Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples

Figure 1 Study design. Bone marrow samples from 7 healthy volunteers were aliquoted and treated by different protocols. The cells were plated for passage culture or CFU-F assay. CFU-F: Colony-forming unit-fibroblast.

Figure 2 Colony-forming unit-fibroblast and mesenchymal stromal cell numbers of different groups. A: CFU-F numbers per 10⁶ nucleated cells from seven bone marrow samples; B-D: Bone marrow samples were diluted and split into 12 aliquots containing 10⁷ nucleated cells. The attached cells at passage 0 (B: × 10⁴), passage 1 (C: × 10⁵) and passage 2 (D: × 10⁵) were counted and summed up. The average and standard errors are shown. CFU-F: Colony-forming unit-fibroblast.

Figure 3 Representative morphological features of the colonies and the attached cells. Bone marrow samples were treated by different protocols and the cells were maintained for 12 d. Colonies of fibroblast-like cells were stained with Wright-Gimsa solution and observed under a microscope with a lower (A) and a higher magnification field (B). CTR: Un-coagulated control; UK: Urokinase-treated clots; CUT: Mechanically-cut clots; CLOT: Untreated clots.

Figure 4 Surface marker analysis of the attached cells by flow cytometry. The attached cells at passage 2 were collected for flow cytometric analysis. The antigenic markers are indicated. UK: Urokinase-treated clots; CUT: Mechanically-cut clots; CLOT: Untreated clots. The results are representative of 7 individual experiments.

Figure 5 Identification of in vitro osteogenesis and adipogenesis. The attached cells at passage 2 were exposed to specific inductive conditions and stained with oil-red (upper panel) or ALP solutions. The results are representative of 7 individual experiments. ALP: Alkaline phosphatase. CTR: Un-coagulated control; UK: Urokinase-treated clots; CUT: Mechanically-cut clots; CLOT: Untreated clots.