**4. Isolation of BMSCs**

**2. Bone marrow stromal cells**

38 Regenerative Medicine and Tissue Engineering

systems for human BMSCs.

BMSCs are a plastic-adherent, non-hematopoietic cell population residing in the bone marrow [16]. As BMSCs are morphologically similar to skin fibroblasts and can be expanded in a culture medium for fibroblasts, they were initially described as stromal fibroblasts [17], though their differentiation potentials are far different from those of skin fibroblasts [18]. While skin fibroblasts are incapable of differentiating into other cell types, BMSCs are capable of differ‐ entiating into cells of multiple mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma [19]. To emphasize this property, BMSCs are also called mesenchymal stem cells or multipotent mesenchymal stromal cells [20], though they can also differentiate into non-mesenchymal (non-mesodermal) cell types such as neurons [21] and insulinproducing cells [22]. Although BMSCs do not possess totipotencies like embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), they are clinically more useful than these totipotent stem cells because they can be easily isolated from a small volume of bone marrow aspirate and do not require gene transfections to demonstrate their differentiation abilities [23]. Thus, BMSCs have attracted significant interest as potent stem cells for use in tissue engineer‐ ing and regenerative medicine of various tissues. In fact, clinical studies have shown that BMSCs are useful for the treatment of bone, cartilage, heart, and the central nervous system [24-27]. In addition, BMSCs recently attracted attention as immuno-modulatory cells useful for the treatment of immue diseases such as graft versus host disease (GVHD) [28, 29].

Therefore, clinical use of BMSCs should increase over the next few years.

BMSCs are present in the bone marrow of humans as well as other animals such as mice, rats, rabbits, dogs, pigs, sheeps, horses, and cows [4, 8, 30 - 35]. As BMSCs seem to be postnatal stem cells that are common among mammalian species, these animals have been used to investigate the origin and *in vivo* functions of BMSCs [36, 37]. In addition, these animal-derived BMSCs are considered useful as a models of human BMSCs because it is not always easy to recruit a sufficient number of human BMSC donors for experimental use. Furthermore, more reliable results can be obtained by using animal-derived BMSCs because experimental animals have uniform genetic backgrounds and are housed under controlled conditions, eliminating behavioral and environmental variations that could influence BMSC properties. In fact, several studies have reported that the characteristics of human BMSCs varied significantly among donors [15, 38, 39], while such variations are not observed in animal-derived BMSCs. There‐ fore, animal-derived BMSCs are considered to be useful alternatives to human BMSCs for laboratory experimentation. However, it remains unknown which animal's BMSCs offer the best model system to represent human BMSCs. In general, donor animals of BMSCs are chosen based on their costs and availabilities. However, it has been shown that there are a number of characteristic differences in the BMSCs among species [40]. Therefore, it is important to consider species difference in addition to the costs and availabilities when selecting model

**3. Animal-derived BMSC as a model of human BMSC**

Since BMSCs form adherent colonies in plastic culture vessels, BMSCs are generally obtained from adherent cultures of untreated whole bone marrow [2 - 4]. However, it has been suggested that this technique is inefficient for the isolation of BMSCs because untreated bone marrow contains a large proportion of erythrocytes and their presence may interfere with the initial colony formation of BMSCs [11 - 13]. As human BMSCs are a rare population in the bone marrow (0.01 - 0.1% of whole marrow), it is possible that the efficacy of initial colony formation directly affects the total yield of BMSCs. Inefficient colony formation may also lead to the reduced potentials of BMSCs because previous studies have shown that BMSCs lose their differentiation abilities depending on the duration of *ex vivo* culture [39]. Accordingly, it is important to investigate whether BMSCs can be more efficiently isolated by the removal of unwanted cells. Both density gradient centrifugation and hemolysis (red blood cell lysis) treatment remove erythrocytes for the efficient isolation of the mononuclear cell fraction of bone marrow. Although both these techniques were originally developed for the isolation of white blood cells such as lymphocytes, they can also be used for the isolation of BMSCs because they are contained within the mononuclear cell fraction. In fact, several studies have used either or both of these techniques for the isolation of BMSCs; they reported that BMSCs were more efficiently isolated by these techniques (Table 1) [12, 32, 34, 43].

However, it remains unknown whether BMSCs isolated by these techniques are identical to those isolated from untreated whole bone marrow because BMSCs are composed of hetero‐ geneous cells with varying growth and differentiation potentials [15]. Thus, the cellular composition of BMSC populations could be dependent upon the isolation technique. Although it remains unknown how many different types of cells constitute the BMSC fraction, at least committed osteogenic cells as well as uncommitted stem cells are present when BMSCs are


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

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