**4.3. Preconditioning of stem cells**

It may result advantageously to precondition MSCs or iPSCs before transplantation according to the particular application or therapy, so that the cells are committed to a particular desired phenotype. Okolicsanyi et al. have more recently shown that heparan sulfate proteoglycans (HSPGs) act as drivers of neural progenitors in expanded bone marrow-derived MSCs [96]. Treatment of MSCs with heparin increased proliferation in addition to the expression of neural markers although these changes were not uniform across growth phases indicating that the direct lineage specifications and functionality of expanded cells might need fine-tuning. Nevertheless, the data sustains that MSCs may provide an abundant source that can be manipulated for purposes of neural repair and regeneration. Biomimetic approaches to exploit the role of HSPGs in neurogenesis, such as synthetic glycopolymers or heparin conjugates, are being developed so that neural differentiation into specific lineages can be controlled and tailored [96, 130].

Technologies, overcoming the need for embryoid body formation and neuronal rosette isolation, was developed by Badja et al. Authors show that after induction the cells express voltage-gated and ionotrophic receptors for GABA, glycine, and acetylcholine (ACh) receptors

Apart from efficient differentiation methods, iPSC generation presents with the limitations of low reprogramming efficiencies (below 0.02%) and genetic modification requirements, as described by Yamanaka et al. [5, 6] and concurrently James Thompson's group [7]; thus, chromosomal instability and tumorigenic potential derived from oncogene overexpression concerns arise for their use in the clinic. An advance for safety is provided by the use of polycistronic plasmids to lead ectopic expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC [118]. Other improvements based on the choice of somatic cell source, choice of reprogramming factors, culture procedures, and delivery methods have been described. For example, reprogramming kinetics and efficiencies vary between somatic cell types, in particular, keratinocytes reprogramed 2 times faster and 100 times more efficient than skin fibroblasts [10], and in general, immature cells are more readily reprogrammed than terminally differentiated cells [119]. The requirement of reprogramming factors also varies according to the cell type, so neural stem cells need only the introduction of OCT4 to be reprogrammed [120]. Reprogramming efficiency can be increased by different methods including adjustment of expression levels of noncoding RNAs, such as microRNAs or lincRNAs [111, 121]. ncRNAs can reduce the amount of reprogramming factors as they specifically target multiple pathways. Traditionally, lentivirus has been the reprogramming vector of choice; other viral vectors such as Sendai and adenovirus to lower transformation risks have been used with lower efficacy [122–124]. Excellent reviews describing more details for reprogramming protocol

In addition to iPSC-derived NPCs, other neural types such as neurons or astrocytes have shown some potential for SCI recovery either to improve synaptic connections or reduce neuropathic pain for the first or to protect the lesion epicenter from infiltrating peripheral inflammatory cells for the second [122, 128]. Peripheral inflammatory cell infiltration can be reduced by the immunoregulatory functions of central nervous system perivascular stromal cells (PSCs). Their low abundance, inaccessibility, and limited proliferation capacity hampers its clinical use. However, PSCs can be successfully generated from iPSCs [129] and expanded *in vitro* without senescing. Thus, SCI stem cell-based therapeutics may benefit including PSCs

It may result advantageously to precondition MSCs or iPSCs before transplantation according to the particular application or therapy, so that the cells are committed to a particular desired phenotype. Okolicsanyi et al. have more recently shown that heparan sulfate proteoglycans (HSPGs) act as drivers of neural progenitors in expanded bone marrow-derived MSCs [96]. Treatment of MSCs with heparin increased proliferation in addition to the expression of neural markers although these changes were not uniform across growth phases indicating that the direct lineage specifications and functionality of expanded cells might need fine-tuning. Nevertheless, the data sustains that MSCs may provide an abundant source that can be manipulated for purposes of neural repair and regeneration. Biomimetic approaches

and recommend the method to model human pathologies [117].

improvements are available [125–127].

122 Cell Culture

as part of combinatorial treatments.

**4.3. Preconditioning of stem cells**

Stem cell transplantation has shown an important limitation due to its poor survival and engraftment at the injured spinal cord, where cells are exposed to hypoxic conditions, nutritional deficiency, or oxidative stress among others. We recently showed that FM19G11, a small chemical, first described as a HIFα protein inhibitor, is able to allow progenitor cells to differentiate into more mature oligodendrocytes under hypoxia without cytotoxic effects at nanomolar doses [IC50 (80 nM)]. Moreover, FM19G11 induces self-renewal by inducing insulin-like signaling pathway and inducing ATP accumulation, activated glucose metabolism with glucose uptake by upregulation of the GLUT4 transporter. The over-induction of AKT/mTOR signaling was directly correlated to the FM19G11-dependent induction of the self-renewal-related markers Sox2, Oct4, Nanog, and Notch1 [130]. Interestingly, the use of a combination of FM19G11 treatment and epSPC transplantation for SCI therapy reduced the glial scar extension and increased the number of neuronal fibers at the epicenter of the lesion. It also increased expression markers for neuronal plasticity and induced oligodendrocyte turnover for potential remyelination [131, 132].

