**5. Conclusions**

major histocompatibility complex (MHC) and costimulatory molecules and the fact that they can suppress the activity of numerous immune cell populations [42–45]. Despite the overall safety reported by a large number of CTs [25, 41, 45], substantial evidence now supports both cell-mediated and humoral immune responses against donor antigens following administration of these cells highlighting that MSCs can be recognized by the host immune system (reviewed by Berglund et al. and Lohan et al.) [137, 138]. On another end, iPSCs are envisioned as a source to eliminate immune rejection; however, this remains theoretical, as therapeutic human trials have yet to be conducted. It will be important to monitor DNA methylation status and gene expression changes that could evoke immune responses in transplanted hosts even if iPSCs are autologously derived. Therefore, the possibility of a therapeutic cell-free

GFs and cytokines packed and secreted by MSCs (secretome) are thought to play a significant role in SCI repair, mainly by lowering pro-inflammatory cytokines (i.e., IL-2 or IL-6 and TNFα) [139]. In fact, Cizkova et al. attributed motor function recovery, attenuated inflammatory response, and spared spinal cord tissue to a molecular cocktail found in the MSCs after transplantation [140]. MSC paracrine secretion or secretome was first described by Haynesworth et al. in 1996 [141]; since then multiple actions are endowed to MSC secretome rather than to their engraftment. Such actions include increased angiogenesis, decreased apoptosis and fibrosis, enhanced neuronal survival and differentiation, restriction of local inflammation, and adjustment of immune responses, effects that translate into induction of regeneration of damaged tissues [142]. Therefore, the therapeutic value of stem cells may mainly derive from the released factors or secretome including soluble and vesicle-packed factors. This latter fraction, termed extracellular vesicles (EVs), is a heterogeneous mix of vesicles including exosomes, a subset of double-membrane vesicles characterized by the expression of a set of markers, including tetraspanins CD9, CD63, and CD81 with attributed intercellular commu-

nication role including the transfer of their cargo (DNA, RNA, and proteins) [143].

The first documented clinical administration of EVs was performed in 2011, by administration of EVs intravenously infused at intervals of 2 or 3 days during a period of 2 weeks to a steroidrefractory GvHD patient who showed declined symptoms and stability for over 4 months [144]. Many preclinical models have shown the benefit of EV-based therapy including longterm neuroprotection. Treatment with MSC-derived EVs promoted long-lasting recovery of cognitive functions in inflammation-induced preterm brain injury [145]. EV-based therapy of SCI in rats showed a reduction of inflammatory response with apparent astrocyte and microglia disorganization in cord tissue up to 10 mm caudal to the injury site as well as locomotor recovery [146]. This illustrates the multiple potential benefits of EV-based therapies to treat neuroimmune defects. EV superiority with respect to cell-based therapeutics resides in its ready availability, ease of storage and distribution, reduced immunoantigenicity, scalability, and possibility of multiple routes of administration. EVs can also be used as delivery particles by directionally packaging molecules of interest from genetically modified cells while avoiding the risk of transfer of transformed live cells and could be obtained from iPSCs as well. Guidelines and recommendations for production, quality assurance, and application of EV-based therapeutics have been provided in an International Society for Extracellular Vesicles (ISEV) and European Network on Microvesicles and Exosomes in Health and

product could be highly relevant on safety terms.

124 Cell Culture

Although CTs have in general evidenced MSC safety, the removal of FBS from clinical-grade stem cell protocols results imperative. The pooling of a large number of donors of cells and human blood fraction-based media through the use of stem cell banks or the use of xeno-free synthetic defined media should translate into allogeneic MSC preparations leading to more homogeneous clinical results. Thus, allowing minimal immune-related safety concerns derived from FBS and unveiling the real therapeutic value of *in vitro* expanded off-the-shelf MSCs.

The iPSC manufacturing technology offers the possibility of developing patient-tailored cell therapies with the consequent safety and immune-related advantages, as genetically identical cells should prevent immune rejection. iPSCs can differentiate into all three germ layers and, by their nature, do not raise bioethical debate. However, safety concerns related to *in vivo* properties of immortal cell types and the use of genetically manipulated cells raise regulation hurdles for their use in the clinic.

Preconditioning of *in vitro* expanded MSCs to ensure cell lineage commitment might result advantageously for improved treatment of particular diseases. Optimizations for the treatment of SCI and other neuroimmune health problems such as ME/CFS remain. Also, EVs and in particular exosome-enriched MSC-derived fractions may eventually become the treatment of choice for cell-based-free therapeutics by themselves or in combination with other clinical treatments once GMP production is optimized.
