Femke Mathot

Chapter 2 32 MSCS IN PERIPHERAL NERVE REPAIR Differentiation of MSCs into Schwann-like cells Theneural inductionofMSCsbychemicals combinedwithgrowth factors is themost established method to obtain Schwann cell-like differentiation. The induction protocol by Kingham and colleagues is widely used and includes two preparation steps with ß-mercaptoethanol (for 24 hours) and all-trans-retinoic acid (for 72 hours). Subsequently the cells are placed in growth medium enriched with a differentiation cocktail containing Platelet-derived growth factor (PDGF-AA), basis fibroblast growth factor (bFGF), Forskolin and a member of the Neuregulin family (Neuregulin-1 ß1, Glial growth factor-2 or Heregulin- ß1). After 2 weeks in differentiation medium, the morphology of MSCs is altered into an elongated spindle shape, characteristic of Schwann cells. Immunohistochemistry and Western blot analysis after differentiation has revealed expression of several phenotype-specific surface markers, including GFAP, S100 and p75-NTR. 2, 4, 5, 11, 17, 23-25 Studies have demonstrated this protocol is also suitable for human MSCs and that the function of those differentiated cells is analogous to Schwann cells. 4, 26 Regardless of the fact that the effect of differentiated MSCs needs to be further examined and confirmed, these findings imply that research on targeted stimulation of MSCs could be applied in humans in the future and therefore has a serious clinical relevancy. The additional timing and cost of differentiation need to be justified prior to human trials. The components of Kingham’s induction protocol each have specific biological functions. Forskolin activates adenylyl cyclase which increases the level of intracellular cyclic adenosine monophosphate (cAMP). cAMP causes an increase in the mitogenic responses of Schwann cells 27 , in response to the mitogenic actions of the growth factors PDGF and bFGF/FGF2. 28 The neuregulin-1 family plays a crucial role in the actual development and evolution of Schwann cells. Their presence activates cascades promoting Schwann cell differentiation and expansion. The level of Neuregulin-1 (NRG1) determines differentiation of Schwann cells into myelinating or non-myelinating cells that are responsible for the development of group C nerve fibers. NRG1 levels determines axon size, which enables the myelinating Schwann cells to optimize myelin sheath thickness. 29, 30 Kingham’s protocol is currently the preferred method to obtain Schwann-like cells from adipose derived MSCs. The effect of altering the dosages of the different components of the differentiation protocol on the ultimate function of the Schwann-like cells is an interesting prospect for future research. As targeted neurogenic stimulation to induce differentiation of MSCs is an expensive, time- consuming and inefficient process requiring several weeks of laboratory-based preparation time, efforts have been made to find alternative approaches. Safford and colleagues used a chemical induction medium with butylated hydroxyanisole, potassium chloride, valproic acid, Forskolin, hydrocortisone and insulin to differentiate murine and human MSCs. Within 5-6 hours following neuronal induction, they observed dramatic cell morphological changes in cytoskeletal organization and membrane appearance in MSC cultures which persisted up to 5 days. Beyond 5 days however, the differentiated MSCs lost viability and perished within 14 days of culture. 10 The induction protocol of Anghileri involves the culturing of MSCs for 72 hours in growth medium with exogenous bFGF (FGF2) and human epithelial