiation

Increasing evidence indicates that the secretome of mesenchymal stem cells (MSCs) has therapeutic potential for the treatment of various diseases, including cartilage disorders. However, the paracrine mechanisms underlying cartilage repair by MSCs are poorly understood. Here, we show that human umbilical cord blood‐derived MSCs (hUCB‐MSCs) promoted differentiation of chondroprogenitor cells by paracrine action. This paracrine effect of hUCB‐MSCs on chondroprogenitor cells was increased by treatment with synovial fluid (SF) obtained from osteoarthritis (OA) patients but was decreased by SF of fracture patients, compared to that of an untreated group. To identify paracrine factors underlying the chondrogenic effect of hUCB‐MSCs, the secretomes of hUCB‐MSCs stimulated by OA SF or fracture SF were analyzed using a biotin label‐based antibody array. Among the proteins increased in response to these two kinds of SF, thrombospondin‐2 (TSP‐2) was specifically increased in only OA SF‐treated hUCB‐MSCs. In order to determine the role of TSP‐2, exogenous TSP‐2 was added to a micromass culture of chondroprogenitor cells. We found that TSP‐2 had chondrogenic effects on chondroprogenitor cells via PKCα, ERK, p38/MAPK, and Notch signaling pathways. Knockdown of TSP‐2 expression on hUCB‐MSCs using small interfering RNA abolished the chondrogenic effects of hUCB‐MSCs on chondroprogenitor cells. In parallel with in vitro analysis, the cartilage regenerating effect of hUCB‐MSCs and TSP‐2 was also demonstrated using a rabbit full‐thickness osteochondral‐defect model. Our findings suggested that hUCB‐MSCs can stimulate the differentiation of locally presented endogenous chondroprogenitor cells by TSP‐2, which finally leads to cartilage regeneration. Stem Cells 2013;31:2136–2148

Mitochondrial dynamics regulate melanogenesis through proteasomal degradation of MITF via ROS‐ERK activation

Mitochondrial dynamics control mitochondrial functions as well as their morphology. However, the role of mitochondrial dynamics in melanogenesis is largely unknown. Here, we show that mitochondrial dynamics regulate melanogenesis by modulating the ROS‐ERK signaling pathway. Genetic and chemical inhibition of Drp1, a mitochondrial fission protein, increased melanin production and mitochondrial elongation in melanocytes and melanoma cells. In contrast, down‐regulation of OPA1, a mitochondria fusion regulator, suppressed melanogensis but induced massive mitochondrial fragmentation in hyperpigmented cells. Consistently, treatment with CCCP, a mitochondrial fission chemical inducer, also efficiently repressed melanogenesis. Furthermore, we found that ROS production and ERK phosphorylation were increased in cells with fragmented mitochondria. And inhibition of ROS or ERK suppressed the antimelanogenic effect of mitochondrial fission in α‐MSH‐treated cells. In addition, the activation of ROS‐ERK pathway by mitochondrial fission induced phosphorylation of serine73 on MITF accelerating its proteasomal degradation. In conclusion, mitochondrial dynamics may regulate melanogenesis by modulating ROS‐ERK signaling pathway.

INPP4B-mediated tumor resistance is associated with modulation of glucose metabolism via hexokinase 2 regulation in laryngeal cancer cells.

Inositol polyphosphate 4-phosphatase type II (INPP4B) was recently identified as a tumor resistance factor in laryngeal cancer cells. Herein, we show that INPP4B-mediated resistance is associated with increased glycolytic phenotype. INPP4B expression was induced by hypoxia and irradiation. Intriguingly, overexpression of INPP4B enhanced aerobic glycolysis. Of the glycolysis-regulatory genes, hexokinase 2 (HK2) was mainly regulated by INPP4B and this regulation was mediated through the Akt-mTOR pathway. Notably, codepletion of INPP4B and HK2 markedly sensitized radioresistant laryngeal cancer cells to irradiation or anticancer drug. Moreover, INPP4B was significantly associated with HK2 in human laryngeal cancer tissues. Therefore, these results suggest that INPP4B modulates aerobic glycolysis via HK2 regulation in radioresistant laryngeal cancer cells.

Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long‐term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood‐derived MSCs (hUCB‐MSCs) associated with cellular senescence using fluorescence‐activated cell sorting analysis and 242 cell surface‐marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB‐MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB‐MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB‐MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence‐associated β‐galactosidase, compared with that observed in hUCB‐MSCs with low‐level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB‐MSCs caused downregulation of other cellular senescence regulators, including Bmi‐1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB‐MSCs.

