Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method

Figure 1 Flow diagram of the “subfractionation culturing method” used to establish mouse clonal mesenchymal stem cell lines. Mouse bone marrow aspirate was mixed with Dulbecco’s modified Eagle’s Medium-low glucose and plated onto a 100-mm cell culture dish. After 2-h incubation, the supernatant only was transferred to a new dish. This procedure was repeated two more times, and then the supernatant was subsequently transferred to cell culture dishes with a 1- or 2-d interval as shown. Each dish was then incubated until single-cell-derived colonies appeared. When colonies of cells were large enough, they were transferred to a six-well plate or 100-mm cell culture dish and then expanded to larger flasks for freezing and further study. Unique mouse clonal mesenchymal stem cell (mcMSC) lines were saved in a mcMSC library.

Figure 2 Pictures of the individual colonies and cell morphology of the established mouse clonal mesenchymal stem cell lines. A: Representative pictures of single colonies obtained from different strains; B: Pictures of mouse clonal mesenchymal stem cells (mcMSCs) established from single colonies at about 70%-80% confluence. Nonclonal MSCs isolated by the conventional method, labeled as the gradient centrifugation method, from the three strains are also shown. Every mcMSCs in the right panel have a fibroblast-like morphology. The cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 5 min. Magnification: 100 ×.

Figure 3 Cell growth of the established mouse clonal mesenchymal stem cell lines and nonclonal mesenchymal stem cells. Cells were plated at 1 × 10³, 5 × 10³, or 1 × 10⁴ cells in a 60-mm dish and incubated in Dulbecco’s modified Eagle’s Medium-low glucose. Cells were trypsinized by incubation with trypsin/ethylene diamine tetraacetic acid and counted twice a week with a haemocytometer for 15 d. GCM: Gradient centrifugation method.

Figure 4 Adipogenic differentiation potential of the established mouse clonal mesenchymal stem cell lines and nonclonal mesenchymal stem cells. A: Oil-red-O staining showed that adipogenically differentiated mouse clonal mesenchymal stem cell lines were positive, tested 4 d after adipogenic induction: a and b: BALB/c gradient centrifugation method (GCM); c and d: BALB/c D1-#2; e and f: BALB/c D2-#1; g and h: C3H GCM; i and j: C3H D2-#1; k and l: C3H D3-#2; m and n: C57BL/6 GCM; o and p: C57BL/6 D3-#1; q and r: C57BL/6 D3-#2; B: Reverse transcriptase-polymerase chain reaction analysis of the adipogenic markers peroxisome proliferator-activated receptor γ2 (PPARγ2), fatty acid-binding protein 4, lipoprotein lipase (LPL), and Col1A1 at days 0 and 4 after adipogenic induction. glyceraldehyde phosphate dehydrogenase was used as an internal control.

Figure 5 Osteogenic differentiation potentials of the established mouse clonal mesenchymal stem cell lines and nonclonal mesenchymal stem cells. A: Alizarin Red S staining showed matrix mineralization in the osteogenically differentiated mouse clonal mesenchymal stem cell lines 21 d after osteogenic induction: a and b: BALB/c gradient centrifugation method (GCM); c and d: BALB/c D1-#2; e and f: BALB/c D2-#1; g and h: C3H GCM; i and j: C3H D2-#1; k and l: C3H D3-#2; m and n: C57BL/6 GCM; o and p: C57BL/6 D3-#1; q and r: C57BL/6 D3-#2; B: Reverse transcriptase-polymerase chain reaction analysis of osteogenic markers, alkaline phosphatase (ALP), Runx-2, Col1A1, and Distal-less homeobox 5 at days 0 and 7 after osteogenic induction. Glyceraldehyde phosphate dehydrogenase was used as an internal control.

Figure 6 Chondrogenic differentiation potential of the established mouse clonal mesenchymal stem cell lines and nonclonal mesenchymal stem cells. A: Histochemical staining with Safranin-O showed chondrogenically differentiated mouse clonal mesenchymal stem cell lines, tested 21 d after chondrogenic induction: a and b: BALB/c gradient centrifugation method (GCM); c and d: BALB/c D1-#2; e and f: BALB/c D2-#1; g and h: C3H GCM; i and j: C3H D2-#1; k and l: C3H D3-#2; m and n: C57BL/6 GCM; o and p: C57BL/6 D3-#1; q and r: C57BL/6 D3-#2; B: Reverse transcriptase-polymerase chain reaction analysis of chondrogenic markers, type II collagen, aggrecan, and type X collagen at days 0 and 21 after chondrogenic induction. Glyceraldehyde phosphate dehydrogenase was used as an internal control.

Figure 7 Immunosuppressive effect of mouse clonal mesenchymal stem cell lines or nonclonal mesenchymal stem cells on T cells. Splenocytes (2 × 10⁵) from BALB/c were stimulated with soluble anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) in the presence of 1 × 10⁴ different mesenchymal stem cells. A: After 48 h, T-cell receptor-activated T-cell clusters were observed by microscopy; B: Interferon (IFN)-γ production was determined by ELISA; C: T-cell proliferation was determined by [³H]-thymidine incorporation after 3 d culture. GCM: Gradient centrifugation method.