Cell surface markers under different culture conditions

The IFATS and ISCT have established minimal criteria for phenotypic identification of adherent stromal/stem cell population, which were first published in 2006 by Dominici et al. (Dominici et al., 2006) and recently updated by Bourin et al. (Bourin et al., 2013). Although this guidance is a valuable tool for cell characterization, the criteria are defined for cells cultured under standard conditions in FBS- supplemented medium, and certain differences may arise if cells are cultured in different serum conditions. Our flow cytometry results demonstrate that the cell surface-marker profile defined by Dominici et al. primarily applies to ASCs cultured under FBS, HS and XF/SF conditions because cell surface-marker expression of ASCs was highly similar between cells grown under different serum conditions.

However, small variations were observed in the expression of CD11a (Integrin alpha), CD14 (lipolysaccharide receptor), CD19 (leukotriene B4 receptor), and CD86 (co-stimulatory molecule for T-cell activation) between cells grown in XF/SF conditions versus serum-containing medium (I). All of these markers are known to interact with immune-related cells, and thus the culture conditions indeed have a potential effect on the immunogenicity of ASCs, as demonstrated in study III.

Of note, the expression of CD54 was altered depending on the serum conditions, and statistically significant higher expression was observed in serum-containing medium versus SF/XF conditions (I, III). The CD54 binds to integrins of type CD11a or CD11b and is reported to be present in endothelial cells, antigen- presenting cells, and certain stromal cells. Furthermore, it was demonstrated that CD54 and CD106 play a key role in mesenchymal stem cell-mediated immunosuppression (Kronsteiner et al., 2011; G. Ren et al., 2010; G. Ren et al., 2011).

These previous published studies are in line with the results of our immunological studies (III) in which FBS expanded ASCs showed high expression of CD54 and

had the strongest immunosuppressive potential. In addition, the low expression of the adhesion molecule CD54 as observed under XF/SF conditions may be reflected by a notably weaker cell adhesion of ASCs. The lower CD54 expression also suggest that a different cell populations could be selected through XF/SF isolation and expansion protocols compared with cells grown in the presence of serum.

Additionally, statistically significant differences were observed for the expression of CD45 with higher expression in XF/SF conditions (III). The CD45 is a receptor- linked protein-tyrosine phosphatase that is shown to be essential for leukocyte differentiation and antigen receptor-mediated signal transduction (Altin and Sloan, 1997; Matsuda et al., 1998). The XF/SF expanded ASCs were less immunosuppressive compared with those in serum-containing medium, which was in line with higher expression of CD45 in XF/SF conditions. Nevertheless, due to a lack of stronger evidence, the immunosuppressive capacity of ASCs should not be evaluated based on the expression of CD45. Further, as stated in the updated guidance by IFATS and ISCT, one of the main differences between SVF cells and ASCs should be the high expression levels of CD45 in SVF cells and a notably low or undetectable level in ASCs (Bourin et al., 2013). This definition applies to ASC cultures in FBS or HS conditions, but slightly higher expression of CD45 appears to be present in defined XF/SF conditions (I, III). Compared with BM-MSCs, CD45 is more readily expressed in ASCs because BM-MSCs do not express CD45 even at low levels (Liao and Chen, 2014; Pachon-Pena et al., 2011). Although CD45 is the classic marker used to identify cells of hematopoietic origin, various isoforms of CD45 exist, and a chosen isoform of CD45 marker may also affect the intensity of the expression (Tchilian and Beverley, 2006). In humans, a high-molecular-weight isoform (CD45RA) is expressed in naive T-lymphocytes, whereas the low-molecular- weight isoform (CD45RO) is expressed after T-cell activation (Tchilian and Beverley, 2006).

Furthermore, the hematopoietic stem cell marker CD34 (Bensinger et al., 1993;

Trischmann et al., 1993) was moderately expressed in both XF/SF and serum- supplemented conditions (I) in contrast to the originally defined criteria (Dominici et al., 2006). Since 2006, similar variable expression for CD34 has been reported by others (Mirabet et al., 2008; Rebelatto et al., 2008), and the expression of CD34 appears to be greatly dependent on the length of the in vitro culture period; it is typically expressed during the early phase of culture, but its expression subsequently decreases after continued cell divisions (Maumus et al., 2011; Mitchell et al., 2006).

We observed a similar phenomenon in our studies (I, III) in which the expressions of both CD34 and CD54 were higher in passage 2 but decreased in later passages,

thus indicating a more homogeneous population. Compared with BM-MSCs, CD34 is more readily expressed on ASCs because BM-MSCs do not express CD34 even during the early phase of culture (Liao and Chen, 2014; Pachon-Pena et al., 2011).

Additionally, CD34 is a marker of endothelial progenitor cells, and moderate expression of CD34 has been reported in endothelial cells (43±19), as analyzed from human umbilical vein endothelial cells (HUVECs) (Huttala et al., 2015).

The typical immunophenotype of ASCs was also maintained in MMC cultures (II) in all of the studied serum conditions of FBS, HS and XF/SF. However, in XF/SF conditions the immunophenotypic analysis was performed after 7 days of exposure to MMC because longer-term culture with MMC was not feasible. The XF/SF cultured ASCs showed poor viability and proliferation capacity under MMC, and as a result the typical immunophenotype of ASC was lost under longer-term exposure to MMC, which was confirmed with one ASC donor cell line. Interestingly, the expression of CD54 was significantly higher under MMC in all of the studied serum conditions. Several explanations might exist for these results. The proliferative capacity of ASCs was significantly decreased under MMC culture, especially in XF/SF conditions, and it could be speculated that the cell viability under MMC media was not 100%. Although use of a cell viability marker during a flow cytometry analysis would have strengthened this result, a uniform cell population was still observed in the forward and side scatter data, which suggested that no substantial amount of dead cells was present in the analysis.

As shown in previous studies, ECM is extensively deposited under MMC conditions (Ang et al., 2014; C. Z. Chen et al., 2011) leading to more mature ECM, which might reflected stronger cell adhesion and higher expression of adhesion molecule CD54. In previous studies, the intra- and extracellular protein organization between +/- MMC cultures were compared, and these studies showed that it is possible to further align the ECM even in the absence of cellular interaction (Zeiger et al., 2012). The ECM in turn affects cell-matrix interactions and promotes cell adhesion, thus exerting an influence on the formation and structure of the cytoskeleton (Zeiger et al., 2012). As shown by previous studies, the matrix deposition is increased under MMC conditions (Ang et al., 2014; C. Z. Chen et al., 2011), and when ECM is mature and extensively deposited, the cell adherence is also strong (C. Z. Chen et al., 2011), as can be observed in the higher expression of adhesion molecule CD54. Thus, higher CD54 expression may be associated with stronger cell adherence because higher CD54 expression levels were observed for MMC exposed cells, as opposed to the lower CD54 levels of weakly attached XF/SF cells.