Goang-Won Cho

Bone Marrow-Derived Stromal Cells from Amyotrophic Lateral Sclerosis Patients Have Diminished Stem Cell Capacity

Human bone marrow stromal cells (BM-SCs) possess the potential to differentiate, self-renew, and produce diverse trophic/growth factors and are an excellent cell therapy tool for degenerative diseases. However, they exhibit different therapeutic efficacies, depending on the health status and age of the cell donor. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron death in the central nervous system. In this study, we isolated BM-SCs from 11 ALS patients and characterized their potential secretory capacity of neurotrophic factors. We identified significant reductions in the expression of Oct-4 and Nanog , and in the trophic factors ANG, FGF -2, HGF, IGF-1, PIGF, SDF-1alpha , TGF-beta, and VEGF, but not in BDNF or ECGF. Migration of ALS-SCs was reduced, although the cells expressed the same markers for human mesenchymal phenotypes. These data suggest that ALS-SCs have diminished capacity as trophic mediators and may have reduced beneficial effects in cell therapy.

The neuroprotective effect of erythropoietin-transduced human mesenchymal stromal cells in an animal model of ischemic stroke.

Erythropoietin (EPO) exhibits diverse cellular functions, including neurotrophic, anti-oxidant, anti-apoptotic, and anti-inflammatory effects in non-hematopoietic tissues. This study evaluated whether bone marrow mesenchymal stromal cells (MSCs) transduced with the EPO gene (EPO-MSCs) promoted neural cell survival and improved neurological deficits caused by ischemic stroke. EPO-MSCs stably produced high levels of EPO (10IU/ml) without any alteration of their mesenchymal phenotype. Both EPO transduction and treatment with 10 international units (IU) of recombinant human EPO (rhEPO) provided protection from H(2)O(2)-induced oxidative injury in human bone marrow mesenchymal stromal cells and in SH-SY5Y cells. EPO-MSCs were more protected than were MSCs treated with 10IU rhEPO (10U-MSCs). We also found that the expression of the neurotrophic factors BDNF, PD-ECGF, HGF, SDF-1alpha, and TGF-1beta increased in EPO-MSCs, while only BDNF and TGF-1beta increased in 10U-MSCs. Implantation of EPO-MSCs in an animal model of ischemic stroke significantly improved neurological function and decreased infarct volumes without affecting hematocrit level. An evaluation of the brain tissue 21days after implantation showed that EPO and phosphorylated Akt (a downstream mediator of EPO) increased only in brains implanted with EPO-MSCs. Transduction of the EPO gene into MSCs induced secretion of EPO and various trophic factors that may provide excellent neuroprotective effects in both in vitro and in vivo models of ischemic stroke.

Anti‐senescence effects of DNA methyltransferase inhibitor RG108 in human bone marrow mesenchymal stromal cells

Alteration of DNA methylation is highly associated with ageing and ageing‐related diseases. Remedy of the altered methylation pattern may provide beneficial efficacy in these diseases. In this study, we used a DNA methyltransferase inhibitor, RG108, to investigate the senescence effects in human bone marrow mesenchymal stromal cells (hBM‐MSCs). First, we determined the optimized dose and time of RG108 treatment in hBM‐MSCs to be 5 µM for 48 H, respectively. Under these conditions, the anti‐senescence genes TERT, bFGF, VEGF, and ANG were increased, whereas the senescence‐related genes ATM, p21, and p53 were decreased. The number of β‐galactosidase‐positive cells was significantly decreased in RG108‐treated MSCs, whereas the rates of MSC migration and cellular protection were increased. We have shown that RG108 significantly induces the expression of TERT by blocking methylation at the TERT promoter region. Thus, these data indicate that an optimized dose of RG108 may improve the cell migration, protection, cellular senescence, which may provide a better efficacy of these cells in stem cell therapy.

Human angiogenin presents neuroprotective and migration effects in neuroblastoma cells.

