Figure 1

Identification of suspension BMMSCs (S-BMMSCs). (A) Hypothetical model indicates that bone marrow all nucleated cells (ANCs) were seeded at 15 × 10⁶ into 100 mm culture dishes and incubated for two days at 37°C with 5% CO2, and subsequently non-attached cells from culture suspension were transplanted into immunocompromised mice subcutaneously using hydroxyapatite tricalcium phosphate (HA) as a carrier for eight weeks. Newly formed bone (B) by osteoblasts (arrow heads) and associated connective tissue (C) were detected in this non-attached cell transplants by H & E staining. Bar = 100 μm. (B) Hypothetical model of isolating S-BMMSCs. BMMSCs usually attach on culture dishes within two days; however, a small portion of BMMSCs in ANCs failed to attach to the dishes and remained in the suspension. The suspensions containing putative non-attached BMMSCs were collected and transferred to the extracellular matrix (ECM) coated dish with generating single colony clusters (CFU-F). These ECM-attached BMMSCs (S-BMMSCs) were sub-cultured on regular plastic culture dishes for additional experiments. (C) The number of plastic attached CFU-F from ANCs (1.5 × 10⁶ cells) is more than seven-fold higher than that derived from BMMSC-ECM adherent S-BMMSCs. (D) Proliferation rates of S-BMMSCs and BMMSCs were assessed by BrdU incorporation for 24 hours. The percentage of positive cells is significantly increased in S-BMMSCs when compared to BMMSCs. (E) S-BMMSCs exhibit a significant increase in population doublings when compared to BMMSCs. The results are representative of five independent experiments. Scale bars = 50 μm. ***P <0.001. The graph bar represents mean ± SD. BMMSCs, bone marrow mesenchymal stem cells; BrdU, bromodeoxyuridine; S-BMMSCs, BMMSCs in suspension; SD, standard deviation.