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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 (GSKƒw-3ƒÒ and downregulation of ƒÒ-catenin was observed in 5-Aza treated MSCs. The promoter region of GSK-3ƒÒ was heavily methylated in control MSCs, but was demethylated by 5-Aza. Overexpression of GSK-3ƒÒ potently induced CM differentiation but that of GSK-3ƒÑƒzƒnthe 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-3ƒÒ is necessary and sufficient for CM differentiation in MSCs. Although specific knock-down of endogenous GSK-3ƒÒ abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3£ facilitated CM differentiation in MSCs. Endogenous GSK-3ƒÒ is found in both cytosol and nucleus in MSCs (mainly in cytosol) but GSK-3ƒÑ is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3ƒÒ failed to stimulate CM differentiation. Downregulation of ƒÒ-catenin mediates GSK-3ƒÒ-induced CM differentiation in MSCs, whereas upregulation of c-Jun plays an important role in mediating CM-differentiation induced by GSK-3ƒÑ 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-3ƒÒ 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-3ƒÒƒnwere survived but MI mice injected with GSK-3ƒÒ 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-3ƒÒ-MSCs. Alternatively, the effects of GSK-3ƒÒƒnoverexpression 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-3ƒÒ was induced. Conversely, when GSK-3ƒÒ was induced, cardiac function was maintained but significantly decreased when GSK-3ƒÒ was not induced, clearly showing the importance of GSK-3ƒÒ in MSCs for cell therapy.
These results suggest distinct roles of GSK-3ƒÑ and GSK-3ƒÒ in CM differentiation of BM-derived MSCs. GSK-3ƒÒ in the cytosol induces CM differentiation through down-regulation of ƒÒ-catenin but GSK-3ƒÑƒnin 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