Human angiogenin (ANG) has been highlighted as an angiogenic factor which supports primary and metastatic tumor growth. Recent genetic studies have shown that ANG is presented as a susceptibility gene for amyotrophic lateral sclerosis (ALS) and ALS-frontotemporal dementia (ALS-FTD). They found several missense mutations, including K40I, which present the weakest functional activity in ANG variants. In this study, we investigate whether human wild type ANG (wANG) and its variant K40I (mANG) maintain their divergent functional capacities in neuronal cells. To evaluate this, SH-SY5Y neuroblastoma cells were transfected with wANG and mANG DNA and identified both wild and mutant ANG are localized to nuclei and have no effects on proliferation. We have shown that human wANG prevented cell death under H2O2-induced oxidative stress in both SH-SY5Y and NSC-34 cells, tested by MTT assay. These effects were more enhanced in motor neuron cell NSC-34. wANG also played a role in cell migration, while mANG decreased these functional activities. Immunoblot analysis revealed that the intracellular signaling of ERK1/2 (at Thr183/Tyr185) was increased following transfection of the wANG gene, and significantly decreased by mANG in neuronal cells. These findings suggest that human ANG plays a critical role in cell protection and migration following alterations in ERK1/2 signaling in SH-SY5Y cells. This may provide the possible relationship between mutations in hANG and other neurodegenerative diseases as well as ALS.

Endogenous ROS levels are increased in replicative senescence in human bone marrow mesenchymal stromal cells.

Cellular senescence is characterized by functional decline induced by cumulative damage to DNA, proteins, lipids, and carbohydrates. Previous studies have reported that replicative senescence is caused by excessive amounts of reactive oxygen species (ROS) produced as a result of aerobic energy metabolism. In this study, we established human bone marrow mesenchymal stromal cells (hBM-MSCs) in replicative senescence after culture over a long term to investigate the relationship between ROS levels and stem cell potential and to determine whether differentiation potential can be restored by antioxidant treatment. Intracellular ROS levels were increased in hBM-MSCs; this was accompanied by a decrease in the expression of the antioxidant enzymes catalase and superoxide dismutase (SOD)1 and 2 and of phosphorylated forkhead box O1 (p-FOXO1) as well as an increase in the expression of p53 and p16, along with a reduction in differentiation potential. When the antioxidant ascorbic acid was used to eliminate excess ROS, the levels of antioxidant enzymes (catalase, SOD1 and 2, p-FOXO1, and p53) were partly restored. Moreover, differentiation into adipocytes and osteocytes was higher in hBM-MSCs treated with ascorbic acid than in the untreated control cells. These results suggest that the decline in differentiation potential caused by increased endogenous ROS production during in vitro expansion can be reversed by treatment with antioxidants such as ascorbic acid.

Recombinant human erythropoietin reduces aggregation of mutant Cu/Zn-binding superoxide dismutase (SOD1) in NSC-34 cells.

Human erythropoietin (hEPO) has multiple actions in non-hematopoietic tissues, including neurotrophic, anti-oxidant, anti-apoptotic, and anti-inflammatory effects. To examine the effect of EPO in an vitro model of amyotrophic lateral sclerosis (ALS), we stably overexpressed wild SOD1 and a mutant form, SOD1/G93A, in NSC-34 motoneuron-like cells. Transformants harboring the wild and mutant forms of SOD1 were selected by G418 selection and immunoblot analysis. RT-PCR analysis showed that cox-2 expression was increased in the NSC-34/mSOD1s, and MTT assays and BrdU-ELISAs revealed reduced cell growth and proliferation in the NSC-34/mSOD1 cell line. Incubation with 5 or 10IU/mL rhEPO increased the viability and decreased the cox-2 expression in the dNSC-34/mSOD1s cells. Immunocytochemical staining with anti-SOD1 antibody revealed the presence of aggregates of mSOD1 protein in dNSC-34/mSOD1 cells. Incubation with10IU/mL rhEPO reduced the proportion of cells containing such aggregates. Our findings suggest that the anti-oxidant and anti-inflammatory effects of EPO increase the survival of NSC-34/mSOD1 cells and reduce aggregation of the mutant SOD1 protein.

Functional Restoration of Amyotrophic Lateral Sclerosis Patient-Derived Mesenchymal Stromal Cells Through Inhibition of DNA Methyltransferase

Abstract Alteration of DNA methylation is highly associated with aging and neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). Remedying these aberrant methylation patterns may serve to improve these diseases. Previously, we reported that human bone marrow mesenchymal stromal cells isolated from ALS patients (ALS-MSCs) have functionally decreased stem cell potency, and excessively express DNA methyltransferases (DNMTs). In this study, we examined the correlation between excessive DNMT expression and functional decline in ALS-MSCs. The DNMT inhibitor RG108 was used for this. RG108-treated ALS-MSCs exhibit increased expression of the anti-senescence genes TERT, VEGF, and ANG, and decreased expression of the senescence-related genes ATM and p21. The activity of SA-β-galactosidase and the expression of senescence proteins p53 and p16 were reduced in RG108-treated ALS-MSCs. The abilities of cell migration and protection against oxidative damage were improved in the treated ALS-MSCs. In neuronal differentiation experiments, the treated MSCs more effectively differentiated into neuron-like cells. These results suggest that ALS-MSC function can be restored by inhibiting excessively expressed DNMTs, an approach that may ultimately provide better efficacy in stem cell therapy.

