**6. The number of uncommitted stem cells contained in BMSCs also varies with the isolation technique**

Although it remains unknown whether BMSCs contain committed progenitors of other lineages, their multi-lineage differentiation potentials are mainly attributed to the presence of uncommitted stem cells among heterogeneous BMSC populations. Therefore, it is important to investigate whether the number of uncommitted stem cells contained in BMSCs varies with the isolation techniques. Note, however, that it is difficult to calculate their numbers accurately because no specific markers for uncommitted stem cells are currently available. However, the abundance of these cells in BMSCs populations can be determined by analyzing the respon‐ siveness to differentiation-inducing media (induction media), since uncommitted stem cells are highly responsive to differentiation stimuli. Thus, BMSCs that are rich in these cells show Isolation of Bone Marrow Stromal Cells: Cellular Composition is Technique-Dependent http://dx.doi.org/10.5772/55543 43

**Figure 3.** Differences among committed osteogenic cell populations from BMSCs isolated without treatment, or after hemolysis, or Ficoll separation. (A) The expression of cell surface alkaline phosphatase (ALP) of non-induced BMSCs was greatest in the untreated group, followed by the hemolyzed group, and lowest in the Ficoll-treated group. (B) Quantitative ALP assays confirmed the lowest ALP activity in the Ficoll-treated group. Data are presented as the means ± standard deviation (n = 3). \*: P < 0.05. (Modified from Agata et al., 2012 [13] with permission).

**Figure 2.** Morphology and expression of cell surface CD54 and CD90 of BMSCs that were isolated from untreated,

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

**6. The number of uncommitted stem cells contained in BMSCs also varies**

Although it remains unknown whether BMSCs contain committed progenitors of other lineages, their multi-lineage differentiation potentials are mainly attributed to the presence of uncommitted stem cells among heterogeneous BMSC populations. Therefore, it is important to investigate whether the number of uncommitted stem cells contained in BMSCs varies with the isolation techniques. Note, however, that it is difficult to calculate their numbers accurately because no specific markers for uncommitted stem cells are currently available. However, the abundance of these cells in BMSCs populations can be determined by analyzing the respon‐ siveness to differentiation-inducing media (induction media), since uncommitted stem cells are highly responsive to differentiation stimuli. Thus, BMSCs that are rich in these cells show

hemolysed, or Ficoll-treated bone marrows. (Modified from Agata et al.*,* 2012 [13] with permission)

hemolysed bone marrow.

42 Regenerative Medicine and Tissue Engineering

**with the isolation technique**

great responsiveness when culture medium is changed from non-induction medium to induction medium. Accordingly, we investigated BMSCs isolated from untreated, hemolysed, or Ficoll-treated bone marrow for their responses to osteogenic induction medium. As shown in Figure 4A, the Ficoll-treated group showed the lowest ALP activity on day seven. However, this group significantly upregulated ALP activity and showed the greatest activity after 14 days of culture in osteogenic medium, though the difference did not reach a statistically significant level.

Since the Ficoll-treated group constantly showed the lowest ALP activity when cultured in non-induction medium (Figure 3B), the ratio of ALP upregulation (ALP activity in osteogenic induction medium/ ALP activity in non-induction medium) was also the greatest in this group. Gene expression analyses of osteopontin and core-binding factor subunit alpha-1 (*Cbfa1*), both of which are indicators of osteogenic differentiation, also showed the greatest responsiveness in the Ficoll-treated group (Figure 3B - 3E). These results indicate that BMSCs isolated from Ficoll-treated bone marrow contain greater numbers or higher concentrations of uncommitted stem cells than those isolated from untreated or hemolysed bone marrow.

**Figure 4.** Differences in the responses to osteogenic induction medium among BMSCs isolated from untreated, hemo‐ lysed, or Ficoll-treated bone marrow. (A) ALP activities in osteogenic induction medium. (B) Gene expression of osteo‐ pontin in non-induction medium. (C) Gene expression of osteopontin in osteogenic induction medium. (D) Gene expression of *Cbfa-1* in non-induction medium. (E) Gene expression of *Cbfa-1* in osteogenic induction medium. Data are presented as the means ± standard deviation (n = 3). \*: P < 0.05, \*\*: P < 0.01. (Modified from Agata et al*.,* 2012 [13] with permission)
