Min-Soo Seo

Functional recovery and neural differentiation after transplantation of allogenic adipose-derived stem cells in a canine model of acute spinal cord injury

In this study, we evaluated if the implantation of allogenic adipose-derived stem cells (ASCs) improved neurological function in a canine spinal cord injury model. Eleven adult dogs were assigned to three groups according to treatment after spinal cord injury by epidural balloon compression: C group (no ASCs treatment as control), V group (vehicle treatment with PBS), and ASC group (ASCs treatment). ASCs or vehicle were injected directly into the injured site 1 week after spinal cord injury. Pelvic limb function after transplantation was evaluated by Olby score. Magnetic resonance imaging, somatosensory evoked potential (SEP), histopathologic and immunohistichemical examinations were also performed. Olby scores in the ASC group increased from 2 weeks after transplantation and were significantly higher than C and V groups until 8 weeks (p < 0.05). However, there were no significant differences between the C and V groups. Nerve conduction velocity based on SEP was significantly improved in the ASC group compared to C and V groups (p < 0.05). Positive areas for Luxol fast blue staining were located at the injured site in the ASC group. Also, GFAP, Tuj-1 and NF160 were observed immunohistochemically in cells derived from implanted ASCs. These results suggested that improvement in neurological function by the transplantation of ASCs in dogs with spinal cord injury may be partially due to the neural differentiation of implanted stem cells.

Metformin represses self-renewal of the human breast carcinoma stem cells via inhibition of estrogen receptor-mediated OCT4 expression.

Metformin, a Type II diabetic treatment drug, which inhibits transcription of gluconeogenesis genes, has recently been shown to lower the risk of some diabetes-related tumors, including breast cancer. Recently, “cancer stem cells” have been demonstrated to sustain the growth of tumors and are resistant to therapy. To test the hypothesis that metformin might be reducing the risk to breast cancers, the human breast carcinoma cell line, MCF-7, grown in 3-dimensional mammospheres which represent human breast cancer stem cell population, were treated with various known and suspected breast cancer chemicals with and without non-cytotoxic concentrations of metformin. Using OCT4 expression as a marker for the cancer stem cells, the number and size were measured in these cells. Results demonstrated that TCDD (100 nM) and bisphenol A (10 µM) increased the number and size of the mammospheres, as did estrogen (10 nM E2). By monitoring a cancer stem cell marker, OCT4, the stimulation by these chemicals was correlated with the increased expression of OCT4. On the other hand, metformin at 1 and 10 mM concentration dramatically reduced the size and number of mammospheres. Results also demonstrated the metformin reduced the expression of OCT4 in E2 & TCDD mammospheres but not in the bisphenol A mammospheres, suggesting different mechanisms of action of the bisphenol A on human breast carcinoma cells. In addition, these results support the use of 3-dimensional human breast cancer stem cells as a means to screen for potential human breast tumor promoters and breast chemopreventive and chemotherapeutic agents.

Histone deacetylase controls adult stem cell aging by balancing the expression of polycomb genes and jumonji domain containing 3

Aging is linked to loss of the self-renewal capacity of adult stem cells. Here, we observed that human multipotent stem cells (MSCs) underwent cellular senescence in vitro. Decreased expression of histone deacetylases (HDACs), followed by downregulation of polycomb group genes (PcGs), such as BMI1, EZH2 and SUZ12, and by upregulation of jumonji domain containing 3 (JMJD3), was observed in senescent MSCs. Similarly, HDAC inhibitors induced cellular senescence through downregulation of PcGs and upregulation of JMJD3. Regulation of PcGs was associated with HDAC inhibitor-induced hypophosphorylation of RB, which causes RB to bind to and decrease the transcriptional activity of E2F. JMJD3 expression regulation was dependant on histone acetylation status at its promoter regions. A histone acetyltransferase (HAT) inhibitor prevented replicative senescence of MSCs. These results suggest that HDAC activity might be important for MSC self-renewal by balancing PcGs and JMJD3 expression, which govern cellular senescence by p16INK4A regulation.