Trichostatin a modulates intracellular reactive oxygen species through SOD2 and FOXO1 in human bone marrow‐mesenchymal stem cells

Engraft cells are often exposed to oxidative stress and inflammation; therefore, any factor that can provide the stem cells resistance to these stresses may yield better efficacy in stem cell therapy. Studies indicate that histone deacetylase (HDACs) inhibitors alleviate damage induced by oxidative stress. In this study, we investigated whether regulation of reactive oxygen species (ROS) occurs through the HDAC inhibitor trichostatin A (TSA) in human bone marrow‐mesenchymal stem cells (hBM‐MSCs). Intracellular ROS levels increased following exposure to hydrogen peroxide (H2O2), and were suppressed by TSA treatment. Levels of the antioxidant enzyme superoxide dismutase 2 (SOD2) increased following treatment with 200 nM TSA and to a lesser level at 1–5 μM TSA. Cell protective effects against oxidative stress were significantly increased in TSA‐MSCs after treatment with low doses of TSA (50–500 nM) and decreased with high doses of TSA (5–10 μM). Consistent results were obtained with immunoblot analysis for caspase3. Investigation of Forkhead box O1 (FOXO1), superoxide dismutase 2 (SOD2), and p53 levels to determine intracellular signaling by TSA in oxidative stress‐induced MSCs demonstrated that expression of phosphorylated‐FOXO1 and phosphorylated‐SOD2 decreased in H2O2‐treated MSCs while levels of p53 increased. These effects were reversed by the treatment of 200 nM TSA. These results suggest that the main function of ROS modulation by TSA is activated through SOD2 and FOXO1. Thus, optimal treatment with TSA may protect hBM‐MSCs against oxidative stress. Copyright

Proteomic analysis reveals KRIT1 as a modulator for the antioxidant effects of valproic acid in human bone-marrow mesenchymal stromal cells

Abstract Valproic acid (VPA) protects human bone marrow-mesenchymal stromal cells (hBM-MSCs) against oxidative stress and improves their migratory ability through increasing the secretion of trophic factors. This suggests that VPA may be an excellent candidate for improving stem cell function. However, the molecular mechanisms of VPA in BM-MSCs are not known. In this study, we used a proteomic approach to investigate VPA-associated targets under oxidative stress conditions. Krev/Rap1 interaction Trapped-1 (KRIT1), a modulator for the homeostasis of intracellular reactive oxygen species (ROS), was identified as a target protein by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analyses. The up-regulation of KRIT1 and its target proteins (SOD2 and FoxO1) with VPA treatment of hBM-MSCs was revealed by qPCR and immunoblot analysis. Damage from oxidative stress was reduced in VPA-pretreated BM-MSCs, which was also confirmed by qPCR and immunoblot analysis. In addition, increased in intracellular ROS by H2O2 were also reduced by VPA pretreatment in BM-MSCs. This suggests that VPA reduces intracellular ROS level by the modulation of KRIT1 and its correlated proteins, FoxO1, SOD2, and cyclin D1. Thus, this study is the first to provide evidence that VPA modulates KRIT1 and intracellular ROS in BM-MSCs.

Development of a Cellular Tau Enzyme-Linked Immunosorbent Assay Method for Screening GSK-3β Inhibitors

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase also known as tau protein kinase I, has been implicated in the pathogenic conditions of Alzheimers disease. Many investigators have focused on GSK-3 inhibitor as a therapeutic drug. In this study, we established a cell-based assay for the screening of novel GSK-3β inhibitors. For this purpose, four-repeat tau cDNAs were stably expressed in human embryonic kidney 293 (HEK293) cells (HEK293-Tau). The proliferation of HEK293-Tau cells was no different from that of HEK293 cells, as measured by the bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA). The concentration-dependent reduction of tau phosphorylation by GSK-3 inhibitors, LiCl, Chir98023, and SB415286, was examined by immunoblot analysis and Tau ELISA (in situ ELISA). Highly consistent data were obtained, suggesting that this novel ELISA method is highly reproducible. Using this ELISA strategy, we isolated a few candidate compounds, including compounds 114 and 149, from several hundreds of synthetic agents and demonstrated that such candidates protect nerve growth factor-differentiated PC12 cells against amyloid-β-induced cell death. These data indicate that this Tau ELISA method in HEK293-Tau cells may be a suitable cell-based assay system to screen for GSK-3β inhibitors.