MiR p downregulated in OM MSCs has been

MiR-140-5p (downregulated in OM-MSCs) has been reported to inhibit the expression of CXCL12 (Nicolas et al., 2008), which has been shown to promote oligodendroglial cell maturation in vitro (Göttle et al., 2010; Kadi et al., 2006) and myelination in the demyelinated cuprizone model (Patel et al., 2012). Given that miR-140-5p is downregulated in OM-MSCs, it was possible that CXCL12 expression correlated with their increased myelinating capabilities. We demonstrated that OM-MSCs secreted significantly greater amounts of CXCL12, confirmed its pro-myelinating effect using myelinating co-cultures, and using miR-140-5p mimic/antagomir, we demonstrated an inverse relationship of mRNA for secreted CXCL12. Moreover, CM from MSCs transduced with the miR-140-5p antagomir and mimic affected CNS myelination in vitro, indicating that the pro-myelinating effect of OM-MSCs was due, at least in part, to CXCL12 secretion controlled by miR-140-5p. This reveals an important role for MSC-secreted CXCL12 in myelination. It is well understood that CXCL12 is vital in controlling hematopoietic stem cell and progenitor function within the human and rodent BM (Isern et al., 2014; Greenbaum et al., 2013) and that there are certain cell types that secrete high levels of CXCL12: CXCL12-abundant reticular (CAR) cells, nestin-GFP + stromal cells, and leptin receptor + stromal cells. However, what is not known currently is whether CD271 selection isolates which cell population from the BM, although a recent report has demonstrated nestin gene expression after CD271 purification (Li et al., 2014). Since we have previously shown our BM-MSCs to be only 50% nestin positive, it would suggest we are harvesting only some of the nestin-positive antibiotics previously shown to secrete CXCL12 (Isern et al., 2014; Greenbaum et al., 2013). CD271 may also isolate a population that does not produce CXCL12 or produces it in only low amounts, unlike in the OM where it exclusively isolates the nestin-positive MSCs. However, a direct correlation between nestin, CD271 expression, and CXCL12 secretion has yet to be established in OM-MSCs. To elucidate CXCL12 mechanism of action, we determined the cellular expression of its receptors (Lipfert et al., 2013). Both OPCs and microglia strongly expressed multiple isoforms of CXCR4, which may reflect various post-transcriptional modifications that have been shown to exist previously (Sloane et al., 2005; Carlisle et al., 2009). CXCR7 expression could be detected only weakly, suggesting that the predominant receptor type is CXCR4. OPCs responded to CXCL12 by enhanced process branching and membrane formation, however myelin marker differentiation remained unchanged. Since a prominent activity for chemokines is to regulate leukocyte trafficking, which is considered to occur through actin cytoskeleton modulation (Thelen and Stein, 2008), it could be that CXCL12 regulates OPC actin cytoskeleton during process extension in myelination. Since microglia express CXCR4, it is possible that CXCL12 may indirectly influence myelination through their activity. Microglia are both a source and/or target for CXCL12 (Albright et al., 1999; Lee et al., 2002), which promotes their migration and proliferation. Microglia treated with CXCL12 or OM-, not BM-MSC-CM, upregulated arginase I expression, which resembles polarization seen in anti-inflammatory macrophages (Murray et al., 2014). Conversely, iNOS expression was induced by BM-MSC-CM but not CXCL12 or OM-MSC-CM. These differences suggest that OM-MSCs induce microglia to polarize predominantly to a more anti-inflammatory phenotype, in contrast to BM-MSC-CM, which appears to shift them toward a pro-inflammatory phenotype. Although the anti-inflammatory phenotype is thought to play a role in myelination via activin A (Miron et al., 2013), there was no difference in activin A levels in CM of both MSCs or an upregulation of it in treated microglia, suggesting that this is not the mechanism.