Implication of NOD1 and NOD2 for the Differentiation of Multipotent Mesenchymal Stem Cells Derived from Human Umbilical Cord Blood

Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are known to trigger an innate immune response against microbial infection. Although studies suggest that activation of TLRs modulate the function of mesenchymal stem cells (MSCs), little is known about the role of NLRs on the MSC function. In this study, we investigated whether NOD1 and NOD2 regulate the functions of human umbilical cord blood-derived MSCs (hUCB-MSCs). The genes of TLR2, TLR4, NOD1, and NOD2 were expressed in hUCB-MSCs. Stimulation with each agonist (Pam3CSK4 for TLR2, LPS for TLR4, Tri-DAP for NOD1, and MDP for NOD2) led to IL-8 production in hUCB-MSC, suggesting the expressed receptors are functional in hUCB-MSC. CCK-8 assay revealed that none of agonist influenced proliferation of hUCB-MSCs. We next examined whether TLR and NLR agonists affect osteogenic-, adipogenic-, and chondrogenic differentiation of hUCB-MSCs. Pam3CSK4 and Tri-DAP strongly enhanced osteogenic differentiation and ERK phosphorylation in hUCB-MSCs, and LPS and MDP also slightly did. Treatment of U0126 (MEK1/2 inhibitor) restored osteogenic differentiation enhanced by Pam3CSK4. Tri-DAP and MDP inhibited adipogenic differentiation of hUCB-MSCs, but Pam3CSK4 and LPS did not. On chondrogenic differentiation, all TLR and NLR agonists could promote chondrogenesis of hUCB-MSCs with difference in the ability. Our findings suggest that NOD1 and NOD2 as well as TLRs are involved in regulating the differentiation of MSCs.

Isolation and characterization of equine amniotic fluid-derived multipotent stem cells.

BACKGROUND AIMS Amniotic fluid (AF) is a well-known source of stem cells. However, there have been no reports regarding equine AF stem cells. We have isolated equine AF-derived multipotent stem cells (MSC) (eAF-MSC) and show that these cells exhibit self-renewal ability and multilineage differentiation. METHODS AF was obtained from thoroughbred mares and mononuclear cells (MNC) were isolated by Ficoll-Paque density gradient. We measured the cumulative population doubling level (CPDL) and characterized the immunophenotype by flow cytometry. To investigate differentiation ability, a trilineage differentiation assay was conducted. RESULTS eAF-MSC could be isolated and the proliferation level was high. eAF-MSC presented typical MSC phenotypic markers, as determined by flow cytometry. Moreover, eAF-MSC showed a trilineage differentiation capability. CONCLUSIONS Equine AF is a good source of MSC. Furthermore, eAF-MSC may be useful as a cell therapy application for horses.

Human umbilical cord blood-derived mesenchymal stem cells protect against neuronal cell death and ameliorate motor deficits in Niemann Pick type C1 mice.

Niemann Pick disease type C1 (NPC) is an autosomal recessive disease characterized by progressive neurological deterioration leading to premature death. In this study, we hypothesized that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have the multifunctional abilities to ameliorate NPC symptoms in the brain. To test this hypothesis, hUCB-MSCs were transplanted into the hippocampus of NPC mice in the early asymptomatic stage. This transplantation resulted in the recovery of motor function in the Rota Rod test and impaired cholesterol homeostasis leading to increased levels of cholesterol efflux-related genes such as LXRα, ABCA1, and ABCG5 while decreased levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase were observed in NPC mice. In the cerebrum, hUCB-MSCs enhanced neuronal cell survival and proliferation, where they directly differentiated into electrically active MAP2-positive neurons as demonstrated by whole-cell patch clamping. In addition, we observed that hUCB-MSCs reduced Purkinje neuronal loss by suppression of inflammatory and apoptotic signaling in the cerebellum as shown by immunohistochemistry. We further investigated how hUCB-MSCs enhance cellular survival and inhibit apoptosis in NPC mice. Neuronal cell survival was associated with increased PI3K/AKT and JAK2/STAT3 signaling; moreover, hUCB-MSCs modulated the levels of GABA/glutamate transporters such as GAT1, EAAT2, EAAT3, and GAD6 in NPC mice as assessed by Western blot analysis. Taken together, our findings suggest that hUCB-MSCs might play multifunctional roles in neuronal cell survival and ameliorating motor deficits of NPC mice.

