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"GSK-3] MEDIATES CARDIOMYOCYTE DIFFERENTIATION OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS AND IMPROVES CELL THERAPHY EFFECTS AFTER MYOCARDIAL INFARCTION"

by
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.
Professor
Dept. of Cell Biology and Molecular Medicine

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


Abstract

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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