Femke Mathot

2 Stem cell differentiation in peripheral nerve repair 33 growth factor as mitogenic factors to facilitate formation of cellular spheres. The spheres were induced to differentiate in media containing BDNF and all-trans-retinoic acid. After four weeks of culture, only half of the MSCs demonstrated the characteristic neuronal morphology, which expressed nestin and neuronal markers MAP-2 and NeuN, but lacked the expression of glial markers S100 and GFAP. 31 Ahmadi and colleagues compared the method of Anghileri to the chemical induction protocol of Woodburry and colleagues. Woodburry included an optimization step in which MSCs are initially induced by addition of ß-mercaptoethanol (BME) for 24 hours followed by induction of neural differentiation using dimethylsulfoxide (DMSO), BME and butylated hydroxyanisole for 1 to 5 days. Ahmadi noted the differentiation protocol of Anghileri significantly improved MSC survival and increased MSC viability, indicating the use of potentially toxic substances (e.g., DMSO and BME) may not be necessary and could be avoided for MSC differentiation. 32, 33 Thaler and colleagues confirmed the toxic effect of DMSO by demonstrating DMSO can initiate epigenetic changes which increased cell apoptosis. 34 Despite the attempts to equal the efficiency of Kingham’s differentiation protocol by alternative chemical induction methods, none have resulted in high percentages of viable Schwann-like cells. In an ideal scenario, MSCs can be differentiated by natural, non-toxic compounds that are largely available, cost- effective and which do not influence the viability of MSCs. In an effort to find a method meeting the requirements listed above, studies have been performed on the effect of nerve tissue/nerve leachate to cell cultures, co-culture of MSCs with Schwann cells and the electrical stimulation of MSCs. The induction culture medium of Liu and colleagues consisted of 1cm fragments of rat sciatic nerves soaked in normal growth medium (i.e., DMEM and 10% FBS). After 2 days, nerve fragments were removed and adipose tissue derived MSCs were further cultured in the sciatic nerve leachate for another 3 days. Cells adopted a spindle-shape within 48 hours and reflected by expression of S100 and GFAP proteins as was confirmed by immunohistochemistry and western blot analysis, but the nerve autografts required for this protocol would not create a clinically viable therapeutic solution. 18 Liao compared three methods to induce adipose tissue derived MSCs, including (I) neural induction with chemicals only (i.e., media with 2% DMSO for 5 hours), (II) neural induction by chemicals combined with growth factors (i.e., NGF, bFGF/FGF2 and BDNF, as well as the cAMP-related drug Forskolin) for 2 weeks, and (III) neural induction by co-culture of MSCs with Schwann cells. Immunohistochemistry and gene expression analysis showed higher mRNA levels for S100, nestin and GFAP in method II and III compared to method I. Similar to Liu and others, autologous Schwann cells would pose a practical problem for the clinical implementation of method III. 35 Das and colleagues differentiated MSC into Schwann-like cells by electrical stimulation to alter cellular membrane potential. The majority of electrically induced MSCs (>80%) showed Schwann cell markers S100 and p75 and enhanced secretion of NGF compared to chemically induced MSCs or undifferentiated MSCs. 36 Although electrical differentiation is promising and may mimic aspects of normal neuronal cell differentiation, physical methods for differentiation have remained largely unexplored and it remains unclear whether electrical stimulation will have practical benefits compared to differentiation with growth factors.