Volume 9, Issue 1, Pages 69-79 (January 2007) Comparison of cellular functionality of human mesenchymal stromal cells and PBMC  H. Schmal, MD, P. Niemeyer, M. Roesslein, D. Hartl, T. Loop, N.P. Südkamp, G.B. Stark, A.T. Mehlhorn  Cytotherapy  Volume 9, Issue 1, Pages 69-79 (January 2007) DOI: 10.1080/14653240601011557 Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 1 MIP-1α induced a chemotactic response with MSC, with a typical bell-shaped curve and maximal activity at concentrations of 1ng/mL and 10ng/mL. PBMC did not migrate following stimulation with the β-chemokine used. Fluorescence correlates with the number of migrated cells. The HBSS control indicates the background migration of cells. Statistical significance indicates migration compared with background levels (n=6, mean ± SEM). Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 2 The expression of chemokine receptors was analyzed using FACS. CCR-1 was expressed on the surface of MSC (right) (P=0.03) but not on PBMC (left) (P=NS). CCR-2 was not present on either MSC (right) (P=NS) or PBMC (left) (P=NS). CXCR-4 (P=0.000004) and CCR-7 (P=0.002) were highly expressed on PBMC (left) and on a small subpopulation of MSC (right) that expressed CXCR-4 (P=0.008) and CCR-7 (P=NS) (iso – isotype Ab control, mean ± SEM). Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 3 A chemotaxis assay demonstrated serum-induced migration of MSC (left) down to a dilution of 1:100 (P=0.05 vs. background levels). Activation of serum by Z did not induce an enhancement of the chemotactic response of MSC. The assay further demonstrated serum-induced chemotactic migration of PBMC (right) in a dose-dependent manner. This response was enhanced by activation of serum with Z, releasing active C5a (serum without dilution, P=NS; dilution 1:10, P=0.007; dilution 1:100, P=0.025, mean ± SEM). The HBSS control indicates the background migration of cells. Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 4 The C5a-induced chemotactic response of PBMC showed the typical bell-shaped curve, with a maximal activity at a concentration of 10ng/mL (P=0.03 vs. background) and 100ng/mL (P=NS vs. background) C5a, whereas MSC did not migrate following stimulation by complement factor (mean ± SEM). The HBSS control indicates the background migration of cells. Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 5 MSC showed a dose-dependant adhesion to FN. In contrast, PBMC did not show any adhesion to FN, independent of the concentration applied. Pre-incubation with 1ng/mL TNFα for 72 h significantly increased adhesion of MSC to FN by 58.5% (P=0.03). The difference also reached statistical significance at 2µg/mL and 10µg/mL FN (P=0.03) (mean ± SEM). Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 6 FN most efficiently enhanced the attachment of MSC, as shown by an adhesion assay (P=0.0012). This was also shown for FG (P=0.0023), col I (P=0.005) and col II (P=0.006), with declining efficiency (mean ± SEM). Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 7 Regulation of receptor gene expression in MSC and PBMC was determined by real-time PCR. C5aR, C5a receptor; ITGa1, 2,5,10,V, β1-integrin α1,2,5,10,V. Gene expression levels were reported relative to gene expression of the housekeeping gene GAPDH. We found minor expression of α1 and α10 and low level expression of α2 and α5; the differences between MSC and PBMC did not reach statistical significance. In contrast, mRNA expression of αv was 12.2-fold higher in MSC compared with PBMC (P=0.0039). Expression of C5aR mRNA was 4.7-fold higher in PBMC (P<0.0001). Values are expressed as mean ± SEM. Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 8 The chemotactic response of TNFα-stimulated MSC was significantly higher compared with cells without pre-incubation, as shown in a chemotaxis assay (undiluted serum, P=0.02; 1:10 diluted serum, P=0.012) (mean ± SEM). The HBSS control indicates the background migration of cells. Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions

Figure 9 To investigate the ability of TNFα to activate NFκB in MSC, cells were incubated with TNFα at concentrations of 1ng/mL and 10ng/mL for 15 min and 30 min. Using EMSA, it could be shown that exposure of MSC to TNFα caused NFκB activation under all the conditions that were explored. (a) Relative optical densities comparing the different treatment methods. (b) A representative blot. Cytotherapy 2007 9, 69-79DOI: (10.1080/14653240601011557) Copyright © 2007 International Society for Cellular Therapy Terms and Conditions