| Summary: | 博士 === 國立成功大學 === 生物科技研究所碩博士班 === 97 === In vivo, postnatal stem cells are usually quiescent and drug resistant; they can be prompted to proliferate when triggered by proper signals. In vitro, however, stem cells propagate quickly and differentiate spontaneously. Therefore, holding stem cells in vitro with reduced growth and multidrug resistance (MDR) phenotype is an appropriate strategy for keeping them in a primitive state. Because hyaluronan has been recognized as a crucial regulator for maintaining the microenvironments termed stem cell niches, we examined whether hyaluronan induces slow cycling and drug resistance in placenta-derived mesenchymal stem cells (PDMSCs) by comparing hyaluronan-coated surface with tissue-culture polystyrene surface.
The hyaluronan-coated surface significantly downregulated the proliferation of PDMSCs, more of which were maintained in the G0/G1 phases than were cells on the tissue-culture polystyrene surface. Both PKH-26 labeling and BrdU incorporation assays showed that most PDMSCs grown on a hyaluronan-coated surface duplicated during cultivation indicating that the hyaluronan-coated surface did not inhibit PDMSCs from entering the cell cycle. Mitotic synchronization showed that the G1-phase transit was prolonged in PDMSCs growing on a hyaluronan-coated surface. In addition, increases in p27Kip1 and p130 were the crucial factors that allowed hyaluronan to lengthen the G1 phase.
We found that PDMSCs cultured on a tissue-culture polystyrene surface coated with 30 μg/cm2 hyaluronan were more resistant to doxorubicin compared to control PDMSCs. Inhibiting PI3-K/Akt signaling showed that the PI3-K/Akt pathway modulated both P-glycoprotein activity and doxorubicin resistance. In addition, 10 μM verapamil dramatically suppressed the doxorubicin resistance induced by the hyaluronan-coated surface, indicating that P-glycoprotein activity was necessary for MDR. We further demonstraterd that PDMSCs treated with CD44 small interfering RNA (siRNA) and grown on a polystyrene surface coated with 30 μg/cm2 hyaluronan had fewer P-glycoprotein+ cells and lower CD44 expression levels (less than 60% in both cases) compared with PDMSCs not treated with CD44 siRNA and grown on the hyaluronan-coated surface. Moreover, treatment with CD44 siRNA suppressed the hyaluronan-substratum-induced resistance of PDMSCs to doxorubicin.
The data provided herein demonstrated that hyaluronan might be a promising candidate for maintaining stem cells in slow-cycling mode by prolonging their G1-phase transit. In addition, we presume that induction of MDR by hyaluronan would hold PDMSCs in a primitive state by providing the capability to extrude molecules required for differentiation. It seems reasonable to suggest that hyaluronan may cause PDMSCs to enter a program of dormancy, the natural state of stem cells consisting with slow cycling and drug resistance. Therefore, our findings may contribute to a deeper understanding of physiological functions of hyaluronan in stem cell research, and that is valuable for the applications in regenerative medicine.
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