**3. Stem cell therapy for other neuroimmune-related health problems: potential benefits for the treatment of myalgic encephalomyelitis/ chronic fatigue syndrome (ME/CFS)**

instance, therapeutic effects in sensorimotor function recovery in preclinical models [22, 23]

Among these cell types used, mesenchymal stem cells (MSCs) from the adipose tissue, bone marrow [27], umbilical cord, [28] or dental pulp [29], in addition to olfactory ensheathing glial

Preclinical studies have shown that MSCs have potent anti-inflammatory, anti-apoptotic, immunomodulatory, and angiogenic effects post-SCI [33]. MSC transplantation, overall, results in substantially improved locomotor recovery among animal models of SCI [34]. There have also been several clinical trials using autologous bone marrow-derived MSCs. These early studies confirm the safety of different administration protocols using MSCs post-SCI. It seems that sufficient quantities of transplanted allogeneic MSCs combined with immunosuppression prolong the survival of engrafted cells and improve functional and morphological

Transplantation of neural stem/progenitor cells (NSPCs) has shown promising results in the repair and regeneration of lost neural tissues and the associated restoration of neurological deficits [36] with particular benefits among other cell types. The engrafted transplanted NSPCs generate a favorable non-inhibitory environment for functional recovery creating additional paracrine activity modulating the post-SCI inflammatory response, feeding the injured area with growth factors, and rendering additional neurotrophic support by releasing, among others, GDNF. NSPCs include multipotent stem cells present in the ependymal region lining the central canal of the spinal cord (epSPC) [37]. epSPC represents an ideal candidate for stem cell therapy based on noted functional improvements after transplantation and the absence of malignant transformation, offering a safe and relevant cell type for clinical applications. The rationale for the therapeutic application of epSPC for SCI includes the replacement of damaged neurons and glial cells, secretion of trophic factors, regulation of gliosis and scar formation, prevention of cyst formation, and enhancement of axon elongation. After SCI, epSPCs proliferate and migrate to the injured area and produce new oligodendrocyte precursor cells (OPCs) [37]. Acute [38] and chronic [39] transplantation of undifferentiated epSPCs from SCI donors or *in vitro* differentiated OPCs into a rat model of severe spinal cord contusion produced significant locomotion recovery 1 week after injury. Transplantation of epSPCs provides trophic support and positively modulates the local immune response. It reduces purinergic receptor expression associated with neurodegenerative and neuropathic pain, thereby inducing signals promoting neuronal protection and survival with axonal outgrowth [40]. Interestingly, an immortalized human fetal epSPC line (HuCNS-SC) has been the main focus of cell therapies developed by the company Neuralstem, Inc. (USA), and therapies based on this product have been applied to human subjects in a phase II clinical trial. Phase I/II trial has

declared no adverse effects of treatment, with modest functional improvements [41].

Stem cell therapy can contribute to SCI repair not only by restoring the damaged tissue through differentiation and engraftment but also by potentiating endogenous tissue regenerative potential. Both processes are influenced by the action of the immune system controlling local inflammation, and thus stem cell paracrine factor role in this context should be carefully

(OEG) cells [30], Schwann cells [31], or neural precursor cells [32], have been used.

and in several ongoing clinical trials [24–26].

outcomes after SCI [35].

114 Cell Culture

evaluated.

Mesenchymal stromal cells (MSCs) have been used in clinical trials (CTs) for a broad range of immune-related health problems such as acute and chronic inflammatory disorders, autoimmune diseases, and transplant rejection by their potent immunosuppressive and antiinflammatory properties [42–45]. As reviewed by Wang et al., as of April 2016, over 500 MSCrelated clinical trials were registered on the NIH clinical trial database (https://clinicaltrials. gov/). Although the immunomodulatory properties of MSCs have more recently been identified, almost half of the registered CTs (230 or 42% of them) have or are being conducted for immune- or inflammation-mediated diseases (see **Figure 2**) [45].

Multiple sclerosis (MS) and its animal model (experimental autoimmune encephalomyelitis or EAE) associate with CNS inflammation, gliosis, demyelination, and axonal loss. MSCs' pleiotropic properties, including immunomodulation, immunosuppression, neurotrophy, and repair-promotion, make them attractive candidates for the treatment of neurodegenerative diseases, including MS [42–46]. The remyelination benefits reported in MS are largely attributed to paracrine signals and secreted soluble molecules such as tumor growth factor (TGF-β1), interferon (INF)-γ, indoleamine 2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2) [46–48]. On another side, neural precursors obtained from induced pluripotent stem cells (iPSCs) promote the viability of endogenous OPCs facilitating remyelination through the secretion of leukemia inhibitory factor (LIF) in EAE [46, 49–51]. LIF, a member of the IL-6 cytokine family implicated in the pathophysiology of MS, has shown to offer neuroprotection and axonal regeneration as well as prevention of demyelination [49–51].

**Figure 2.** Summary of the number of clinical trials using MSC therapy in immune- or inflammation-mediated diseases, as registered on the website https://clinicaltrials.gov (accessed April 2016). MS, multiple sclerosis; T1DM, type 1 diabetes mellitus; GVHD, graft-versus-host disease; OA, osteoarthritis; IBD, inflammatory bowel disease (a). MSC-derived paracrine factors mediating immunomodulatory functions, particularly toward T lymphocytes, in preclinical animal studies of various immune- and inflammation-mediated diseases (b). Source: Wang et al. [45].

