Femke Mathot

Chapter 4 68 expression of COL3A1 in undifferentiated MSCs (+186.328 ± 50.264; p=0.011), but did not appreciably alter expression of COL1A1 , FBLN1 and LAMB2 . For differentiated MSCs, the interaction resulted in a significant enhanced expression of FBLN1 (1.918 ± 0.386; <0.001) but no changes in the COL1A1 , COL3A1 and LAMB2 gene expression. Gene expression over time The temporal expression of extracellular matrix genes exhibited significant differences between cell type ( figure 4 ). In general, the temporal expression profiles of COL1A1 and COL3A1 in undifferentiated and differentiated MSCs did not differ substantially from each other. ANOVA analysis only demonstrated a significant interaction between and within groups for COL3A1 expression over time (p=0.027). For undifferentiated MSCs, a significant decrease in COL3A1 between 3 days and 7 days after seeding (p=0.050) was observed, while for differentiated MSCs a significant increase between 14 and 21 days after seeding (p=0.007) was measured. There was a significant interaction between and within groups for FBLN1 expression (p<0.001). The FBLN1 expression in both groups started high, slowly decreased in the first week and increased from 7 days onwards. A significant interaction between cell-types (p=0.001) was found for the LAMB2 expression, while no within-group differences were found over time. Overall, the LAMB2 expression in undifferentiated MSCs slowly seemed to climb, reaching maximal levels at 21 days after seeding, while the expression of LAMB2 in differentiated MSCs appeared to decline slowly. Cell regulatory gene expression Baseline gene expression The baseline expression of CCBN2 (1.613 ± 0.580, p=0.049) and CASP3 (0.867 ± 0.081, p<0.001) in undifferentiated MSCs was significantly higher than in differentiated MSCs. After seeding After seeding, the gene expression in undifferentiated MSCs of CASP3 was significantly enhanced (0.983 ± 0.279; p=0.018), while CCBN2 -expression significantly decreased (-1.638 ± 0.501; p=0.040). Seeding of differentiated MSCs resulted in a significant enhanced expression of CASP3 (2.501 ± 0.279; p<0.001) and CCBN2 (1.796 ± 0.501; p<0.001). Gene expression over time Overall, CASP3 expression of both cell-groups was high directly after seeding, but decreased afterwards and mRNA levels remained steady from 7 days onwards. ANOVA analysis showed a significant interaction between culture duration and the mean CASP3 expression (p<0.001), but no significant differences between groups (p=0.404). CCNB2 expression had a significant interaction with the cell-type and culture duration (p<0.001). In general, CCBN2 expression of undifferentiated MSCs was low during the first 3 days after seeding and then slowly increased, reaching a peak after 21 days. In differentiated MSCs there was a peak measured after 1 day and then the expression slowly decreased. All expression values for cell cycle regulatory genes are presented in figure 5 with significant differences between groups as indicated.