Cell Characteristics

10.2.1 Cell Isolation

MSCs isolation is fairly easy, because they can be obtained from a small amount of tissue. For instance, BMSCs are usually cultured from 25 to 50 mL of bone marrow aspirate. The traditional method used to harvest BMSCs is density gradient centrifugation, in which BMSCs concentrated at the high-density interface are collected and seeded onto tissue culture plates and cultured in medium with 10% fetal calf serum. Most of the attached mononuclear cells are MSCs. Some hematopoietic cells can also adhere, but will be washed away over time. Gradually, the morphology of the cells with heterogeneous appearance becomes more and more uniform after serial passages. The typical morphology of MSCs is a spindle-shaped cell body, with a large, round nucleus containing a prominent nucleolus. Some MSCs are polygo-nal in shape, exhibit a few long cell processes, and are characterized by small numbers of Golgi apparati, rough endoplasmic reticulum, mitochondria, and polyribosomes present in cytoplasm.

MSCs from tissues other than the bone marrow can be isolated via enzymatic digestion and selected by their adherence to plastic tissue culture plates. They all show phenotypic heterogeneity initially, and become homogenous after a few passages in culture (Huang et al., 2008, Katz et al., 2005, Nomura et al., 2007). Alternative protocols for MSCs isolation have been tested to achieve more purified stem cell populations (Smith et al., 2004). However, plastic attachment is still considered to be the most reliable method.

10.2.2 Colony Forming Unit Assay

When MSCs were plated at low cell seeding density, individual precursor can proliferate to form a colony. The number of colony forming units (CFUs) reflects the number of progenitor cells present in the cell population, and in turn the mesenchymal tissue potential. For human MSCs, each colony is generated by a single cell. Although more than one colony can be generated by a single mouse or rat MSCs because they can detach and reseed in the plate (Javazon et al., 2001). CFU of fibroblasts measur-ing assay is considered to be a gold standard in vitro assay to identify MSCs (Friedenstein et al., 1970).

A refined method for colony-forming ability of MSCs is single-cell colony forming unit assay. That is the method to seed the cells into individual wells of a microtiter plate to ensure that each observed colony was generated by a single cell, which showed a smaller variation than the standard CFU assay (Pochampally, 2008). Several groups estimated that 0.01% to 0.0001% of nucleated marrow cells are MSCs in the bone marrow (Castro-Malaspina et al., 1980, Perkins and Fleischman, 1990). Most of the MSCs derived from tissues other than bone marrow can form colonies in vitro. However, CFU assay has not been routinely successful for the detection of MSCs in peripheral blood and cord blood (Deans and Moseley, 2000).

10.2.3 Cell Surface Marker and Flow Cytometry

Recently, significant increase in knowledge on MSC surface markers has been gathered using microar-ray and cell-sorting methods, including fluorescence activated cell sorting and magnetic bead sorting.

However, additional data still need to be accumulated in order to complete the cell surface antigenic profile. To date, no specific single marker of MSCs has been identified. Reasons for this include the fact that current protocols for MSCs isolation result in highly heterogeneous cell populations, and also that MSCs isolated from different tissues represent highly variable cell populations.

It is generally accepted that MSCs lack typical hematopoietic cell surface antigens, such as CD14, CD31, CD34, and CD45. However, CD34 expression was observed in MCSs harvested from some mice strains (Peister et al., 2004). MSCs also lack the expression of CD 11b (Integrin alpha M), CD79alpha or CD19, and HLA-DR. BMSCs also do not express the co-stimulatory molecules CD80, CD86, CD40, the adhesion molecules CD31 (platelet/endothelial cell adhesion molecule [PECAM]-1), CD18 (leuko-cyte function-associated antigen-1 [LFA-1]), or CD56 (neuronal cell adhesion molecule-1). Other than the three markers, CD73, CD90, and CD105, identified by the Mesenchymal and Tissue Stem Cell Committee of the International Society, BMSCs also express STRO-1, CD105 (SH2), CD73 (SH3/4), CD44, CD71, and CD90 (Thy-1), as well as the adhesion molecules CD106 (vascular cell adhesion mol-ecule [VCAM]-1), CD166 (activated leukocyte cell adhesion molmol-ecule [ALCAM]), intercellular adhe-sion molecule (ICAM)-1, and CD29 (Pittenger et al., 1999). Mesenchymal stem cells are also known to express numerous receptors important for cell adhesion with hematopoietic cells. Eleven common genes were identified as stemness genes because there are highly expressed in undifferentiated and de-differentiated MSCs by comparing the gene profiles of undifferentiated and de-differentiated MSCs to differentiated osteoblasts, chondrocyte, and adipocytes (Song et al., 2006).

10.2.4 Self-Renewal Capacity

One of the two main characteristics of stem cells is their capacity of self-renewal. There are two types of stem cell division, symmetric and asymmetric (Morrison and Kimble, 2006). Asymmetric replication is a defining characteristic of stem cells, where each stem cell divides into one daughter cell with a stem cell fate, and one daughter cell with a differentiated cell fate. Symmetric division of one stem cell will produce two identical daughter cells, both of which can retain their stem cell characteristics (Knoblich, 2008). In this way, MSCs can maintain the cell number and keep their self-renewal potential though symmetric and asymmetric division (Ramasamy et al., 2007). It has been shown that embryonic stem cells (ESCs) have unlimited self-renewal capacity, but there is no evidence that the self-renewal capacity of MSCs is comparable to that of ESCs, the pluripotent stem cells derived from the inner cell mass of the blastocyst from an early-stage embryo (Thomson et al., 1998). Self-renewal potential of bone mar-row-derived MSCs has been confirmed by numerous research groups, but with highly variable results (Bruder et al., 1997, Colter et al., 2000).