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multiorgan system disease, often devastating, for which no single diagnostic test yet exists. The diagnosis of ME/CFS is based on exclusion, meaning other medical conditions, including psychiatric disorders, must be first ruled out. The disease is characterized by profound fatigue and disability lasting for at least 6 months, episodes of cognitive dysfunction, sleep disturbance, autonomic abnormalities, chronic or intermittent pain syndromes, microbiome abnormalities [52], cerebral cytokine dysregulation [53, 54], natural killer cell dysfunction [55], and other symptoms that are made worse by exertion of any kind [56, 57]. The Institute of Medicine (IOM) recently published an update of the diagnostic criteria recommended for CFS [56, 57]. The estimated worldwide prevalence of ME/CFS is 0.4–1%. The disease predominantly affects young adults, with a peak age of onset of between 20 and 40 years, and women, with a female-to-male ratio of 6:1 [58]. Although the etiological agent of ME/CFS remains unknown, the many hypotheses raised based on patient testimonies and clinical observations seem to lead to pathological immune system malfunctioning as one major factor. Autoimmune features on one side [59] and latent infection of unknown microorganisms, with a chronically activated immune system leading to inflammatory type situations, on another [60] have led our group to propose that stem cellbased therapeutics, as evidenced for MS, might be of benefit to these patients as well. The World Health Organization (WHO) has classified ME/CFS as a neurological disorder (International Classification of Diseases, Tenth Revision, Clinical Modification or ICD-10-CM R53.82; G93.3 if post-viral) based on the cognitive and other neurologic associated symptoms these patients suffer from. The neurological symptoms, however, could be explained by microglial activation and the lower-than-normal production of cortisol and adrenocorticotropic hormone (ACTH) these patients show, causing serotonin and corticotropin (CRH) deregulation [61]. A decrease in cortisol production by adrenal glands in turn can influence immune system activity [62]. MSC therapeutics could, at least partially, restore normal immune and, perhaps, neural functioning. Preclinical safety studies, however, should precede CT in ME/CFS.

harmonization, particularly across lab comparisons. Despite this limitation, consensus good manufacturing procedures (cGMP) for large-scale clinical-grade MSC have been developed [67–69], based on original low-scale lab preparation methods consisting of tissue trimming, enzyme-based (collagenase) dissociation, cell filtration, and cell-type selection through adher-

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

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

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Although the numbers of CT with MSCs are already considerable and increasing, only 13 human MSC-based products count with marketing authorization. As shown in **Table 1**, nine are developed for allogeneic therapies and only four for autologous. The main source for MSC manufacturing is the bone marrow, followed by adipose tissue, although others such as umbilical cord, cord blood, placental tissue, and Wharton's jelly are being explored. However, as ASCs (adipose stromal stem cells) possess similar therapeutic potential other than bone marrow MSCs as described by the ISCT and the International Federation of Adipose Therapeutics and Science (IFATS), and since they are obtained by minimally invasive procedures from a generally undesired tissue, the fat, they may shortly become the main choice of adult stem cells for clinical applications. In fact, as reported by Nordberg and Loboa, clinical trials using ASC raised from 18 to 152 in less than 5 years (2010 to the first quarter of 2015) [72]. Standard procedures based on single-use bioreactors yield superior quantities and quality of cells when compared to traditional planar multilayer cultivation systems, such as CELLstack,

Efficient manufacture of MSC-based products also takes costs into account. Either allogeneic or autologous therapies involve cGMP upstream processing (USP) through master and working cell banks (MCB and WCB, respectively) and downstream processing (DSP) events, a

These manufacturing processes are tightly regulated by the Advanced Therapeutic Medicinal Product (ATMP) path [73], the European Medicines Agency (EMA) in Europe, the Center for Biologics Evaluation and Research/Food and Drug Administration (FDA) in the USA, and the Central Drugs Standard Control Organization in Asia (readers are directed to selected

A potential formulation to standardize cell source has been proposed by Yi et al. who using GMPs could expand clonal MSCs from a single colony-forming unit (CFU)-derived colonies

Typically, the conventional media used for clinical production of MSCs are the common, defined Dulbecco's Modified Eagle Medium (DMEM) and Minimum Essential Medium (MEM) basal media supplemented with 10–20% fetal bovine serum (FBS), due to limitations of human alternatives and to cost reasons, although FBS is not cGMP compliant. FBS is prone to batch-to-batch variation and to contamination with prions, viral and zoonotic agents [76]. Thus, most clinical trials (phases I to III) used ASCs or other MSCs produced in the presence of FBS, some of them reporting immunogenic effects in patients, elicited by components of FBS (antibodies against components of FBS, Arthus, and anaphylactic reactions) [77–79]. In addition, the immune responses elicited by FBS could turn into rejection of the transplanted cells in cell-based therapies restricting their therapeutic efficacy. FBS-free alternatives can be basically grouped into serum-free (SF) medium containing animal-derived or human serum albumin and growth factors (GFs). Among human alternatives, the use of autologous products obviates the need for infectious or other pathological agent testing but limits the

derived from a small amount of bone marrow to treat a number of patients [75].

ence to plastic and extended survival *in vitro* [70, 71].

HYPERStack, and CellFactories (Corning, Nalge) [67].

reviews for further legal regulatory details) [67, 73, 74].

summary of which are shown in **Figure 3**.
