Quality control of mesenchymal stem cells is an important step before their clinical use in regenerative therapy. Among various characteristics of mesenchymal stem cells, reproducibility of population compositions should be analyzed according to characteristics, such as stem cell contents and differentiation stages. Such characterization may be possible by assessing the expression of several surface markers. Here we report our attempts to utilize antibody arrays for analyzing surface markers expressed in mesenchymal stem cell populations in a high-throughput manner. Antibody arrays were fabricated using a glass plate on which a micropatterned alkanethiol monolayer was formed. Various antibodies against surface markers including CD11b, CD31, CD44, CD45, CD51, CD73, CD90, CD105, and CD254 were covalently immobilized on the micropatterned surface in an array format to obtain an antibody array. To examine the feasibility of the array, cell binding assays were performed on the array using a mouse mesenchymal stem cell line. Our results showed that cell binding was observed on the arrayed spots with immobilized antibodies which exhibited reactivity to the cells in flow cytometry. It was further found that the density of cells attached to antibody spots was correlated to the mean fluorescent channel recorded in flow cytometry. These results demonstrate that data obtained by cell binding assays on the antibody array are comparable to those by the conventional flow cytometry, while throughput of the analysis is much higher with the antibody array-based method than flow cytometry. Accordingly, we concluded that the antibody array provides a high-throughput analytical method useful for the quality control of mesenchymal stem cells.
We investigated the effects of carbon ion and gamma-irradiation on osteoblastic MC3T3-E1 cells by comparing mRNA expression levels for RANKL and osteoprotegerin by RT-PCR. MC3T3-E1 cells were irradiated with 2, 4, or 6 Gy of carbon ions or gamma-rays, and total RNA was harvested 1, 2, 3, 5, or 7 days after irradiation. The RANKL mRNA/OPG mRNA ratio in carbon ion-irradiated MC3T3-E1 cells was lower, while in gamma-irradiated MC3T3-E1 cells this ratio was higher than in non-irradiated cells. To evaluate osteoclastogenesis of MC3T3-E1 cells, carbon ion- or gamma-irradiated cells were co-cultured with non-irradiated cells from murine bone marrow. Staining for tartrate-resistant acid phosphatase (TRAP) in co-cultures showed that carbon ion irradiation suppressed osteoclastogenesis. This result is consistent with the lower RANKL/OPG mRNA ratio for carbon ion-irradiated cells. These results suggest that carbon ion irradiation acts primarily on osteoblastic cells, leading to a decrease in the RANKL/OPG mRNA ratio. This effect, in turn, leads to a decrease in osteoclastogenesis and osteoclast activity, which results in an increase in bone volume.
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