**5. The number of committed osteogenic cells contained in BMSCs varies with the isolation technique**

Committed osteogenic cells can be defined as a cell population that is capable of forming bone without osteogenic induction. Because of the presence of this cell population, *in vivo* trans‐ plantation of untreated whole bone marrow to ectopic sites usually results in the formation of new bone [45]. If this cell population is decreased or lost by the hemolysis or density gradient centrifugation steps, new bone formation may not be observed in the transplants. On the contrary, if this cell population is enriched by these techniques, more significant bone forma‐ tion should be observed. Therefore, we investigated the *in vivo* bone-forming ability of three populations: marrow that was untreated, marrow that was hemolysed with ammonium chloride, and marrow that was fractionated by density-gradient-centrifugation over Ficoll® (Ficoll-treated). As shown in Figure 1A, the percentage of bone-forming transplants (trans‐ plants containing ectopic bone/ total transplants) was the lowest in the Ficoll-treated group. The amount of new bone formation, which was scored on a semi-quantitative scale from zero to three (Table 2), was also lowest in the Ficoll-treated group (Figure 1B). The hemolysed group also showed less bone-forming ability than did the untreated group, though its ability was still greater than that of the Ficoll-treated group (Figure 1 A and 1B).

**Figure 1.** *In vivo* bone-forming ability of untreated, hemolysed, or Ficoll-treated bone marrow. (A) The percentage of bone-forming transplants (transplants containing ectopic bone/ total transplants), which was calculated from the re‐ sults of seven independent experiments, was greatest in the untreated group, followed by the hemolyzed group, and lowest in the Ficoll-treated group. (B) The amount of new bone formation (total bone score/ total transplants) was greatest in the hemolyzed group, followed by the untreated group, and lowest in the Ficoll-treated group (n = 7). (Modified from Agata et al., 2012 [13] with permission)

isolated from untreated whole bone marrow [44]. Changes in the relative sizes of these two cell populations greatly influence the characteristics of BMSCs. In other words, a greater number of committed osteogenic cells makes the BMSC fraction more osteogenic, while a greater number of uncommitted stem cells makes them more stem-cell like. Thus, we investi‐ gated differences in the cellular composition of BMSCs isolated from untreated, densitygradient-centrifuged, and hemolysed bone marrow, with a special reference to committed osteogenic cells and uncommitted stem cells. For these experiments, rat bone marrow was used

**Table 1.** Removal of erythrocytes by hemolysis or density gradient centrifugation may enable the efficient isolation of

instead of human bone marrow to avoid the influence of variations among donors.

**with the isolation technique**

40 Regenerative Medicine and Tissue Engineering

BMSCs.

**5. The number of committed osteogenic cells contained in BMSCs varies**

Committed osteogenic cells can be defined as a cell population that is capable of forming bone without osteogenic induction. Because of the presence of this cell population, *in vivo* trans‐ plantation of untreated whole bone marrow to ectopic sites usually results in the formation of new bone [45]. If this cell population is decreased or lost by the hemolysis or density gradient centrifugation steps, new bone formation may not be observed in the transplants. On the contrary, if this cell population is enriched by these techniques, more significant bone forma‐ tion should be observed. Therefore, we investigated the *in vivo* bone-forming ability of three


**Table 2.** Bone score of each sample was determined from the percentage of the area containing bone (new bone area/ total area) (Modified from Agata et al., 2012 [13] with permission)

As these results showed that Ficoll-treated bone marrow contains fewer committed osteogenic cells than either untreated or hemolysed bone marrow, we next investigated whether BMSCs isolated from Ficoll-treated bone marrow actually contains lower numbers of committed osteogenic cells. Untreated, hemolysed, or Ficoll-treated rat bone marrow was plated on cell culture dishes, and adherent colony-forming cells were expanded as BMSCs. Although these BMSCs did not show significant differences in their morphology or their expression of cellsurface CD54 and CD90 (Figure 2), they showed a significant difference in the expression of cell-surface alkaline phosphatase (ALP) (Figure 3A). The difference in ALP expression was also confirmed by quantitative ALP assays (Figure 3B).

**Figure 2.** Morphology and expression of cell surface CD54 and CD90 of BMSCs that were isolated from untreated, hemolysed, or Ficoll-treated bone marrows. (Modified from Agata et al.*,* 2012 [13] with permission)

Since these BMSCs were simply cultured in non-induction medium, the expression of cell surface ALP directly indicates the number of committed osteogenic cells contained in each BMSC. Therefore, it can be concluded that BMSCs isolated from Ficoll-treated bone marrow contain lower numbers of committed osteogenic cells than those isolated from untreated or hemolysed bone marrow.
