**1. Introduction**

Bone marrow contains a colony-forming, fibroblast-like cell population called bone marrow mesenchymal stem cells or bone marrow stromal cells (BMSCs) [1, 2]. Since BMSCs are capable of differentiating into multiple lineages (osteogenic, chondrogenic, adipogenic, neurogenic, and myogenic lineages), they have attracted significant interest as useful somatic stem cells for use in tissue engineering and regenerative medicine [3 - 7]. As BMSCs adhere to tissue culture-treated plastic, they are usually isolated by adherent cultivation of untreated whole bone marrow [8 - 10]. However, this technique may be inefficient for the isolation of BMSCs because untreated bone marrow contains a large proportion of erythrocytes and their presence may interfere with the initial adherence of BMSCs. The removal of unwanted high density blood cells by density gradient centrifugation increases the number of colony-forming units (CFUs) in primary BMSC culture [11]. Removal of erythrocytes by hemolysis treatment is also effective at increasing the number of CFUs [12]. However, recent studies have shown that BMSCs isolated by these techniques are different from those isolated by adherent culture techniques [13]. Since BMSCs consist of a heterogeneous mixture of cells with varying potentials at different stages of differentiation, the characteristics of the cultured cells depend on the initial composition of the cell population [14, 15]. Therefore, the final cellular compo‐ sition of BMSCs will vary significantly with the isolation technique used. Few studies have focused on the importance of the initial cellular composition of isolated BMSCs. In this chapter, possible differences in the cellular composition of BMSCs isolated from untreated, hemolysed, or density gradient fractionated bone marrow will be discussed. Furthermore, the optimal technique for the isolation of BMSCs for use in tissue engineering and regenerative medicine will be discussed from a clinical point of view.

© 2013 Agata; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Bone marrow stromal cells**

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.

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

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 systems for human BMSCs.

Considering their costs and availabilities, mice are more attractive candidates than other laboratory animals. However, rat BMSCs are used as a model of human BMSCs in our laboratory because mouse BMSC characteristics differ from those of human BMSCs. For example, mouse BMSCs need the support of feeder cells for their stable growth, while human BMSCs are able to grow in a feeder cell-independent manner [40]. Responses to differentiation stimuli are also different. While human BMSCs are readily induced to differentiate into the osteogenic lineage by dexamethasone, mouse BMSCs are less responsive to dexthamethasone treatment [41]. Although the reasons why mouse BMSCs differ from human BMSCs remain unknown, it has been suggested that mouse BMSCs are very rare in the bone marrow and need support by other cells for their growth and differentiation [40]. On the contrary, rat BMSCs can be easily isolated from bone marrow and they are able to grow without feeder cells, as do human BMSCs [13]. In addition, rat BMSCs are able to differentiate into multiple lineages under induction protocols used for human BMSCs [42]. Therefore, we believe that rat BMSCs offer a more appropriate model of human BMSCs, though fewer reagents and antibodies are available for rat cells than for mouse cells.