In addition to preconditioning toward enhancing cell survival and proliferation or inducing differentiation into particular cell types, various treatments have shown to impact MSC secretome which could be advantageous for particular therapies. For example, the treatment of MSCs with IL-1β increases the expression levels of a number of cytokines and chemokines as well as induces the expression of cell adhesion molecules improving the migration ability of preconditioned cells to the site of inflammation *in vivo* [133]. Preconditioning protocols typically include physical treatments such as different degrees of hypoxia, mechanical stretching, application of electromagnetic fields or mimicking of three-dimensional environments on one side, and chemical or pharmacological treatments, including herbal medicines or natural extracts on another. For a recent quite complete review of preconditioning treatments of MSCs and their effects, readers are directed to the review by Hu and Li [134]. It is interesting to note that preconditioning of MSCs with low-dose lipopolysaccharide (LPS), a major component of Gram-negative bacteria, preserves mitochondrial membrane potential inhibiting cytochrome c release in hypoxia serum-deprived cultured cells [135], suggesting that a mild local infection could in fact potentiate stem cell treatment. Therefore, it should be taken into account that patients undergoing stem cell therapies are often subjected to additional pharmacological treatments and exposed to particular environmental factors which may impact the performance of the introduced stem cells at the post-implant level. To circumvent the uncertainty associated with these hard-to-control variables, genetic modification of MSCs toward the production of defined immunoregulatory effects or homing molecules is starting to be explored. For example, EAE was shown to be consistently attenuated by using engineered MSCs with CNS-homing ligand genes along with overexpression of IL-10 [136].

#### **4.4. Extracellular vesicle-based therapeutics**

Although autologous MSCs constitute a safer choice in terms of avoiding unwanted immune responses, donor comorbidities may hamper the use of their own stem cells. Expanded allogeneic MSCs were initially believed to be immune privileged due to their low expression of 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 product could be highly relevant on safety terms.

Disease (ME-HaD) position paper [147]. Also, the International Council for Harmonization of Technical Requirements for Pharmaceuticals of Human Use (ICH) mission guidelines to ensure the production of safe and effective high-quality medicines can be accessed on the

Culturing Adult Stem Cells for Cell-Based Therapeutics: Neuroimmune Applications

http://dx.doi.org/10.5772/intechopen.80714

125

Although clinical trials using EVs are still seldom, several companies have already engaged in EV or secretome production. A list of these companies with the corresponding links to their web pages can be found in the recent review by Gimona et al. [148]. This review includes a further in-depth review of clinical-grade EV production current status and remaining challenges. Involvement of biobank networks with pharmaceuticals may be relevant for granting

Lastly, it should be mentioned that EVs can also be used as a sensor of stem cell plasticity or other cell features as they reflect characteristics of the cell of origin [150] constituting a helpful

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

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

This work was supported by the Universidad Católica de Valencia San Vicente Mártir Research

Grant 2018-121-001 and MINECO (MAT2015-66666-C3-2-R FEDER).

following link: http://www.ich.org/products/guidelines.

standardized GMP production consistency of EVs [149].

**5. Conclusions**

hurdles for their use in the clinic.

**Acknowledgements**

treatments once GMP production is optimized.

tool to develop optimized differentiation and preconditioning protocols.

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 communication 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 Disease (ME-HaD) position paper [147]. Also, the International Council for Harmonization of Technical Requirements for Pharmaceuticals of Human Use (ICH) mission guidelines to ensure the production of safe and effective high-quality medicines can be accessed on the following link: http://www.ich.org/products/guidelines.

Although clinical trials using EVs are still seldom, several companies have already engaged in EV or secretome production. A list of these companies with the corresponding links to their web pages can be found in the recent review by Gimona et al. [148]. This review includes a further in-depth review of clinical-grade EV production current status and remaining challenges. Involvement of biobank networks with pharmaceuticals may be relevant for granting standardized GMP production consistency of EVs [149].

Lastly, it should be mentioned that EVs can also be used as a sensor of stem cell plasticity or other cell features as they reflect characteristics of the cell of origin [150] constituting a helpful tool to develop optimized differentiation and preconditioning protocols.