10.2.5 Cell Differentiation Capacity

Multilineage differentiation potential is another specific property of MSCs. MSCs can differentiate into all of the cell types of the tissues or organ that they were originally harvested from. More recently, reports indicate that MSCs can cross lineage boundary and differentiate into a variety of tissue-specific cell types, a capacity termed “plasticity” (Herzog et al., 2003, Zipori, 2005). MSCs can give rise to all somatic cells in variety of tissues and organs, such as the brain, lung, liver, spleen, kidney, blood, and skin when injected into blastocysts (Jiang et al., 2002). The in vitro and in vivo differentiation potential of bone marrow-derived MSCs has been studied most extensively. Other than the identical tri-differ-entiation (osteo/adipo/chondro) potentials, many studies also demonstrate that bone marrow-derived MSCs can differentiate into various functional cell types in all three germ layers, including mesoderm,

10-4 Tissue Engineering ectoderm, and endoderm, both in vitro and in vivo. The specific cell types into which BMSCs have differentiated included, but not limited to, cardiomyocytes (Makino et al., 1999, Psaltis et al., 2008, Valarmathi et al., 2010), lung epithelial cells (Popov et al., 2007), endothelial cells (Oswald et al., 2004), hepatocytes (Kang et al., 2005, Ke et al., 2008), pancreatic islets (Eberhardt et al., 2006), bladder cells (Tian et al., 2010), and neurons (Cho et al., 2005). Recent reports also suggest the potential of BMSCs for hematopoietic cell differentiation (Zhao et al., 2008, Rafii and Lyden, 2003). Human BMSCs were able to differentiate into site-specific cells after intraperitoneal injection (Liechty et al., 2000). After intravenous infusion, MSCs can be detected in a variety of tissues (Devine et al., 2003), especially at sites of inflam-mation or injury (Mouiseddine et al., 2007). The mechanism behind this BMSC localization remains unclear, although one theories is that MSCs migrate via the stromal cell-derived factor 1/chemokine CSC receptor 4 pathway (Shi et al., 2007, Dar et al., 2006).

The differentiation capabilities of MSCs derived from tissues other than bone has been verified by various studies. However, no report indicates that the differentiation potential of MSCs is as strong as ESCs. Moreover, debate of the differentiation potential of MSCs continues, due to the heterogeneous nature of these cells.

A number of possible mechanisms could explain the plasticity of MSCs. One possibility is transdif-ferentiation, by which a cell directly differentiates into another cell type, under the influence of environ-mental cues. Another theory is de-differentiation, where a differentiated cell can revert back to a more primitive differentiation stage, and then can be induced to form another differentiated cell lineage (Song et al., 2006). For example, Song and Tuan (2004) have proved that cloned osteoblasts can differentiate into chondrocytes or adipocytes. Delorme et al. (2009) reported that cloned MSCs that were differen-tiated into vascular smooth muscle cells, can in turn form adipocytes, osteoblasts, or chondrocytes.

However, no definitive evidence for either theory has been found, and the molecular basis for plasticity remains poorly understood. Plasticity may be related to lineage priming, as differentiation in the primed lineages would not entail resetting the entire molecular program, but rather the up-regulation of only a few of the differentiation program components (Delorme et al., 2009).

10.2.6 The MSC Niche

It is well known that MSCs can be released from their local resident and participate in the tissue repair or regeneration under the stimulation of specific signals. In vivo, BMSCs are widely dispersed and the adjacent extracellular matrix is populated by a few reticular fibrils but is devoid of the other types of collagen fibrils (Brighton and Hunt, 1997). However, the exact anatomical location of MSCs remains uncertain because there is no identified single marker for stem cells. Current evidences suggest that those MSCs are resident in a common stem cell microenvironment within different tissues, which is termed niche (Bianco and Robey, 2001, Gronthos et al., 2003, da Silva Meirelles et al., 2006). MSCs can maintain their stem cell properties in those niches. More and more evidences indicated that those stem cell niches are situated in close proximity within the vascular wall (Covas et al., 2008).

MSCs in the bone marrow form the niches which can support hematopoietic stem cell maintenance and differentiation in the bone marrow by producing matrix to provide physical support and secreting growth factors to offer signals.

The origin of the MSCs is under debate. One of the theories is all the MSCs from different tissue actually circulating cells derived from the bone marrow. Because tissue-specific MSCs display many common characteristics attributed to bone marrow-derived MSCs although they are vary in phenotype, proliferation rate and differentiation potentials, which suggest all those cells share a similar ontogeny.

Another argument is that the tissue-specific MSCs are actually pericytes (Crisan et al., 2008b). Pericytes is also referred as Rouget or mural cells, mesangial cells in the kidney, or Ito cells in the liver (Kuhn and Tuan, 2010). Pericytes reside in close proximity to endothelial cells in capillaries (Andreeva et al., 1998), and similar to MSCs, have the multilineage capacity to differentiate into adipogenic, osteogenic, and chondrogenic cell lineages (Doherty et al., 1998, Crisan et al., 2008a, Farrington-Rock et al., 2004).

Furthermore, both types of cells express similar markers, such as STRO-1, CD146, and α-smooth muscle actin (Shi and Gronthos, 2003).