Isolation and characterization of canine amniotic membrane-derived multipotent stem cells.

Recent studies have shown that amniotic membrane tissue is a rich source of stem cells in humans. In clinical applications, the amniotic membrane tissue had therapeutic effects on wound healing and corneal surface reconstruction. Here, we successfully isolated and identified multipotent stem cells (MSCs) from canine amniotic membrane tissue. We cultured the canine amniotic membrane-derived multipotent stem cells (cAM-MSCs) in low glucose DMEM medium. cAM-MSCs have a fibroblast-like shape and adhere to tissue culture plastic. We characterized the immunophenotype of cAM-MSCs by flow cytometry and measured cell proliferation by the cumulative population doubling level (CPDL). We performed differentiation studies for the detection of trilineage multipotent ability, under the appropriate culture conditions. Taken together, our results show that cAM-MSCs could be a rich source of stem cells in dogs. Furthermore, cAM-MSCs may be useful as a cell therapy application for veterinary regenerative medicine.

HMGA2 regulates the in vitro aging and proliferation of human umbilical cord blood-derived stromal cells through the mTOR/p70S6K signaling pathway ☆

The human high-mobility group protein A2 (HMGA2) protein is an architectural transcription factor that transforms chromatin structure by binding to DNA. Recently, it has been reported that HMGA2 is highly expressed in fetal neural stem cells and has the capacity to promote stemness. However, there is currently no information available on the functional significance and molecular mechanisms of the cellular in vitro aging and proliferation of human umbilical cord blood-derived stromal cells (hUCBSCs). In the present study, we evaluated the direct effects of HMGA2 on the cellular aging and proliferation of hUCBSCs and investigated potential regulatory mechanisms responsible for the corresponding functions. We found that the overexpression of HMGA2 enhanced proliferation and reduced or even reversed the in vitro aging process of hUCBSCs. This effect was accompanied by the increased expression of cyclin E and CDC25A and the significantly decreased expression of cyclin-dependent kinase inhibitors. Furthermore, HMGA2 inhibition compromised cell proliferation and adipogenic differentiation in early-stage hUCBSCs. From the molecular/cellular functional analysis of microarray data, we found that HMGA2 overexpression induced a PI3K/Akt/mTOR/p70S6K cascade, which in turn suppressed the expression of p16(INK4A) and p21(CIP1/WAF1) in hUCBSCs. These results provide novel insights into the mechanism by which HMGA2 regulates the in vitro aging and proliferation of hUCBSCs.

Characterization and clinical application of mesenchymal stem cells from equine umbilical cord blood.

Tendinitis of the superficial digital flexor tendon (SDFT) is a significant cause of lameness in horses; however, recent studies have shown that stem cells could be useful in veterinary regenerative medicine. Therefore, we isolated and characterized equine umbilical cord blood mesenchymal stem cells (eUCB-MSCs) from equine umbilical cord blood obtained from thoroughbred mares during the foaling period. Horses that had tendinitis of the SDFT were treated with eUCB-MSCs to confirm the therapeutic effect. After eUCB-MSCs transplantation, the core lesion in the SDFT was found to decrease. These results suggest that transplantation using eUCB-MSCs could be another source of cell treatment.

bFGF enhances the IGFs-mediated pluripotent and differentiation potentials in multipotent stem cells

It has widely been reported that basic fibroblast growth factor (bFGF) promotes proliferation of human stem cells and contributes to the maintenance of their self-renewal capability through repeated replications. In contrast to embryonic stem cells (ESCs), the effects of growth factors on adult stem cells are poorly understood. In human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs), bFGF is associated with an increased number of proliferating cells. Furthermore, expression levels of ESC markers were increased after treatment with bFGF. bFGF also increased the expression of FGFR, which in turn increased expression of insulin-like growth factor (IGFs). Since IGFs exert autocrine and paracrine effects on stem cells, bFGF-mediated release of IGFs from hUCB-MSCs might enhance FGFR1 and IGF1R expression in neighboring cells. These receptors could subsequently regulate the effects of bFGF and IGFs in adult stem cells. These results suggest that positive feedback regulation of bFGF and IGFs leads to proliferation of hUCB-MSCs.