Determinantes y permutaciones

Considerable studies have been conducted aiming to identify certain MSC markers for detection, isolation, characterisation and evaluation of human MSCs populations. Amongst these efforts is the rise of monoclonal antibodies as reagents for detecting and isolation of human MSCs. An example of these antibodies is Stro-1 a monoclonal antibody which can react with non-haematopoietic progenitor stromal cells derived from human bone marrow (Simmons and Torok-Storb, 1991). Also, the differentiation potential of MSCs is consistent with Stro-1 since MSCs positive for this marker become osteocyte, adipocyte, chondrocyte, smooth muscle cells and HSC-supporting fibroblasts (Kolf et al., 2007). However, Gronthos and colleagues suggested Stro-1 is an unlikely generic MSCs biomarker for two main reasons: first; it is not secreted by MSCs exclusively, second; it is gradually lost during MSC in vitro expansion (Gronthos et al., 2003). Therefore, Stro-1 expression as an MSC identification marker should be in conjunction with other markers such as vascular cell adhesion molecule-1 (VCAM-1) which is involved in MSC chemotaxis, adhesion, and signal transduction (Carter and Wicks, 2001) and CD106 which could be an indicator for increasing frequency of CFU-F (Gronthos et al., 2003). Another example of reagents for MSC detection are the SH-2n SH3 and SH4 antibodies

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which do not react with haematopoietic cells (Haynesworth et al., 1992; Kolf et al., 2007). A further example is the SB-10 antibody which reacts specifically with CD166 (activated leukocyte-cell adhesion molecule, ALCAM). In addition, SB-10 can react with an antigen expressed on undifferentiated MSC cells and once these undifferentiated MSCs initiate osteogenesis, this antigen disappears and is replaced by alkaline phosphatase (Bruder et

al., 1998). For more details about MSC positive markers, see (Table 1.1). 1.4.3.1 POSITIVE MARKERS

The International Society for Cellular Therapy (ISCT) has minimised the criteria for defining human MSCs to be positive for CD73 (SH3/4), CD90 and CD105 (SH2) (Oswald

et al., 2004; Dominici et al., 2006; Gimble et al., 2008; Hocking and Gibran, 2010; Blaber et al., 2012; Wang et al., 2012a). However, other CD marker profile have been expressed

by MSCs such as CD106, CD120a, CD124 (Baron and Storb, 2012), CD29 (b1-integrin), CD13, CD44, CD71 (Oswald et al., 2004; Honczarenko et al., 2006; Wu et al., 2007; Motaln et al., 2010; Parekkadan and Milwid, 2010; Baron and Storb, 2012), CD166 (ALCAM) (Motaln et al., 2010; Parekkadan and Milwid, 2010; Baron and Storb, 2012), CD146, CD58, CD54 (intercellular adhesion molecule-1 [ICAM]-1), CD49e (a5-integrin) and CD10 (Motaln et al., 2010; Parekkadan and Milwid, 2010) and CD271 (Quirici et al., 2002; Motaln et al., 2010; Parekkadan and Milwid, 2010). Also, MSCs strongly express typical surface antigens such as stem cells antigen-1 (Sca-1) (Wu et al., 2007), HLA class I (HLA-I) and they can be induced by IFN-γ to express HLA-II (Baron and Storb, 2012). Additionally, they can express combination of chemokine receptors for example, CXCR 1, 2, 3, 4 and 5, CCR 1, 4, 7, 9 and 10 and CX3CR1 (Patel et al., 2013). It is therefore difficult to identify human MSCs with the use of specific markers.

1.4.3.2 NEGATIVE MARKERS

As per the ISCT, the minimal criteria for defining h-MSCs pertaining the negative markers are CD14, D19, CD34, CD45, CD11b, CD79 alpha and HLA-DR (Oswald et al., 2004; Dominici et al., 2006; Gimble et al., 2008; Hocking and Gibran, 2010; Blaber et al., 2012; Wang et al., 2012a). Yet, there is an agreement that h-MSCs do not express CD3 (Oswald

et al., 2004; Wu et al., 2007), CD14, CD34, CD45 (Baron and Storb, 2012), CD31, an

endothelial and haematopoietic cells marker and CD117, a haematopoietic stem/progenitor cell biomarker (Kolf et al., 2007; Motaln et al., 2010; Parekkadan and

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Milwid, 2010; Torsvik et al., 2010), CD15, CD33 (Oswald et al., 2004; Motaln et al., 2010; Parekkadan and Milwid, 2010), CD38 (Oswald et al., 2004), CD40 (Rastegar et al., 2010), CD80, CD86 (Motaln et al., 2010; Parekkadan and Milwid, 2010; Rastegar et al., 2010). Moreover, h-MSCs do not express CD106 (vascular cell adhesion molecule [VCAM]-1), CD62E, CD62L, CD62P, CD50, CD49b, CD49d, CD49f, CD25, CD16, CD11a, CD8, glycophorin A and cadherin V (Motaln et al., 2010; Parekkadan and Milwid, 2010). Accordingly, the criteria mentioned by Rastegar and colleagues that MSCs negative for CD40, CD80, CD86 and MHC II make these cells attractive candidate for treating human diseases especially allogenic stem cell transplantation without immunosuppression (Rastegar et al., 2010).

