90 Additionally, MSC can assimilate proteins and pieces of membrane from other cells, requiring more definitive proof of “trans-differentiation” of MSC to neurons, particularly in co-cultures and in vivo where MSC have extensive contact with neural tissue. Recent studies have shown that RNA species and proteins can be passed from mammalian donor cell to target cell, through gap junc- tions, exosomes, virtosomes, or tunneling nanotubules [47–49]. RNA interference (RNAi) molecules were found to be secreted in microvesicles, which later fused with other cell [50–53]. Ratajczak et al. [54] described horizontal transfer of mRNA from cell to cell through embryonic stem cell-derived microvesicles. Virtosomes were recently described to be complexes of RNA– lipoprotein, which can readily enter other cells where they can modify the biology of the recipient cells [47]. Tunneling nanotubes have also been described for trans- ferring RNA species and organelles between cells [48]. Our group is currently exploring an MSC-based platform for producing and delivering RNAi moieties targeted to the huntingtin mRNA as a potential therapeutic avenue for treating HD [55]. We chose to explore human MSC, which we and others have previously shown to be excellent in vivo delivery vehicles for enzymes and proteins, as the cell type to produce and convey RNAi [2, 17]. Transfer of organelles as large as mitochondria from MSC to damaged cells has also been described [56]. Therefore, reports of MSC trans-differentiating to neural phenotypes, particu- larly after co-cultivation with neurons, must be cautiously interpreted. However, the ability of MSC to migrate to areas of damage in the brain and to secrete neurorestor- ative factors is significant and could be harnessed to treat neurodegenerative disease. Expression of Neuroregulatory Factors by MSC Understanding the innate capacity of MSC to influence neural cell growth, survival, and neurite extension is currently a very important field of study. Detailed determi- nation of which factors MSC express, under which conditions, is key. Crigler et al. [23] demonstrated that specific subpopulations of human MSC expressed BDNF and beta-NGF, but not neurotrophins NT-3 or NT-4. The authors used a co-culture assay to show that BDNF expression levels correlated with the ability of MSC subclones to induce survival and neurite outgrowth in the SH-SY5Y neuroblastoma cell line. The effects were only partially inhibited by a neutralizing anti-BDNF antibody, indicating that other factors secreted by the MSC also had neuroregulatory effects. The authors identified production of other neurite-inducing factors, axon guidance, and neural cell adhesion molecules, which contributed to the capacity Mol Neurobiol (2012) 45:87–98 of the MSC to induce neuronal cell survival and nerve regeneration. Wilkins et al. [57] have also demonstrated that MSC secrete BDNF and that addition of anti-BDNF neutralizing antibodies attenuated their neuroprotective effects. These studies help to at least partially determine the mechanisms of MSC-mediated neural survival and also validate the important role of BDNF in neuronal protection. In addition to neurotrophic factors, the extracellular matrix (ECM) molecules produced by MSC have also been shown to support neural cell attachment, growth, and axonal extension [58]. Neurons on ECM derived from MSC formed more complex neurite networks than those cultured on poly-D-lysine [58]. Croft et al. [59] showed that soluble factors produced by MSC promoted the survival of neurons in culture and promoted axonal growth in neuronal progeny. The authors proposed mechanisms to manipulate MSC in vitro prior to transplantation that could potentially further enhance the endogenous neurogenic response to injury. Different extracellular matrix scaffolds have also been examined to enhance survival of stem cells after implantation to damaged areas of the brain, and some ECM molecules can synergize with the neurorestorative effects of MSC [60]. However, a highly important consideration is to not use matrix proteins such as fibronectin that could enhance fibroblastic differentiation from MSC, which could risk scarring and microglial activation. Therefore, such scaffolds for implantation must be carefully tested in animal models of HD before consideration for future clinical application. MSC provide a very promising platform from which to produce factors for neural regulation. Viral vectors under inducible promoters can be used to customize MSC for the treatment of specific diseases, as described in more detail below. Overexpression of BDNF is a particularly promising modification for HD. A traditional gene therapy approach, where the neurons would be directly infected in vivo by live lentiviral or AAV vectors carrying BDNF would suffer from a number of safety concerns. Integrating virus can pool at the injection site, superinfecting neighboring cells and limiting distribution beyond a small area. Viral integrations can be controlled ex vivo with MSC and limited to one to two viral integrants per MSC genome, as suggested by the FDA for stem cell gene therapy trials. Using MSC as the delivery vehicle, a “suicide gene” such as thymidine kinase can also be used to eliminate a graft if anything went wrong. This would not be possible with vector-mediated delivery since the “suicide approach” would destroy the neuron into which the gene had integrated. The natural reparative characteristics of MSC can act synergistically with the growth factors that they produce. MSC could potentially migrate to injured cells to deliver BDNF locally.