Femke Mathot

4 Interaction between MSCs and a nerve allograft 71 DISCUSSION In this study, the gene expression profiles of undifferentiated and differentiated MSCs before and after seeding on a decellularized nerve allograft were analyzed. There is little consensus about which stem cell types are most optimal for use in clinical human peripheral nerve regeneration studies due to the ambiguity in their mechanism of action. We examined the expression of a large panel of genes that have a known role in nerve regeneration and compared their expression in undifferentiated MSCs to differentiated MSCs, before and after they interacted with the ECM of decellularized nerve allografts. Interaction between MSCs and the nerve allograft Compared to undifferentiated MSCs, the differentiation of MSCs into Schwann-like cells led to significantly enhanced mean expressions of the neurotrophic genes PMP22 and PTN , the angiogenic gene VEGF1 and extracellular matrix gene COL3A1 . Expression of other neurotrophic genes (i.e., NGF and GDNF ), the extracellular matrix gene FBLN1 , and cell growth and survival related genes CCBN2 and CASP3 was significantly decreased after differentiation. When comparing the gene expression of both undifferentiated and differentiated cell states before and after the introduction to the ECM of decellularized nerve allografts, it was demonstrated that the interaction initially led to enhanced expression of VEGF1 , COL3A1 and CASP3 in undifferentiated MSCs and increased mRNA levels of NGF , GDNF , VEGF1 , FBLN1 , CASP3 and CCBN2 in differentiated MSCs. Our results corroborate studies that describe interactions between the ECM and MSCs which led to significant changes in their gene expression profiled and the observation that differentiation of MSCs leads to enhanced expression of VEGF1 . 26, 69 Possible explanations for the reduced expression of NGF and GDNF in undifferentiated MSCs directly after seeding may include the lack of surgical host factors and the original (motor) function of the nerves used. 70, 71 Gene expression over time The short-term measurements (immediate post seeding, 1 day and 3 days after seeding) indicate differentiated MSCs express high levels of neurotrophic ( NGF , GDNF , GAP43 ), angiogenic ( VEGF1 ), tissue supportive ( FBLN1 ) and cell regulatory genes ( CASP3 , CCNB2 ) which decrease over time. Undifferentiated MSCs appear to have a smaller role in the short term and only express high levels of VEGF1 and CASP3 . The effect of undifferentiated MSCs are seen in the long term (7, 14 and 21 days after seeding) by expressing enhanced levels of neurotrophic ( NGF , GDNF , PMP22 ), extracellular matrix ( FBLN1 , LAMB2 ) and cell regulatory genes ( CCNB2 ). In this later phase, the expression of factors from differentiated MSCs is less than undifferentiated MSCs. The expression of tissue supportive genes COL1A1 and COL3A1 remained stable over time in both cell types. The gene expression cascades presented here suggest that a combination of both differentiated and undifferentiated cells would lead to an optimal regenerative micro- environment for decellularized nerve allografts, by providing growth factors that stimulate axon ingrowth after implementation.