Conditioned Media from Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Inhibits Melanogenesis by Promoting Proteasomal Degradation of MITF

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) secrete various beneficial molecules, which have anti-apoptotic activity and cell proliferation. However, the effect of hUCB-MSCs in melanogenesis is largely unclear. In this study, we show that conditioned media (CM) derived from hUCB-MSCs inhibit melanogenesis by regulating microphthalmia-associated transcription factor (MITF) expression via the ERK signalling pathway. Treatment of hUCB-MSC-CM strongly inhibited the alpha-melanocyte stimulating hormone-induced hyperpigmentation in melanoma cells as well as melanocytes. Treatment of hUCB-MSC-CM induced ERK1/2 activation in melanocytes. In addition, inhibition of ERK1/2 suppressed the anti-pigmentation activity of the hUCB-MSC-CM in melanocytes and in vitro artificial skin models. We also found that the expression of MITF was appreciably diminished while expression of phosphorylated MITF, which leads to its proteasomal degradation, was increased in cells treated with hUCB-MSC-CM. These results suggested that hUCB-MSC-CM significantly suppresses melanin synthesis via MITF degradation by the ERK pathway activation.

Autocrine Action of Thrombospondin‐2 Determines the Chondrogenic Differentiation Potential and Suppresses Hypertrophic Maturation of Human Umbilical Cord Blood‐Derived Mesenchymal Stem Cells

Previous studies have shown that mesenchymal stem cell (MSC)‐based therapies have varying efficacies for the treatment of various diseases, including cartilage defects. In this study, we demonstrated that the chondrogenic differentiation potential of human umbilical cord blood‐derived MSCs (hUCB‐MSCs) obtained from different individual donors varies, and we investigated the molecular basis for this variation. Microarray gene expression analysis identified thrombospondin‐2 (TSP2) as a candidate gene underlying the interindividual variation in the chondrogenic differentiation potential of hUCB‐MSCs. To assess the association between TSP‐2 and the differentiation potential, we evaluated chondrogenic differentiation of hUCB‐MSCs treated with TSP2 siRNA. In addition, we studied the effect of supplementing exogenous recombinant TSP‐2 on TSP2 siRNA‐treated hUCB‐MSCs. We found that TSP‐2 autocrinally promoted chondrogenic differentiation of hUCB‐MSCs via the Notch signaling pathway, which was confirmed in MSCs from other sources such as bone marrow and adipose tissue. Interestingly, we observed that TSP‐2 attenuated hypertrophy, which inevitably occurs during chondrogenic differentiation of hUCB‐MSCs. Our findings indicated that the variable chondrogenic differentiation potential of MSCs obtained from different donors is influenced by the TSP‐2 level in the differentiating cells. Thus, the TSP‐2 level can be used as a marker to select MSCs with superior chondrogenic differentiation potential for use in cartilage regeneration therapy. Stem Cells 2015;33:3291–3303

Beclin-1 knockdown shows abscission failure but not autophagy defect during oocyte meiotic maturation

ABSTRACT Cytokinesis is the final step in cell division that results in the separation of a parent cell into daughter cells. Unlike somatic cells that undergo symmetric division, meiotic division is highly asymmetric, allowing the preservation of maternal resources for embryo development. Beclin-1/BECN1, the mammalian homolog of yeast Atg6, is a key molecule of autophagy. As part of a class III phosphatidylinositol 3-kinase (PI3K-III) complex, BECN1 initiates autophagosome formation by coordinating membrane trafficking. However, emerging evidence suggests that BECN1 regulates chromosome segregation and cytokinesis during mitosis. Thus, we investigated the function of BECN1 during oocyte meiotic maturation. BECN1 was widely distributed during meiotic maturation forming small vesicles. Interestingly, BECN1 is also detected at the midbody ring during cytokinesis. Depletion of BECN1 impaired the cytokinetic abscission, perturbing the recruitment of ZFYVE26 at the midbody. Similar phenotypes were observed when PI3K-III activity was inhibited. However, inhibition of autophagy by depleting Atg14L did not disturb meiotic maturation. Therefore, our results not only demonstrate that BECN1 as a PI3K-III component is essential for cytokinesis, but also suggest that BECN1 is not associated with autophagy pathway in mouse oocytes.