Isolation and Characterization of Side Population Cells from Neonatal Murine Epidermis
Date Issued
2009
Date
2009
Author(s)
Wu, Chi-En
Abstract
Previous researchers had published the results which the harvested rate of epidermal SP cells are low, distributed among 0.3% to 1.4% when stained with the best condition: 5 µg/mL Hoechst 33342 for 90 min, for the isolation of SP cells. In addition, the cell cycle stage of SP cells are analyzed and demonstrated that they exhibit lower percentage of G2/M phase cell than non-SP cells. Furthermore, they express some markers of epidermal stem cell as Sca-I, α6-integrin and β1-integrin etc. Logically assume that they are epidermal stem/progenitor cells and belong to deep quiescent cells in vivo. Concern with diversified efficiency and dead cell rate of isolated SP cells, to develop more suitable conditions of isolating SP cells from murine epidermis is of great worth. Thus, the main objective of this study is to improve the efficiency of isolating SP cells derived from neonatal murine epidermis and clarify their particular characterizations.or further research, to decrease the damage of staining procedure is a dramatic topic. Therefore, the first experiment is to improve the harvested rate of SP cells and attempt to decrease the propidium iodide (PI) positive rate after the harmful staining procedure. Epidermal cells were isolated from neonatal mice skin (Day 1-3) and stained with different concentrations of dye and stop reaction in specific time points. The result reveals that the SP cells harvested rate is 4.94 ± 0.6% in 3.5 µg/mL treated group, which is more stable than other groups. In treated time assay, the SP cell rate gradually increased following with time elapsing till 90 min and significant difference (P<0.05) was obtained comparing with 30, 45 and 60 min treated group, but has not significant difference with 120 min one. In the respect of live cell rate assay, the dead cell rate is trending down when concentration of dye is increasing. The extremely significant difference was observed between 3.5 µg/mL and 5 µg/mL treated groups (P<0.01). Furthermore, the 90 min treated group exhibits lower dead cell rate than 120 min one. Obviously, the appropriate condition that 3.5 µg/mL Hoechst 33342 treat primary cells for 90min was used for following trials.he second experiment is to analyze cell cycle stage of epidermal SP cells and non-SP cells, and verify whether epidermal SP cells possess the characteristics of stem/progenitor cells. The result shows that S and G2/M phases of SP cells (9.68% ± 1.87%) are significantly lower than epidermal non-SP cells (19.43% ± 2.40%) (P<0.05). Besides, surface marker analysis confirmed the sorted epidermal SP cells express higher levels α6-integrin and β1-integrin which are epidermal stem cell markers than non-SP cells. Furthermore, Sca-1, a progenitor cell marker, is expressed in SP cells, exclusive of non-SP cells. In addition, those cells are no contamination with blood and bone marrow derived cells based on the results show that the SP cells are negative expressions of CD45 and CD34. After examine cell cycle and cell surface marker, we use epidermal SP cells to trans-differentiate into adipocyte in vitro. The result shows that epidermal SP cells gradually change their morphology in differentiation medium between 12 days. We use Oil Red O to stain the cells after culture for 12 days, partial cells had give rise into adipocyte and expressed red after staining.aken together, a more efficient method for the isolation of epidermal SP cells have been established and verify harvested SP cells possess some characteristics resembling to epidermal stem cells whether in cell cycle, surface markers expression and adipocyte differentiation. These results suggest that the established conditions of isolating epidermal SP cells that can express stem cell characteristic.
Subjects
Stem cells
side population
murine epidermis
Type
thesis
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