1.4.3.3 CONTROVERSIAL MARKERS

Human MSCs populations express considerable variability in their cell surface markers. There is therefore no unique surface antigen specific to MSCs (Quirici et al., 2002; Hocking and Gibran, 2010). Mabuchi and others (Mabuchi et al., 2013) reported that most CD markers expressed by h-MSCs such as CD49a, CD73, CD105, CD106, CD271, MSC antigen-1, Stro-1, and SSEA-4 are not specific for MSCs; rather, they are widely expressed in stromal cells. CD106 is also considered as a positive marker for h-MSCs while other researchers have shown that h-MSC do not express CD106 (Motaln et al., 2010; Parekkadan and Milwid, 2010; Baron and Storb, 2012). Additionally, other CD markers such as CD271/NGFR (Quirici et al., 2002), CD10, CD13, CD29, CD44, CD90/Thy-1, CD105, foetal liver kinase 1 (Flk-1) /CD309 and Sca-1 (Honczarenko et al., 2006) are often expressed on the MSC cell surface, but they lack specificity or consistent expression. Researchers have therefore reported an enormous variation in positive markers and each group of co-workers have used different subsets of markers. The lack of specific markers for h-MSCs represents the challenge of MSC identification (Kolf et al., 2007) and attempts to investigate the true identity and function of these cells is still problematic (Mabuchi et al., 2013).

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Positive Markers References Negative Markers References

CD73 CD90 CD105 (Oswald et al., 2004; Dominici et al., 2006; Gimble et al., 2008; Hocking and Gibran, 2010;

Blaber et al., 2012; Wang et al., 2012) CD14 CD34 CD45 (Oswald et al., 2004; Dominici et al., 2006; Gimble et al., 2008; Hocking and Gibran, 2010;

Baron and Storb, 2012; Blaber et al., 2012; Wang et al., 2012) CD13 (Oswald et al., 2004; Honczarenko et al., 2006; Wu et al., 2007; Motaln et al., 2010; Parekkadan and Milwid, 2010)

D19 CD11b CD79a HLA-DR (Oswald et al., 2004; Dominici et al., 2006; Gimble et al., 2008; Hocking and Gibran, 2010;

Blaber et al., 2012; Wang et al., 2012) CD29 CD44 CD71 (Oswald et al., 2004; Honczarenko et al., 2006; Wu et al., 2007; Motaln et al., 2010; Parekkadan and Milwid, 2010;

Baron and Storb, 2012)

CD31 CD117

(Kolf et al., 2007; Motaln et al., 2010; Parekkadan and Milwid, 2010)

CD38 (Oswald et al., 2004) CD40 (Rastegar et al., 2010) CD106 CD120a CD124 HLA-I HLA-II

(Baron and Storb, 2012)

CD15 CD33

(Oswald et al., 2004; Motaln et al., 2010; Parekkadan and Milwid, 2010;

CD3 Oswald et al., 2004; _{Wu et al., 2007)}

CD106 CD62E CD62L CD62P CD50 CD49b CD49d CD49f CD25 CD16 CD11a CD8 glycophorin A cadherin V (Motaln et al., 2010; Parekkadan and Milwid, 2010)

CD166 Parekkadan and Milwid, 2010; (Motaln et al., 2010;

Baron and Storb, 2012) CD146 CD58 CD54 CD49e CD10 (Motaln et al., 2010; Parekkadan and Milwid, 2010)

CD271 (Quirici et al., 2002; Motaln et al., 2010;

Parekkadan and Milwid,2010)

Sca-1 (Wu et al., 2007)

Chemokine Receptors CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR4, CCR7, CCR9, CCR10, CX3CR1 (Patel et al., 2013) CD80 CD86 (Motaln et al., 2010; Parekkadan and Milwid, 2010;

Rastegar et al., 2010)

Table 1.1 Positive and negative markers of MSCs.

MSCs are able to express a wide range of cluster of differentiation (CD) markers and many chemokine receptors. These markers enable MSCs to bind to a variety of growth factors, cytokine and chemokines and their activating ligands thereby MSCs participate in a wide range of cellular activities and treating many pathological conditions. On the other hand, MSCs determined as negative haematopoietic stem cell markers.

In addition to the phenotypic characterisation of MSCs, they could be characterised according to their differentiation potential.

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