Background: Mouse bone marrow-derived mesenchymal stem cells (MSCs) cannot be easily harvested in matured mice because of the low MSC frequency, contamination of hematopoietic cells and the mouse age, while the MSCs may modulate the function of immune cells in various of mouse models. This study was designed to establish a simple and easy method of culturing MSCs from neonatal mouse, and elucidate their morphological characteristics and immunological function.

Methods: Bone marrow (BM) cells were obtained from C57BL/6 neonatal mouse. The filtered BM cells were cultured for 24h for MSCs adherent tightly to plastic culture dishes. The MSCs were cultured up to passage 9. The MSCs of passage 4 were used to identify surface markers by flow cytometry, including CD29/CD105 and CD34/MHC-II, and to assess the multilineage differentiation capacity of these cells, including adipogenic and osteogenic inductions. The same procedure was performed using the BM cells from matured mice. Dendritic cells (DCs) were obtained from BALB/c mice and then cocultured with the neonatal MSCs at a 1:1 ratio, following analysis of CD86 and MHCIIon DCs by flow cytometry. Another coculture system of C57BL/6 T cells and the neonatal MSCs at a 1:1 ratio were stimulated with anti-CD3/CD28 beads for 4 days, and the T cell proliferation was analyzed using CFSE assay.

Results: Immumophenotypic analysis showed that the neonatal MSCs were positive for CD29/CD105 and negative for CD34/MHC-II, and the MSCs exhibted multipotency through their ability to differentiate into adipocytes and osteocytes. Meanwhile, the MSCs from matured mice failed to proliferation for the further experiments. After coculture with the neonatal MSCs, CD86 and MHC class IIwere down-regulated and this result was found to be statistically significant compared with no MSC intervention(p<0.05). CFSE assay showed that the neonatal MSCs strongly inhibited lymphocyte proliferation, with a proliferation ratio of 5.44% CD4⁺T cells and 2.43% CD8⁺ T cells, compared with 44.5% CD4⁺T cells and 58.7% CD8⁺ T cells in the absence of the neonatal MSCs.

Conclusion: We form a reliable method to harvest MSCs from neonatal mice. The MSCs present superior multilineage differentiation capacity, while the MSCs from matured mice fail to proliferation. Neonatal MSCs exhibit capacity to suppress DC maturation and T-cell proliferation.

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