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Jaeyeaon Cho
Cell Biology and Molecular Medicine

B.S. 1999, Inha University, Korea
M.Sc. 2004, New Jersey Institute of Technology

Thesis Advisor: Junichi Sadoshima, Ph.D., M.D.
Dept. of Cell Biology and Molecular Medicine

Tuesday, January 26, 2010
12:00 p.m., MSB G-609b


Irreversible damages are followed after myocardial infarction (MI) due to a limited proliferation ability of cardiomyocyte (CM). Therefore, cardiac regeneration using stem cells has been suggested as a potential therapy to prevent heart failure and mesenchymal stem cells (MSCs) is one of the suggested cell sources for MI treatment. However, injected MSCs showed a limited survival and rare CM differentiation and therefore, therapeutic effects existed only transiently. The aim of this study was to identify a clue to induce CM differentiation of MSCs and improve cell therapy effects for MI treatment.
5-Azacytidine (5-Aza), a DNA demethylating agent, has been reported to induce expression of cardiac-specific genes and we also confirmed that 5-Aza induces expression of cardiac genes such as Nkx2.5 and -MHC in mouse bone morrow-derived MSCs. In addition to induction of cardiac genes, a significant upregulation of glycogen synthase kinase (GSKw-3 and downregulation of -catenin was observed in 5-Aza treated MSCs. The promoter region of GSK-3 was heavily methylated in control MSCs, but was demethylated by 5-Aza. Overexpression of GSK-3 potently induced CM differentiation but that of GSK-3уznthe other isoform of GSK-3, induced markers of neuronal and chondrocyte differentiation. GSK-3 inhibitors, including LiCl, SB 216743, and BIO, abolished 5-Aza-induced upregulation of CM-specific genes, suggesting that GSK-3 is necessary and sufficient for CM differentiation in MSCs. Although specific knock-down of endogenous GSK-3 abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3 facilitated CM differentiation in MSCs. Endogenous GSK-3 is found in both cytosol and nucleus in MSCs (mainly in cytosol) but GSK-3 is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3 failed to stimulate CM differentiation. Downregulation of -catenin mediates GSK-3-induced CM differentiation in MSCs, whereas upregulation of c-Jun plays an important role in mediating CM-differentiation induced by GSK-3 knockdown.
It was examined whether ex vivo-modified GSK-3] overexpressing MSCs (GSK-3]-MSCs) improves cardiac dysfunction after MI. Either saline or LacZ transduced MSCs (LacZ-MSCs) injected MI mice showed significant dilation of left ventricle (LV) and pathological hypertrophy. However, cardiac dysfunction shown in saline and LacZ-MSCs groups were significantly reduced in GSK-3]-MSCs group. To examine the underlying therapeutic mechanisms, CM differentiation and paracrine factors were examined in MSCs with/without GSK-3] overexpression. On 12 weeks, GFP-MSCs without treatment were not found but a significant number of GFP-MSCs with GSK-3] overexpression were found. MSCs carrying a genetic marker, Nkx2.5 promoter linked with LacZ gene, showed that GSK-3] overexpressing MSCs differentiate into CM but MSCs without GSK-3] overexpression does not differentiate into CM. Using angiogenesis PCR array and RT-PCR, vascular endothelial growth factor A (Vegfa), were identified to be upregulated in the peri-infarct area of GSK-MSC mice using angiogenesis PCR array. GSK-3 directly increased protein/mRNA expression of Vegfa in MSCs in vitro, suggesting that up-regulation is cell autonomous. All MI mice injected with MSCs overexpressing GSK-3҃nwere survived but MI mice injected with GSK-3 overexpressing but Vegfa knock-downed showed a significantly low survival rate and cardiac dysfunction, suggesting that Vegfa signaling is one of important therapeutic mechanisms induced by GSK-3-MSCs. Alternatively, the effects of GSK-3҃noverexpression were further examined using Dox-regulatable GSK-3] expression system in MSCs. Cardiac function was significantly decreased without GSK-3] inducion but recovered when GSK-3 was induced. Conversely, when GSK-3 was induced, cardiac function was maintained but significantly decreased when GSK-3 was not induced, clearly showing the importance of GSK-3 in MSCs for cell therapy.
These results suggest distinct roles of GSK-3 and GSK-3 in CM differentiation of BM-derived MSCs. GSK-3 in the cytosol induces CM differentiation through down-regulation of -catenin but GSK-3уnin the nucleus inhibits through down-regulation of c-Jun. ex-vivo overexpression of GSK-3] in MSCs enhances the efficacy of the cell therapy in MI mice through CM differentiation as well as stimulation angiogenesis via upregulation of Vegfa. Also, In situ Dox-regulatable expression of GSK-3] facilitates the therapeutic effect of MSCs injection and suggests the importance of GSK-3] overexpression

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