MSC are initially recognized in the late 1960s by Friendenstein and colleagues, as an adherent, non-phagocytic, fibroblast-like population that could regenerate rudiments of normal bone in vitro and in vivo (Friedenstein et al. 1970; Friedenstein et al. 1974a; Friedenstein et al. 1974b). The group identified a homogenous spindle-shaped adherent cell population when they cultured whole bone marrow (BM) in vitro. Then, this assay was developed into colony forming unit-fibroblast (CFU-F) assay which is the standard method to identify MSC. Later, Pittenger et al showed that MSC differentiate into lineages of mesenchymal tissue, including bone, cartilage, fat, tendon, muscle, and marrow stromal (Pittenger et al. 1999). This multilineage differentiation ability has been further exploited in tissue engineering. MSC attract much interest of scientists when their immunomodulatory functions were described (Bartholomew et al. 2002; Di Nicola et al. 2002; Tse et al. 2003).
2.2.1 Sources of MSC
MSC can be isolated from various sources and wide range of species. Among, the most extensively defined and investigated sources are human (Pittenger et al. 1999) and murine MSC (Nadri and Soleimani 2007; Anjos-Afonso and Bonnet 2008). In addition, MSC also have been derived from various animal including non-human primates (Ke et al. 2009), dogs (Neupane et al. 2008), pigs (Bosch et al. 2006), cows (Bosnakovski et al. 2005), chicken (Khatri et al. 2009) and horses (Arnhold et al. 2007). This provides a large animal model to test MSC therapeutic potential in both human and veterinary medicine. In humans, MSC have been identified and described in variety of tissues. Among, adult bone marrow (BM) is the most common source of MSC and extensively studied. The frequency of MSC in BM is low and estimated to constitute about 0.01-0.001% of the nucleated cells. This instigates the efforts to explore the alternative sources of MSC. In line with, MSC have been isolated from foetal tissues (Campagnoli et al. 2001) and other adult tissues including granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (Kassis et al. 2006), adipose tissue (Locke et al. 2009), appendices (De Coppi et al. 2006), scalp tissue (Shih et al. 2005), cardiac tissue (Tateishi et al. 2007) and placenta (Barlow et al. 2008). Besides, MSC also have been isolated from umbilical cord blood (Lee et al. 2004b) and normal adult peripheral blood despite the low yield (Zvaifler et al. 2000). Surprisingly, MSC also present in human endometrium and menstrual blood recently (Hida et al. 2008; Gargett et al. 2009). The findings confirm MSC stromal support and mobilisation properties. Of all the sources, the frequency of MSC is higher in the tissues of the early life/newly formed (foetal tissues, cord blood, placenta) than adult tissues suggesting that they may play an important role in early tissue formation.
2.2.2 Isolation of MSC
In general, MSC can be isolated by standard cell culture method by exploiting the plastic adherence property of MSC. Though, it is not sufficient to achieve high purity as the adherent cells fraction may contain other cell types. Various approaches have been tested to enrich MSC population in vitro. Several groups employed positive selection in isolation of MSC using monoclonal antibodies labelling such as Stro-1 (Simmons and Torok-Storb 1991), CD133 (Tondreau et al. 2005), CD271(Poloni et al. 2009) and CD105 (Roura et al. 2006). On the other hand, instead of positive selection, MSC can be purified by depleting other cells in the samples (Lee et al. 2004b; Tondreau et al. 2005). Besides, modification of culture media formulation including serum pre-selection, conditioned media, and addition of growth supplement may also selectively promote MSC growth in culture.
2.2.3 In vitro Characteristic of MSC
MSC colonies can be isolated by plastic adherence after collection/extraction of nucleated cells from the primary source. At early phase of culture, the colonies are usually heterogeneous given the fact that other cell types presence in the sample. Over the time, about 2-3 passage, non-MSC will be washed away, leaving exclusively adherent, fibroblast-like cells due to the high rate of clonally expansion of MSC. Although purity of MSC is definite at later passage of culture, these single-cell-derived colonies are morphologically heterogeneous (Mets and Verdonk 1981; Colter et al. 2000). Collectively, the studies identified two different MSC with inimitable appearance and unique properties. These subpopulations are: (1). small, spindl-shaped and rapidly renewing cell; (2). larger flatter slow-growing cell.
Due to their self-renewal ability, MSC can be maintained for 50-100 passages. Nevertheless, this is depending on the origin of the species (Bianchi et al. 2003; Meirelles Lda and Nardi 2003; Yanada et al. 2006). Throughout the cultivation, in vitro senescence of MSC is noticeble. Senescent MSC reduce their proliferation ability; show accumulation of cells with large and mature character as well as decline in multipotentiality of differentiation (D’Ippolito et al. 1999). Telomere shortening in human cells can induce replicative senescence which blocks the cell division. The mechanism is controlled by activity of telomerase which is highly expressed in tumour cells and embryonic stem cells. Telomerase enables indefinite proliferation of these cells. However, most somatic cells lack of telomerase activity or have a low level of this enzyme. Several studies showed there is no or low telomerase activity in MSC using highly sensitive assay (Banfi et al. 2002; Yanada et al. 2006). This denoted that MSC senescence is associated with shortening of telomere length. In addition, the origin of MSC also play role in determining their senescence rate. MSC from old donor may encounter difficulties in initial cultivation and accelerated senescence in vitro (Stenderup et al. 2003; Shamsul et al. 2004).